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Available online at www.sciencedirect.com Journal of Food Engineering 85 (2008) 222–231 www.elsevier.com/locate/jfoodeng Microstructure affects the rate of chemical, physical and color changes during storage of dried apple discs Nuria C. Acevedo a, Vilbett Briones b, Pilar Buera a,*,1, José M. Aguilera b a Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria (1428), Ciudad de Buenos Aires, Argentina b Department of Chemical and Bioprocess Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile Received 16 April 2007; received in revised form 19 June 2007; accepted 28 June 2007 Available online 8 August 2007 Abstract Blanching, freezing and drying induce major changes in the physical properties of processed foods. Microstructural changes induced by these processes in apple discs were related to the degree and kinetics of browning and to fracture mechanics after drying and later storage at 70 °C under a wide range of relative humidity (RH). Blanched and unblanched apple discs were dehydrated by vacuum drying or freeze-drying to induce the formation of different structures, then equilibrated from 33% to 75% RH and stored at 70 °C in order to promote browning. Color changes on the surface of apple discs were analyzed non-invasively by image analysis using a computerized vision system. Pre-treatments and drying conditions modified the structural characteristics of apple discs, which in turn, changed sorption properties, texture hardness and browning development. Microstructural changes (e.g., loss of cellular integrity) promoted higher browning rates, the rate and degree of browning was analyzed. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Apple; Microstructure; Browning; Kinetics; Hardness; Freeze-drying; Vacuum drying; Color 1. Introduction Food materials science attempts to develop structure– property relationships in foods. In particular, it has been demonstrated that many desirable traits of food such as texture, color, or flavour, depend on the way foods are structured (Aguilera, 2005). In the case of plant food tissues (e.g., fruits and vegetables) pre-treatment such as blanching and processing (canning, freezing, dehydration, etc.) induce significant changes in the microstructure whose effects on product properties are starting to be elucidated. Browning in dehydrated fruits could be caused by enzyme action, taking place in early stages of processing, previously to polyphenoloxidase inactivation, or by Maillard reactions or non-enzymatic browning (NEB), during * 1 Corresponding author. Tel./fax: +54 11 4576 3366. E-mail address: [email protected] (P. Buera). Member of CONICET, Argentina. 0260-8774/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2007.06.037 drying and the later storage. Browning discoloration lowers the quality value of products. Some of the deleterious effects that these reactions have in foods are the undesirable brown discoloration, textural changes, off-flavours, decreased solubility and loss of nutritional value. The effect of temperature and relative humidity (RH) on NEB rate has been extensively investigated in blanched and dehydrated systems. Mechanisms and factors influencing NEB have been reviewed by Labuza and Baisier (1992). Although the effect of phase changes and transitions on NEB kinetics has been reported (Buera & Karel, 1995), the impact of structural characteristics has not yet been analysed. The quantitation of brown coloration appearance is usually made spectrophotometrically after extraction of pigments in a solvent (Lievonen & Roos, 2002), or instrumentally by hand-held colorimeters (Krokida, Maroulis, & Saravacos, 2001; Lopez et al., 1997). These methods provide an average color value which is inappropriate to N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 discriminate the changes of color patterns in foods such as sliced apples (Hatcher, Symons, & Manivannan, 2004). Alternatively, computer vision is becoming an established tool to assess quality traits of foods based on its simplicity, analysis of the whole image and non-destructive nature. Changes in average color and color patterns of whole apple slices during dehydration were effectively determined by this technique (Fernández, Castillero, & Aguilera, 2005). The objective of the present work was to assess if the changes induced on tissue structure by different pre-treatments and drying methods were reflected in the development and kinetics of brown discoloration and in hardness changes during the later storage of dehydrated apple discs under different conditions. 2. Materials and methods 2.1. Materials Granny Smith apples were purchased from a local market (Santiago, Chile) and stored at 4 °C until the moment of the experiment. 2.2. Sample preparation Apple discs (22 mm diameter and 0.5 mm thickness) were obtained from a cylinder cut parallel to the main axis of the fruit using a cork borer and sliced by two parallel knives. Cellular debris on cut surfaces were removed by rinsing with distilled water. In order to avoid enzymatic browning the cut materials were submerged in a 1% (w/ w) ascorbic acid solution until blanching or freezing (approx. 1 min). In order to induce structural changes in apple tissue, apple discs with different pre-treatments and dehydration procedures were studied, leading to the following types of samples and nomenclature: Vacuum-dried (VD) Blanched/vacuum-dried (B/VD) Fast-frozen/freeze-dried (FF/FD) Slow-frozen/freeze-dried (SF/FD) A fifth group of samples (FD/P) was included for comparative purposes in the form of a powder by grinding slow-frozen freeze-dried discs in a mortar before thermal treatment (data from Acevedo, Schebor, & Buera, 2006). Steam treatment in B/VD samples had the objective of denaturing cell membranes and also this pre-treatment inactivated the enzymes causing enzymatic browning. It was accomplished by holding apple discs over a metal grid, exposing them for 3 min to live steam and immediately after submerging the samples in cold water until the dehydration process. The freeze-drying process requires freezing the sample as a previous procedure. Two freezing rates (fast and slow freezing, FF and SF, respectively) were used to freeze unblanched apple discs (samples FF/FD and SF/FD) in 223 order to induce changes in apple structural characteristics. Samples FF/FD were frozen by immersion in liquid nitrogen immediately before the freeze-drying process, while samples SF/FD were frozen at approximately 20 °C in a home freezer for 12 h and then freeze-dried. Vacuum drying was performed in a vacuum oven (model 5831, Napco, Winchester, VA) over desiccant (silica gel) at 50 °C and 25 in Hg vacuum for 4 h. Residual water (approx. 2–3%) was removed by incubation over P2O5 in vacuum desiccators at 20 °C for 12 h. The freeze-drying process lasted 48 h. A Labconco 4.5 (Kansas, USA) freeze-dryer was used which operated at 70 °C, at a chamber pressure of 6 103 mbar. After drying, apple discs (15 samples per treatment) were equilibrated at 20 °C over appropriate saturated salt solutions in vacuum desiccators. Saturated solutions of magnesium chloride (MgCl2), magnesium nitrate (MgNO3), sodium bromide (NaBr), sodium chloride (NaCl) salts were used for 33%, 52%, 64% and 75% relative humidity, respectively (Greenspan, 1977). 2.3. Methods The water content was determined by difference in weight before and after vacuum drying over desiccant (silica gel) at 70 °C for 24 h or 48 h. The remaining water in the samples was removed in vacuum desiccators over desiccant (P2O5) at 20 °C for 12 h. Water content was expressed as mass of water per mass of solids (in dry basis, d.b.), as the average of two measurements. Soluble solids, measured as sugar, were determined in apple juice using a refractometer (ATAGO Co. Ltd., model NAR-3T, Japan) and expressed as °Brix. The pH of apple juice was measured with a pHmeter (model 320, Hanna Instruments, Portugal). Titratable acidity was determined by titration against a 0.1 N NaOH solution to pH 8.1 and the acidity was expressed as percentage citric acid (AOAC, 1984). 2.4. Environmental scanning electronic microscopy (ESEM) The microstructure of the cross-section of samples from all four pre-treatments was examined using ESEM (LEO 1420VP, Carl Zeiss SMT AG, Oberkochen, Germany) without metal coating. At least duplicate specimens were viewed at different magnifications and images of representative areas saved for further analysis. 2.5. Computer vision system (CVS) The CVS used consisted of three elements: a lighting system, a digital camera and a personal computer. The lighting system included four D65 lamps inside a dark chamber. This is particularly important because the color of the sample will depend on the light spectrum generated by the source of illumination. The angle between the camera axis and the light source was 45° in order to capture 224 N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 the fuzzy reflection responsible for color which is produced at this angle (Papadakis, Abdul-Malek, Kamden, & Yam, 2000). The digital camera, a Power Shot A70 (Canon Inc., Tokyo, Japan), is positioned above the base on which the samples are placed. Samples were transferred from the forced air oven to the dark box and images acquired every 2 h during the whole storage period. The iris was operated in manual mode, with the lens aperture at f = 8 and speed 1/6 (no zoom, no flash) to achieve high uniformity and repeatability. The calibration of the camera and the parameters used for image capture are described in Briones and Aguilera (2005). Images of apple discs had a resolution of 2048 1536 pixels. Images were saved on a PC (Pentium III, 30 GB, 300 MHz) in uncompressed JPG format using Canon’s Remote Capture program (Canon Inc., USA). 2.6. Storage and color measurement Apple discs equilibrated to different RH were placed inside rubber o-rings which in turn were sandwiched between two glass plates held hermetically with metal clamps to avoid water loss, as depicted in Fig. 1. The glass sample holders were then placed in an air-convection oven kept at 70 ± 1 °C to observe changes during high temperature storage at constant water content. The glass plates containing six discs were removed every 2 h to acquire images and placed back in the oven. CVS permitted acquiring information for the whole pieces directly inside the glass plates and then calculate an average color for the entire image. Color images of apple disc were digitized into pixels of 24 bits containing levels of the three primary colors: red, green and blue. Then RGB values were converted to the L, a and b color values using a program Adobe PhotoshopÒ 7.0 (Adobe Systems Inc., San Jose, CA). L, a and b are not standard color values however they can be converted to CIELab (or L*, a*, b* space) using mathematical formulas described by Papadakis et al. (2000). The CIELab space is commonly used in research and quality control of foods. This color space is device-independent, creating consistent colors regardless of the device used to acquire the image. L* is the luminance or lightness component, which ranges from 0 to 100 from black to white, while Glass plates Apple disc clip Glass plates o-rings clip o-ring Fig. 1. Sample holder for apple disc thermal treatment and color measurement. Entrance of probe head Screw system Apple disc Fixed to TA.XT2 Fig. 2. Scheme of test cell for measurements of maximum force at fracture (hardness). N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 a* (green to red) and b* (blue to yellow) are the two chromatic components. The color function selected to follow browning changes was DL*, which is defined as L0 L , where L0 is the lightness before the high temperature storage and L* is the corresponding value at a given storage time. DL* is sometimes referred to as browning index. 2.7. Force at fracture (hardness) measurements Force at fracture was measured in apple discs before and after storage at 70 °C by puncture test using a texture analyzer TA.XT2 (Texture Technologies Corp., Scarsdale, NY). A special test cell (Fig. 2) was designed so that apple discs (five samples per treatment) were tightly held by a bolted metal ring while penetrated through the center by a cylindrical probe of 2 mm diameter. Crosshead speed was 1 mm/s and force–displacement curves were recorded as the plunger descended through the apple disc to the point of fracture and the maximum force recorded as hardness. The reported values of hardness correspond to the average of the individual measurements of five apple discs corresponding to each of the four sample types and the respective RH. The calculated confidence interval ranged between 5% and 10%. 3. Results and discussion 3.1. Effect of pre-treatment and drying Fresh Granny Smith apples had 12.6 ± 0.2 °Brix and 84.4 ± 0.2% water (wet basis) at the initial time of the experiment. Fig. 3 shows the water adsorption isotherms (20 °C) for dehydrated apple discs equilibrated at different relative humidities. Samples belonging to groups VD, B/ VD and SF/FD showed similar water sorption isotherms, which were also comparable to those of the powdered freeze-dried (P/FD) apple samples analyzed in a previous work (Acevedo et al., 2006). FF/FD samples (fast-frozen/ 225 freeze-dried), however, were more hygroscopic and adsorbed more water than the rest of the samples, particularly at RH > 33%. It is known that fast-frozen, freezedried materials have higher porosity (more and smaller pores) than similar slow-frozen materials and consequently more effective sites for water adsorption (Aguilera & Stanley, 1999), thus, a high hygroscopicity in these samples was to be expected. The water adsorption isotherms shown in Fig. 3 were fitted to the GAB equation using the least square method for minimizing the absolute differences between measured and calculated water content values, and the water content at the monolayer (mm) was calculated (van den Berg & Bruin, 1981). The following equation (Eq. (1)) was used to evaluate the sorption surface area (Gregg & Sing, 1982): A ¼ mm Na=M ð1Þ where A is the surface area of adsorbent, N is the Avogadro’s number (6.02 1023 molecules/mol); M is the molar mass of water (18 g/mol); a is the area of one water molecule (10.6 1020 m2). The specific surface area of adsorption places plays an important role in determining the water binding properties of materials (Tolaba, Peltzer, Enriquez, & Pollio, 2004), and the obtained values are shown in Table 1. The powdered (P/FD) samples had the highest monolayer value, and correspondingly, the highest surface area, followed by the FF/FD samples. The rest of the samples had lower effective surface for adsorption. During fast-freezing many small water crystals are rapidly formed which leave a very porous material after water sublimation, hence freeze-dried fruit products have more surface area for water adsorption (Tsami, Krokida, & Drouzas, 1998). Therefore, after freeze-drying the apple matrices FF/FD presented larger surface/volume ratio than those SF/FD, which explains their higher capacity to adsorb water. Structural changes after drying were observed by ESEM and shown in Fig. 4a–d. A higher cellular collapse and tissue disruption were observed in vacuum-dried apple discs Table 1 Sorption parameters corresponding to the different apple samples Fig. 3. Water adsorption isotherm (20 °C) for dehydrated apple discs equilibrated at different relative humidity. The results from freeze-dried and powdered samples are shown in dotted lines. System mma (g water/g solids) Ab (m2/g) %Ec VD B/VD FF/FD SF/FD FD/Pd 0.0624 0.0669 0.0715 0.0658 0.0836 220.90 236.83 253.1 232.93 295.94 3.4 2.7 2.7 5.7 2.5 a mm is the monolayer water content calculated through the GAB equation. b A is the effective surface for water sorption calculated as A = mmNa/ M. c %E is the goodness of fit as applied to the experimental adsorption P isotherms of apple systems, calculated as %E = 100/n (mi mi*)/ mi, where n is the number of experimental points, mi is the measured water content and mi is the calculated content. d Results from freeze-dried and powdered samples (FD/P) were taken from Acevedo et al. (2006). 226 N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 Fig. 4. ESEM micrographs of cross-sections of apple discs. (a–d) After drying: (a) vacuum-dried, VD samples, (b) blanched and vacuum dried, B/VD samples, (c) fast-frozen and freeze-dried, FF/FD samples and (d) slowly frozen and freeze-dried, SF/FD samples. (e–h) After storage at 70 °C: (e) VD samples, (f) B/VD samples, (g) FF/FD samples, and (h) SF/FD samples. (samples VD and B/VD) than in freeze-dried apple discs (samples FF/FD and SF/FD). The freezing rate is a variable recognized as responsible for tissue damage (Fuchigami, Kato, & Teramoto, 1997). It is generally accepted that high freezing rates retain the quality (and thus, the structure) better than slow freezing rates (Partmann, 1975). Slow freezing rate causes severe changes in product microstructure, SF/FD samples presented broken surfaces, as shown in Fig. 4d, probably due to the higher ice crystals formed during slow freezing before freeze-drying. This is due the mechanical stress which is provoked by extracellular ice formation leading to membrane rupture and to the cryoconcentration phenomena, which promotes membrane denaturation and cell wall degradation. Membrane rupture N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 results in enzyme and/or chemical activity that also contributes to the mechanical damage (Fuster, Prestamo, & Cano, 1994; Tregunno & Goff, 1996). The different freezing rate in samples FF/FD and SF/FD caused a significant influence in water sorption properties, as previously discussed, and this change was also manifested at microscopic level by ESEM observation (Fig. 4a–d). Samples SF/FD presented deformed stretched pores due to the higher ice crystals formed during slow freezing, which presumably caused the broken surfaces visually observed. ESEM images of the different dried samples subjected to further high temperature storage at 70 °C are also shown in Fig. 4e–h. After storage total loss of cellular integrity was observed in discs VD and B (Fig. 4e and f), while in samples FF/FD and SF/FD (Fig. 4g and h) structural changes were less drastic and remnants of void cells could be observed. It is interesting to note that although thermal treatment extended structural modifications, the most important differences occurred already among the different pre-treatments and/or dehydration methods. 3.2. Color development Fig. 5 depicts a gallery of images of dehydrated apple discs equilibrated at different RH as they underwent storage at 70 °C (note: at each RH the same specimen was photographed through time). Specimens at the initiation of storage (time 0) in Fig. 5 are representative of the variation in color and size after each drying treatment. Vacuumdried samples presented darker colors and more irregular borders and shapes, evidencing a certain degree of shrinkage. Freeze-dried apple discs were appreciably lighter in 227 color, rounder and larger at the beginning of storage compared to vacuum-dried samples. Browning increased with storage time at 70 °C, and was more evident at 64% and 75% RH, but no major changes in shape or size of specimens were appreciated upon further heating. Vacuum drying led to appreciable darkening of samples during storage compared to freeze-drying. In summary, visual observation of samples in Fig. 5 shows that the treatment that better preserved structure and color was FF/FD, followed by SF/FD, and that the appearance of VD samples were the most affected. It is interesting to note that at the beginning of storage blanched samples were lighter than unblanched samples and that browning during the storage period was less intense. This suggests that not only NEB but also enzymatic browning (EB) played a major role in color changes during storage. In fact, the color difference at each time (and constant RH) between VD and B/VD may be taken as an EB contribution to browning. Fig. 6 (a–d) shows the changes in lightness (DL*) or browning index versus storage time at a given RH for all samples. Data from samples powdered after freeze-drying were also included for comparative purposes (dotted lines). It has to be kept in mind that the index DL* measures differences in color with respect to time 0 and is not an absolute color value. An almost linear trend for DL* increase was observed as a function of time for all groups but was more pronounced at higher RH. The browning index, however, was different depending on the pre-treatment and/or drying conditions of the samples. For example, at a given time, vacuum-dried samples had developed higher browning indexes than freeze-dried samples, which retained a light color until the end of the storage period. After drying Fig. 5. Gallery of images of dehydrated apple discs during storage at 70 °C as a function of RH: (a) vacuum-dried, VD samples, (b) blanched and vacuumdried, B/VD samples, (c) fast-frozen and freeze-dried, FF/FD samples and (d) slowly frozen and freeze-dried, SF/FD samples. 228 N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 Fig. 6. Change in color lightness (DL*) of apple discs stored at 70 °C as a function of time. Samples: VD (h), B/VD (*), FF/FD (N), SF/FD (d) and FD/ P (s), dotted line. (a) 33% RH, (b) 52% RH, (c) 64% RH and (d) 75% RH. Results from freeze-dried and powdered samples are shown in dotted lines. VD samples were darker than B/VD samples, and after storage they presented a browning index 25–42% higher than B/VD samples. As mentioned before, the larger browning indexes developed in VD samples are possibly the consequence of the contribution of enzymatic browning occurring in these non-blanched samples. Rate constants for non-enzymatic browning development were calculated using linear regression analysis and DL* as response variable. The regression coefficients of the pseudo zero order reaction rate constants (slope of the lines in Fig. 5a–d) varied from 0.89 to 0.99. Fig. 7 shows the rate constants of browning for samples VD, B/VD, FF/FD and SF/FD and also for the powdered samples (FD/P) as a function of RH. Powdered samples showed the highest browning rates at all RH, probably due their higher proportion of broken cells that may increase the effective release of reacting species and their diffusion, thus accelerating the Maillard reaction. VD samples exhibited higher browning rates than those of the B/VD group (Fig. 7), which can be attributed to the effects of blanching causing inhibition of the enzymes responsible for enzymatic browning. Other enzymes, involved in the release of reducing sugars from pectins, may also affect browning of Fig. 7. Browning rate of apple discs stored at 70 °C as a function of RH. Group VD (h), Group B/VD (*), Group FF/FD (N), Group SF/FD (d) and Group FD/P (s). Results from freeze-dried and powdered samples are shown in dotted lines. samples, as well as protein aggregation and solute mobilization during blanching (Greve, McArdle, Gohlke, & Labavitch, 1994). N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 Up to a RH of 40%, FF/FD samples showed higher browning rates than SF/FD samples, after which the rates were almost similar. This result can be explained on the basis of the effect of freezing rate on porosity and the sorption properties of the freeze-dried samples. The higher water sorption capacity of FF/FD samples (see Fig. 3) compared to that of SF/FD samples may also be a factor preventing browning development, since it is known that water exerts an inhibitory effect on the Maillard reaction (Acevedo et al., 2006). These results suggest that one way to control browning is to preserve the structure of apple discs. The extreme case is the freeze-dried powdered sample where all structure has been obliterated and exhibited the highest browning rate. It is interesting to note that independently of the pre-treatment and drying method, the browning rate followed parallel trends as increasing RH. There are evidences that the maximum browning rate in vegetable tissues occurs around 70% RH (Acevedo et al., 2006; Hendel, Silveira, & Harrington, 1955; Lee, Chung, Kim, & Yam, 1991; Rapusas & Driscoll, 1995). Thus, a decrease in browning rate is expected at RH values higher than 75%, as was experimentally obtained for powdered apple samples (FD/P) at 85% RH (Fig. 7). 229 3.3. Hardness of samples Hardness values (force at fracture) obtained by the puncture test are plotted in Fig. 8 as a function of RH for the four sample groups. Grey bars represent data collected before storage of the samples while black bars correspond to data obtained after 36 h of storage at 70 °C. Samples dried under vacuum at 50 °C (VD and B/VD) showed higher hardness values than FD samples at similar RH. Storage induced no change or a slight increase in hardness of VD samples except at RH = 33%, where the hardness of the stored samples appears to be larger than initially. Maximum hardness was always observed at the lowest RH (33%). Bourne (1986) showed that hardness of apple tissue increased logarithmically as aw decreased (up to aw values of 0.12), similar to what occurs in protein gels (Beveridge, Arntfield, Ko, & Chung, 1980). Fracture of dried food products is a complex phenomenon that depends largely on the material itself, the plasticizing effect of water as well as on the presence and propagation of microscopic cracks induced by processing (Castro & Aguilera, 2007). Based on these facts and the previous structural information, the following interpretation of hardness results can be made: dehydrated cell walls Fig. 8. Force at fracture (hardness, Newton) of apple discs equilibrated at different RH after drying (t = 0 h) and after storage at 70 °C (t = 36 h). Segments over bars represent confidence intervals. 230 N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 Table 2 Effect of induced structural damage on browning and hardness development during thermal treatment of dried samples rehumidified at 33% RH System Structural damage after dryinga Browning rateb (DL*/h) L0 c L *d Hie (N) Hff (N) VD B/VD FF/FD SF/FD FD/P ++ +++ +/ + NDg 0.89 0.46 0.70 0.28 0.90 51.8 ± 0.1 60.2 ± 0.1 73.3 ± 0.1 70.1 ± 0.1 80.8 ± 0.1 28.3 ± 0.1 42.9 ± 0.1 55.8 ± 0.1 56.7 ± 0.1 66.4 ± 0.1 16. ± 3 17. ± 3 7. ± 2 12. ± 2 NDg 32. ± 2 24. ± 3 8. ± 1 12. ± 1 NDg a Degree of structural damage, as evaluated by ESEM and correlated with visual macroscopically perceived changes. (/+) slightly damaged structure, (+) low degree of structural change; (++) moderate structural changes; (+++) greatly damaged structure. b Browning rate during heat treatment at 70 °C. c L0 : initial lightness, after drying. d L*: final lightness after 36 h of thermal treatment at 70 °C. e Hi: hardness value after drying. f Hf: hardness value after heat treatment at 70 °C. g ND: not determined. are the common material providing structural integrity and mechanical resistance to all the dried apple discs. Due to shrinkage (see Fig. 5) VD samples became denser than FD samples, thus a larger fracture force was to be expected. As water content increases (i.e., higher RH) water plasticizes the cell walls and the material, which is strong brittle at RH = 33%, becomes softer and more pliable, thus hardness decreases. Since water content is constant during storage the only possible effect of storage at 70 °C on hardness is that due to exposure to this temperature. Consequently, with the exception of the sample VD (at RH 33%) there are no observable differences in sample hardness between the initiation and the end of storage, which is to be expected because major temperature effects on hardness are not expected to occur at 70 °C. Some authors have argued that blanching prior to dehydration causes disruption of cell membranes and a concomitant faster and more complete drying resulting in a harder material (Lazar & Rasmussen, 1964), but this phenomenon could not be statistically demonstrated here because hardness differences between blanched and unblanched samples were not significant. Beveridge and Weintraub (1995) reported that blanching did not affect the texture of airdried apple samples equilibrated in a range between 70% and 77% RH. Freeze-drying introduces another variable beyond shrinkage which is porosity and cell wall damage due to the formation of ice crystals during freezing. Freeze-dried apple discs (FF/FD, fast-frozen and SF/FD, slow-frozen) showed lower hardness than VD samples. The hardness values obtained for FF/FD apple discs were the lowest of all samples and showed no appreciable differences as a function of RH. On the other hand, the SF/FD samples showed a decrease in hardness as the RH increased (both, before and after storage). Unexpected low hardness values for FF/FD samples at low aw may only be explained by the presence of more fragile cell walls pierced by small ice crystals. During slow freezing on the other side, water can migrate and large ice crystals are generated extracellularly. Cell wall cracking and piercing by ice crystals has been observed in frozen apple tissue by Fuster et al. (1994) and Tregunno and Goff (1996). Table 2 summarizes the results for samples humidified to 33% RH, which is a representative condition for dehydrated products. The degree of structural damage was evaluated through ESEM (Fig. 4a–d), and qualitatively correlated to macroscopically observed changes (Fig. 5). As shown in Table 2, in the fast-frozen freeze-dried samples, in which the global quality (structure, hardness and color) was better preserved, the rate of browning development was higher than in SF/FD and B/VD samples, probably because their higher area for water sorption (shown in Table 1) favors the reaction (being more water attached to the active sites in the solids, its inhibitory is less evident). However, the final color of the freeze-dried samples was lighter than that of the vacuum-dried ones because of the better conservation of structure (Table 2). After drying or storage at 70 °C hardness values were also higher for the vacuum-dried samples than for the freeze-dried ones, in parallel to color changes and shrinkage. It is to be noted that the hardness values of freeze-dried samples were not affected by the heat treatment at 70 °C. 4. Conclusions All treatments to which the apple discs were subjected caused structural modifications at the macroscopic (i.e., size, shape) and microstructural levels (i.e., cell walls and cell membrane). These structural changes affected the water sorption properties and possibly led to the release of reacting species involved in browning, thus facilitating their diffusion and reaction. Changes were also directly related to the final texture of the product. Color measurements through the CVS system provided reproducible values of browning changes without invasion of the sample. Particularly interesting is the fact that pre-treatment or processes affected effective surface for water adsorption. Since water plays an inhibiting role in the Maillard reaction, a higher surface for water adsorption restricts water availability at a microscopic level promoting a higher reaction rate. This N.C. Acevedo et al. / Journal of Food Engineering 85 (2008) 222–231 observation is also in agreement with the findings of White and Bell (1999), although their interpretation pointed out to the presence of preferential sites of reactivity for the Maillard reaction in the porous material. The better preserved structure of the freeze-dried samples avoided hardness development due to shrinkage and collapse during storage at 70 °C. The effects of suprastructural changes on the rate of chemical reactions were scarcely explored in the literature concerning food systems. This paper shows that the matrix structure and its dynamics modifications play a decisive role in determining the kinetics of physico-chemical phenomena. 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