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Fig. 2. Young’s modulus obtained by a compression test of apple slices (2 cm diameter and 0.8 cm height) after vacuum-impregnation with PME (90 nkat g~') and calcium (0.5% (w/w) CaCl, 2H20 solution) (a) or no treatment (control) (b). The test was performed after the slices were maintained at 40°C for 20min and then pasteurized. Errors bars correspond to standard deviation (n = 20). Salvatori, Castro, & Almazora, 2004). structure was Young’s modu This preserved associated with a significantly higher us for pasteurized app impregnated with PME and calcium peaches (Javeri Degraeve et al. e cubes vacuum- han for controls (p < 0.05; Fig. 2). The higher mechanical rigidity of apple cubes infiltrated with PME and calcium ous reports on the firming effect of this agrees with previ- process on canned et al., 1991) and pasteurized strawberries , 2003). This firming of with the PME and calcium infiltration is attributed to the tissue associated of the methyl ester groups hydrolysis on Thom, 1973; Saurel, 2004). pectin and the interaction of the free carboxylic acid groups hus liberated with calcium (Grant, Morris, Rees, Smith, &

Figure 2 Young’s modulus obtained by a compression test of apple slices (2 cm diameter and 0.8 cm height) after vacuum-impregnation with PME (90 nkat g~') and calcium (0.5% (w/w) CaCl, 2H20 solution) (a) or no treatment (control) (b). The test was performed after the slices were maintained at 40°C for 20min and then pasteurized. Errors bars correspond to standard deviation (n = 20). Salvatori, Castro, & Almazora, 2004). structure was Young’s modu This preserved associated with a significantly higher us for pasteurized app impregnated with PME and calcium peaches (Javeri Degraeve et al. e cubes vacuum- han for controls (p < 0.05; Fig. 2). The higher mechanical rigidity of apple cubes infiltrated with PME and calcium ous reports on the firming effect of this agrees with previ- process on canned et al., 1991) and pasteurized strawberries , 2003). This firming of with the PME and calcium infiltration is attributed to the tissue associated of the methyl ester groups hydrolysis on Thom, 1973; Saurel, 2004). pectin and the interaction of the free carboxylic acid groups hus liberated with calcium (Grant, Morris, Rees, Smith, &

Related Figures (9)

Fig. 1. ESEM micrographs of parenchyma tissue of apple (core of apple cubes). After vacuum-impregnation with PME (450 nkat g~') and calcium (1% (w/w) CaCh, 2H20 solution), followed by incubation for 20 min at 40 °C (a) or no treatment (control) (b). Impregnated and control samples are heated at 85 °C for 15 min.
Fig. 1. ESEM micrographs of parenchyma tissue of apple (core of apple cubes). After vacuum-impregnation with PME (450 nkat g~') and calcium (1% (w/w) CaCh, 2H20 solution), followed by incubation for 20 min at 40 °C (a) or no treatment (control) (b). Impregnated and control samples are heated at 85 °C for 15 min.
Fig. 3. Carbon CPMAS spectra of re-hydrated alcohol-insoluble residue (AIR) from apple tissues non-impregnated (B) and vacuum-impregnated with PME and calcium, maintained at 40 °C for 20 min and then pasteurized (A). Cl—C6 correspond to signals attributed to cellulose, P1—P6 to signals attributed to pectins (P6 being the carbon of the methyl ester). The impact of PME and calcium infiltration in apple cube was also assessed by solid state '*C NMR spectros- copy of the alcohol-insoluble material. CPMAS (cross- polarisation magic angle spinning) NMR spectra are shown in Fig. 3B and A, respectively. They were character- istic of cell wall residues containing cellulose (C1—C6 sig- nals) and pectins (P1—P6). Larger signals were observed for the vacuum-impregnated samples (Fig. 3A), resulting in a lower spectral resolution compared to the non-impreg- nated samples (Fig. 3B). Since the spectra have been acquired under the same conditions, this observation is directly related to a higher anisotropy for tissues vacuum- impregnated with PME and calcium. Anisotropy observed by NMR increases with disorder (for crystallised samples) and/or molecular rigidity. The lower NMR resolution in the case of the enzyme infiltrated apple tissues (Fig. 3A) is attributed to an increase in the molecular rigidity of cell wall polysaccharides. The degrees of methyl-esterification of pectin in apple cubes vacuum-infiltrated with water or PME and
Fig. 3. Carbon CPMAS spectra of re-hydrated alcohol-insoluble residue (AIR) from apple tissues non-impregnated (B) and vacuum-impregnated with PME and calcium, maintained at 40 °C for 20 min and then pasteurized (A). Cl—C6 correspond to signals attributed to cellulose, P1—P6 to signals attributed to pectins (P6 being the carbon of the methyl ester). The impact of PME and calcium infiltration in apple cube was also assessed by solid state '*C NMR spectros- copy of the alcohol-insoluble material. CPMAS (cross- polarisation magic angle spinning) NMR spectra are shown in Fig. 3B and A, respectively. They were character- istic of cell wall residues containing cellulose (C1—C6 sig- nals) and pectins (P1—P6). Larger signals were observed for the vacuum-impregnated samples (Fig. 3A), resulting in a lower spectral resolution compared to the non-impreg- nated samples (Fig. 3B). Since the spectra have been acquired under the same conditions, this observation is directly related to a higher anisotropy for tissues vacuum- impregnated with PME and calcium. Anisotropy observed by NMR increases with disorder (for crystallised samples) and/or molecular rigidity. The lower NMR resolution in the case of the enzyme infiltrated apple tissues (Fig. 3A) is attributed to an increase in the molecular rigidity of cell wall polysaccharides. The degrees of methyl-esterification of pectin in apple cubes vacuum-infiltrated with water or PME and
Methyl-esterification degree of pectins in non-impregnated, vacuum- impregnated with water or with a 450 nkat PME g' solution apple cubes The two last samples were thermally treated for PME activation (40 °C, 20 min) and subsequently pasteurized (85 °C, 15 min).
Methyl-esterification degree of pectins in non-impregnated, vacuum- impregnated with water or with a 450 nkat PME g' solution apple cubes The two last samples were thermally treated for PME activation (40 °C, 20 min) and subsequently pasteurized (85 °C, 15 min).
Fig. 5. Mean immunofluorescence PME labelling intensity measured in the different zones of apple cubes vacuum-infused either only with water (control) or with PME (PME).
Fig. 5. Mean immunofluorescence PME labelling intensity measured in the different zones of apple cubes vacuum-infused either only with water (control) or with PME (PME).
Fig. 4. Immunofluorescent labelling of PME with anti-A. niger PME antibodies in apple parenchyma tissue: PME vacuum-infused (d, e, f); water-infused -ontrol (a, b, c); superficial zone (0-2 mm depth) of apple tissue cubes (a, d); intermediate zone (2-4 mm depth) (b, e), core zone (more than 4 mm depth in upple cubes) (c, f).
Fig. 4. Immunofluorescent labelling of PME with anti-A. niger PME antibodies in apple parenchyma tissue: PME vacuum-infused (d, e, f); water-infused -ontrol (a, b, c); superficial zone (0-2 mm depth) of apple tissue cubes (a, d); intermediate zone (2-4 mm depth) (b, e), core zone (more than 4 mm depth in upple cubes) (c, f).
Fig. 6. Immunofluorescence labelling of methyl-esterified homogalacturonan with JIM7 in apple parenchyma tissue: PME vacuum-infused (d, e, f); water- infused control (a, b, c); superficial zone (0-2 mm depth) of apple tissue cubes (a, d); intermediate zone (2-4 mm depth) (b, e), core zone (more than 4 mm depth in apple cubes) (c, f).
Fig. 6. Immunofluorescence labelling of methyl-esterified homogalacturonan with JIM7 in apple parenchyma tissue: PME vacuum-infused (d, e, f); water- infused control (a, b, c); superficial zone (0-2 mm depth) of apple tissue cubes (a, d); intermediate zone (2-4 mm depth) (b, e), core zone (more than 4 mm depth in apple cubes) (c, f).
Fig. 7. Mean immunofluorescence labelling intensity with JIM7 measured in the different zones of apple cubes vacuum-infused with either only water (control) or with PME (PME).
Fig. 7. Mean immunofluorescence labelling intensity with JIM7 measured in the different zones of apple cubes vacuum-infused with either only water (control) or with PME (PME).
Fig. 8. Immunogold labelling of cell walls with JIM7 (methyl-esterified homogalacturonans) in apple cubes vacuum-infused only with water (a, c and f) or with A. niger PME solution (b, d and g). (a) and (b) primary cell wall, (c) and (d) cell wall region lining intercellular space, (f) and (g) cell junction. j cell junction, cw cell wall, e intercellular space. At the tissue and cell levels (light microscopy), water infused samples, used as controls, showed an important and homogeneous fluorescence resulting from JIM7 anti- bodies binding in all the slices that are collected at different depths in apple cubes (Fig. 6a—c). By contrast, PME- infused samples showed fewer homogeneous fluorescent patches in all observed zones (Fig. 6d—-f). PME induces the hydrolysis of methyl esters on pectin and the vacuum- impregnation results in a homogenous pectin demethyl- esterification in the entire apple cubes. This qualitative observation is supported by a similar decrease in fluores- cence intensity resulting from JIM7 antibodies binding in all zones, including the apple cube core (Fig. 7). Thereby, vacuum-impregnation constitutes a valuable process for placing an enzyme in contact with its substrate homoge- nously in the fruit tissue (Guillemin et al., 2006).
Fig. 8. Immunogold labelling of cell walls with JIM7 (methyl-esterified homogalacturonans) in apple cubes vacuum-infused only with water (a, c and f) or with A. niger PME solution (b, d and g). (a) and (b) primary cell wall, (c) and (d) cell wall region lining intercellular space, (f) and (g) cell junction. j cell junction, cw cell wall, e intercellular space. At the tissue and cell levels (light microscopy), water infused samples, used as controls, showed an important and homogeneous fluorescence resulting from JIM7 anti- bodies binding in all the slices that are collected at different depths in apple cubes (Fig. 6a—c). By contrast, PME- infused samples showed fewer homogeneous fluorescent patches in all observed zones (Fig. 6d—-f). PME induces the hydrolysis of methyl esters on pectin and the vacuum- impregnation results in a homogenous pectin demethyl- esterification in the entire apple cubes. This qualitative observation is supported by a similar decrease in fluores- cence intensity resulting from JIM7 antibodies binding in all zones, including the apple cube core (Fig. 7). Thereby, vacuum-impregnation constitutes a valuable process for placing an enzyme in contact with its substrate homoge- nously in the fruit tissue (Guillemin et al., 2006).
Fig. 9. Immunogold labelling of cell walls with 2F4 (blocks of non-methyl-esterified galacturonic acid units) in apple cubes vacuum-infused only with water (a, b and c) or with an A. niger PME solution (d, f and g). a and b primary cell wall, c and d cell wall region lining intercellular space, e and g cell function. According to the decrease in pectin methyl-esterification and to the modification of pectin epitopes distribution in apple tissue and cell walls after PME infiltration, it is clear that the enzyme was active wherever found in the tissue. This result agrees with the Banjongsinsiri et al. (2004b) observations on A. niger PME vacuum-infused eggplant slices. Vacuum-impregnation resulted in a higher penetra- tion and in a more homogeneous distribution of the To complete this analysis, the formation of unesterified galacturonic blocks consecutive to the PME activity was also checked at the cell wall level (electron microscopy; Fig. 9). For this purpose, sections were immunolabelled with the 2F4 monoclonal antibody-recognizing calcium- dimerized oligogalacturonides of nine sugar residues (Lin- ers et al., 1989, 1992). In water-infused samples (Fig. 9a, c and f), the 2F4 epitope occurred mainly at tricellular
Fig. 9. Immunogold labelling of cell walls with 2F4 (blocks of non-methyl-esterified galacturonic acid units) in apple cubes vacuum-infused only with water (a, b and c) or with an A. niger PME solution (d, f and g). a and b primary cell wall, c and d cell wall region lining intercellular space, e and g cell function. According to the decrease in pectin methyl-esterification and to the modification of pectin epitopes distribution in apple tissue and cell walls after PME infiltration, it is clear that the enzyme was active wherever found in the tissue. This result agrees with the Banjongsinsiri et al. (2004b) observations on A. niger PME vacuum-infused eggplant slices. Vacuum-impregnation resulted in a higher penetra- tion and in a more homogeneous distribution of the To complete this analysis, the formation of unesterified galacturonic blocks consecutive to the PME activity was also checked at the cell wall level (electron microscopy; Fig. 9). For this purpose, sections were immunolabelled with the 2F4 monoclonal antibody-recognizing calcium- dimerized oligogalacturonides of nine sugar residues (Lin- ers et al., 1989, 1992). In water-infused samples (Fig. 9a, c and f), the 2F4 epitope occurred mainly at tricellular
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ChemistryMedicinePectin MethylesteraseFood Additive
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