E o o S M a b c a A R R 2 A A K M S M S E R 1 O w a f o O e s o h 0 food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 Contents lists available at ScienceDirect Food and Bioproducts Processing j ourna l ho me page: www.elsev ier .com/ locate / fbp ffects of calcium lactate and ascorbic acid on smotic dehydration kinetics and metabolic profile f apples ilvia Tappia,∗, Maria A. Maurob, Urszula Tylewicza, Nicolò Dellarosaa, arco Dalla Rosaa,c, Pietro Rocculi a,c Department of Agricultural and Food Sciences, University of Bologna, Cesena, Italy Department of Food Engineering and Technology, São Paulo State University (UNESP), São José do Rio Preto, Brazil Interdepartmental Centre for Agri-Food Industrial Research, University of Bologna, Cesena, Italy r t i c l e i n f o rticle history: eceived 14 July 2016 eceived in revised form 4 January 017 ccepted 26 January 2017 vailable online 16 February 2017 eywords: inimally processed apples ucrose ass transfer olutes impregnation ndogenous metabolic activity espiration rate a b s t r a c t The influence of the addition of calcium lactate (CaLac) and ascorbic acid (AA) to sucrose (Suc) osmotic solutions on osmotic dehydration kinetics and endogenous metabolic heat production of apple tissue was evaluated. Our research goal was to characterize mass trans- fer and endogenous metabolic phenomena of the tissue to obtain minimally processed apples. The presence of CaLac and AA in solution affected the mass transfer of water and solutes, which was attributed to the changes in the cellular structure and thus to spaces available for solute transport. The metabolic heat production in samples treated in sucrose solutions was slightly lower than in untreated samples, and it was further reduced with CaLac addition. However, samples impregnated with AA exhibited a higher heat production due to a metabolic response of the tissue to AA treatment. When combined with CaLac, the heat production decreased to a level lower than untreated samples, except for those that were treated for 120 and 240 min (higher impregnation), achieving the highest heat production values. These results confirm previous findings, suggesting that AA solution can promote a stress response on specific fresh-cut vegetable tissues, as well as an increase of their endogenous metabolic activity, as confirmed by the higher O2 consumption observed with the head space gas determination. © 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. its efficiency and improve the quality of the final products (Ahmed et al., 2016). . Introduction smotic dehydration (OD) of plant foods is a concentration process in hich water is removed from the plant tissue to a hypertonic solution nd solutes flow from the solution into the food. The water removal rom fresh plant tissues is usually greater than the solute gain because f the semi-permeability of the cell membranes (Ahmed et al., 2016). D depends on the tissue structure, which changes according to the nvironment and structure itself. Consequently, the complexity of tructures and properties of plant tissues are challenging factors for ptimizing processes and designing equipment (Fernandez et al., 2004). ∗ Corresponding author. E-mail address: silvia.tappi2@unibo.it (S. Tappi). ttp://dx.doi.org/10.1016/j.fbp.2017.01.010 960-3085/© 2017 Institution of Chemical Engineers. Published by Elsev In addition to the advantages of lowering the water content, the OD modifies the food composition. As a result, impregnation of desir- able solutes can improve the nutritional and sensorial characteristics (Akbarian et al., 2014; Silva et al., 2014b; Barrera et al., 2004). OD is becoming popular as a technique for obtaining minimally processed fruits, improving their quality and stability, and most recently, this method has been combined with other innovative techniques, such as pulsed high electric field, high hydrostatic pressure, ultrasound, cen- trifugal force, vacuum and gamma and irradiation, which can enhance ier B.V. All rights reserved. http://www.sciencedirect.com/science/journal/09603085 www.elsevier.com/locate/fbp http://crossmark.crossref.org/dialog/?doi=10.1016/j.fbp.2017.01.010&domain=pdf mailto:silvia.tappi2@unibo.it dx.doi.org/10.1016/j.fbp.2017.01.010 2 food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 The type of solute used in the osmotic solution is a fundamental issue, because beyond affecting the dehydration kinetics and process cost, it impacts the organoleptic and nutritional properties of the final product. Sucrose (Suc) is considered by many authors to be the optimal osmotic agent because it is associated with a higher efficiency than glucose (Saputra 2001), reducing enzymatic browning and aroma losses (Cortellino et al., 2011; Qi et al., 1998; Lenart, 1996). OD with calcium in solution has been used to increase the firmness of plant tissue and enhance the process efficiency, restricting the sugar gains and increasing the water losses (Ferrari et al., 2010; Mavroudis et al., 2012; Pereira et al., 2006). Calcium can reinforce cell walls by cross linking pectic polymers and is thus able to reduce damage from dehy- dration (Pereira et al., 2006). At the same time, when the concentration increases or as the treatment proceeds, damage to cell membranes may occur, as reported by Anino et al. (2006). Moreover, calcium has been used in osmotic solutions as a method for obtaining nutritionally for- tified products that can increase consumer intake (Silva et al., 2014b; Barrera et al., 2004). The addition of ascorbic acid (AA) to the osmotic solution has been used to reduce enzymatic browning (Robbers et al., 1997; Lenart, 1996) and compensate for the loss of ascorbic acid in fruits during dehydra- tion (Guiamba et al., 2016; Ramallo and Mascheroni, 2010). Various solutes can be added to the osmotic medium to obtain minimally processed products that can be stored at refrigerated tem- peratures. Nevertheless, it is important to consider that in addition to affecting the compositional and nutritional profile, they can affect the tissue metabolism, which can have consequences on the final product stability and shelf-life. Various authors have observed a reduction in the respiration rate of osmotically dehydrated mangoes, strawberries, pineapples and kiwifruit (Castelló et al., 2010; Moraga et al., 2009; Torres et al., 2008). Nevertheless, after a few days of storage, the respiratory quotient is generally observed to increase, as a result of the develop- ment of fermentative routes, which is an optional metabolic pathway triggered by osmotic stress. Salvatori and Alzamora (2000) found that a 25% w/w sucrose solu- tion can cause vesciculation and rupture of cell membranes in apple tissue. According to Mavroudis et al. (2004), few layers of cells on the surface are expected to die upon osmotic treatment, while plasmol- ysis and shrinkage occur in the remaining tissue. In a previous study, the authors found that 40% w/w sucrose treatment generally preserved the viability of apple cells, which only slightly affected the cell struc- ture observed by fluorescence microscopy and the water distribution within the cells, as observed by time domain nuclear magnetic reso- nance (TD-NMR) (Mauro et al., 2016). For different fruit species types, calcium can decrease the metabolic activity of tissue as well as the respiration rate (Castelló et al., 2010; Lester, 1996; Luna-Guzmán et al., 1999), which potentially enhances the product stability during storage, especially considering that a lower respiration rate may lead to a longer shelf life. In addition, Ca2+ can affect the membrane and cell wall structure and functioning (Maurel, 2007; Peiter et al., 2005). On the other hand, the presence of AA can cause serious injury to the cellular structure, as has been previously reported by Mauro et al. (2016), who observed a loss in the capacity to retain FDA colorant due to cell membrane damage following exposure to OD in a sucrose-ascorbic acid solution. As the AA concentration increased from 0 up to 2%, a loss of vitality was detected. Rocculi et al. (2005) found a higher metabolic activity in potato tissue upon dipping treatments with citric and ascorbic acid, suggesting that AA solution can promote a stress response in specific fresh-cut veg- etable tissues, as well as an acceleration of their endogenous metabolic activity, which was confirmed by a higher O2 consumption accord- ing to head space gas determination. Limbo and Piergiovanni (2007) detected an increase in the respiration rate of sliced potatoes that were subjected to dipping treatment with 2.5% AA. However, when the AA concentration was 5%, the respiration rate decreased. Isothermal calorimetry has been recognized as a useful tool for assessing metabolic responses of various plant tissues to wounding stress (Wadsö et al., 2004), dipping treatment (Rocculi et al., 2005), ther- mal treatments (Gómez et al., 2004) and OD (Panarese et al., 2012). Generally, when a tissue is wounded, certain signal paths are trig- gered, and the plant starts a number of protective processes that increase the produced metabolic heat (Wadsö et al., 2004). As reported by Gómez et al. (2004), after wounding, the energy released by the cellu- lar tissue corresponds to the sum of that from the ‘basic’ cell metabolic activity and of that originating from wounding stress that is produced by the cells near the cut surface. Some of the processes that occur after wounding are aimed at membrane restoration and strengthen- ing of cell walls by cells close to the site of injury (Rolle and Chism, 1987). A progressive reduction in the metabolic heat production dur- ing OD in kiwifruit slices was observed by Panarese et al. (2012) using isothermal calorimetry. The authors suggested that the decrease was due to a reduction in the cell viability that was induced by osmotic stress. Finally, the metabolic response of fruit tissues to OD was found to depend on the botanical origin, exerted osmotic pressure (Ferrando and Spiess, 2001; Mavroudis et al., 2004) and physiological state because loss of membrane integrity upon ripening that increases the permeabil- ity then makes the tissue more sensitive to osmotic stress (Panarese et al., 2012). This study evaluated the effects of the addition of calcium lactate (CaLac) and ascorbic acid on sucrose osmotic solutions, mass transfer kinetics and raw endogenous metabolic response (respiration and heat production) of the tissue. The obtained information can be very useful for investigating the potential stability of minimally processed apples. 2. Materials and methods 2.1. Raw materials Apples (Malus domestica Borkh; 30 kg) of the Cripps Pink variety, popularly known by the brand name Pink Lady (de Castro et al., 2008), were bought at the local market and stored at 5 ± 1 ◦C for 2 weeks, during which the experimental research was performed. Apples were characterized by an average weight of 234 ± 18 g and soluble solid content of 13.4 ± 0.3 g/100 g. From the central part of the mesocarp fruit, cylindrical sam- ples (8-mm diameter, 40-mm length) were cut with a manual cork borer and a manual cutter designed for the purpose. For osmotic treatments, commercial sucrose (refined sugar, Eridania Italia Spa, Italy), l-ascorbic acid (Shandong Luwei Pharmaceutical Co., China) and calcium lactate (calcium-l- lactate 5-hydrate powder, PURACAL ® PP Food, Corbion PURAC, Netherlands) were used. 2.2. Osmotic dehydration OD was performed at 25 ◦C using four different osmotic solu- tions (w/w): 40% sucrose (Suc), 40% sucrose + 4% calcium lactate (Suc-CaLac), 40% sucrose + 2% ascorbic acid (Suc-AA) and 40% sucrose + 4% calcium lactate + 2% ascorbic acid (Suc- CaLac-AA). Approximately 100 g of apple cylinders were weighed for each treatment time (0.5, 1, 2 and 4 h) and placed in mesh baskets that were immersed in 4.5 kg of aqueous osmotic solu- tion with a syrup-to-fruit ratio of approximately 15:1 (w/w) to avoid changes in the concentration of the solution during the treatment. Through an impeller of a mechanical stirrer, the cylindrical baskets were continuously rotated. The rotational speed (0.2 g) was experimentally determined to assure negli- gible external resistance to mass transfer. Two baskets were prepared for each process time. After each treatment time, samples were removed from the solution, rinsed with distilled water, blotted with absorbing paper, and weighed. Subsequently, cylinders were placed in glass sealed ampoules to measure the endogenous metabolic heat production with isothermal calorimetry over 16 h, which food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 3 w a l a 2 T s i w b ( 2 A t B t ( a u n fi e C s p d s T D f t a t 2 T a P h m f s p c d H 0 o b d w i ( 2 T a 2 b e as followed by the determination of the O2 and CO2 levels on mpoule headspaces. Total and soluble solid contents were determined in trip- icate immediately after treatment. Samples for calcium and scorbic acid analyses were freeze-dried. .3. Analytical methods he moisture content of fresh and osmotically dehydrated amples was gravimetrically determined, in triplicate, by dry- ng cylindrical apple samples at 70 ◦C until a constant weight as reached. Soluble solid content was determined at 20 ◦C y measuring the refractive index with a digital refractometer PR1, Atago, Japan) that was calibrated with distilled water. .3.1. Ascorbic acid scorbic acid was determined by HPLC analysis according o the method described by Odriozola-Serrano et al. (2007). riefly, approximately 0.5 g of freeze-dried sample was added o 10 ml of meta-phosphoric acid (62.5 mM) and sulfuric acid 5 mM) solution, which was vortexed for 2 min and centrifuged t 10,000 × g for 10 min at 4 ◦C. The supernatant was directly sed for the fresh sample and diluted tenfold for the impreg- ated samples; it was then filtered through a 0.45 �m nylon lter. The HPLC system LC-1500 (Jasco, Carpi, MO, Italy) was quipped with a diode array UV/Vis detector. A reverse-phase 18 Kinetex (Phenomenex Inc., Torrance, CA, USA) stainless teel column (4.6 mm × 150 mm) was used as the stationary hase. A Jasco AS-2055 Plus autosampler was used to intro- uce samples into the column. The mobile phase was a 0.01% olution of sulfuric acid that was adjusted to a pH of 2.6. he flow rate was fixed at 1.0 ml/min at room temperature. ata were processed with ChromNAV software (ver. 1.16.02) rom Jasco. The ascorbic acid content was quantified at 245 nm hrough a standard calibration curve that was set up using an scorbic acid solution between 0.5 to 30 ppm. The determina- ion was performed in triplicate. .3.2. Calcium he calcium concentration was determined using a flame tomic absorption spectrophotometer (Model A Analyst 400, erkin Elmer, Santa Clara, California, USA) with a lumina ollow cathode lamp (Perkin Elmer) based on the adapted ethodology of AOAC (2002). Briefly, approximately 6 g of reeze-dried, untreated samples and 2 g of freeze-dried treated amples, were weighed in a 50 ml glazed, porcelain crucible; laced in a muffle furnace and heated up to 550 ◦C until omplete ignition. After cooling in desiccators, the ash was issolved in 20 ml (fresh samples) or 30 ml (treated samples) of Cl (0.1 M); then, the solutions were appropriately diluted with .1 M HCl. A calibration curve of the absorbance versus ppm f calcium was established using standard calcium solutions etween 2 to 20 ppm. The initial sample level and subsequent ilution permits to obtain solutions with a concentration that as suitable to the standard solutions used for establish- ng a calibration curve of absorbance versus ppm of calcium 2–20 ppm). The determination was performed in triplicate. .3.3. Metabolic heat production wo fresh cylindrical samples (8-mm diameter, 40-mm length) nd three osmotically dehydrated samples were placed in 0 ml glass ampoules and sealed with a teflon coated rub- er seal and an aluminium crimp cap. Three replicates for ach sample were performed. The rate of heat production was continuously measured in a TAM air isothermal calorimeter (Thermometric AB, Järfälla, Sweden) with a sensitivity (pre- cision) of ±10 �W (Wadsö and Gómez Galindo, 2009). This instrument contains eight twin calorimeters in which each sample is inserted with its own reference, and the measured signal is the difference between the sample and reference sig- nals. The reference has to be a material that does not produce any heat, but it is characterized by thermal properties that are similar to the sample. For this, water was chosen as the refer- ence material and its quantity in each reference ampoule (mo w) was previously determined based on the average composition of the samples and on the heat capacities (J g−1 K−1) of water (Cw) and total solids (CTS), as in the following equation: mo w = CTS · mTS + Cw · mw Cw (1) where mTS is the dry matter content (g) and mw is the water content of the fruit sample (g), and the average heat capacity of the total solids of the apple samples was assumed to be 1 J g−1 K−1. The analysis was performed at 10 ◦C for 16 h. The first 4 h of analysis were discarded because of the instability of the signal due to the loading and conditioning of samples. 2.3.4. Respiration rate Immediately after the ampoules were discharged from the calorimeters, the O2 and CO2 percentages were measured in the ampoule headspaces by a check point gas analyser O2/CO2 mod. MFA III S/L (Witt-Gasetechnik, Witten, Germany). The apparatus has a paramagnetic sensor for O2 and a mini-IR spectrophotometer for CO2 detection. The instrument was cal- ibrated with O2 and CO2 air percentages. The respiration rate was calculated as mol of consumed O2 (RRO2 ) or produced CO2 (RRCO2 ) h−1 g−1 according to the following equations: RRO2 = Vhead · (20.8−%O2,head) 100 · P t · m · R · T (2) RRCO2 = Vhead · %CO2,head 100 · P t · m · R · T (3) where Vhead represents the ampoule headspace volume (dm3), %O2,head and %CO2,head refer to molar gases percentages in the ampoule headspace at time t (h), m is the sample mass (g); R is the gas constant (8.314472 dm3 kPa K−1 mol−1), P is the pres- sure (101.325 kPa) and T is the absolute temperature (283.15 K). 2.4. Osmotic dehydration kinetics Mass transfer of water, sucrose, calcium and ascorbic acid dur- ing the osmotic process was modelled according to the model proposed by Peleg (1988) to describe moisture sorption curves and was further used by Palou et al. (1994) to model OD, as follows: �wk = wk,t − wk,0 = − t k1 + k2t (4) where wk is the mass fraction (g g−1 total mass) of the following k species: water (ww), sucrose (wSuc), calcium (wCa2+ ) or ascor- bic acid (wAA) at time 0 (wk,0) and time t (wk,t). The constants of Peleg’s model are k1 [s (g g−1 total mass)−1] and k2 [1 (g g−1 total mass)−1]. This kinetic model permits, by calculating the inverse of the two constants, to obtain the initial (t = 0) rate 4 food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 Fig. 1 – Comparison between the observed (obs) and calculated (calc) mass fraction of water (a), sucrose (b), calcium (c) and ascorbic acid (d) according to Peleg’s model (Eq. (4)), in g g−1 total mass, for the different treatments. of mass transfer (1/k1) and the mass fraction at equilibrium (t → ∞) conditions ( wk,eq = wk,0 ± 1/k2 ) (Sacchetti et al., 2001). 2.5. Statistical analysis and fitting The significance of the treatments was statistically evaluated by analysis of variance (ANOVA) and comparison of means using the Tukey’s post-hoc test that was applied at a 5% level of significance. The Peleg’s model was fitted to the experimental data using the Levenberg–Marquardt algorithm for the least-square esti- mation of the non-linear parameters (Marquardt, 1963). The fitting efficiency was evaluated by the coefficient of determi- nation (R2) and the relative root mean square error (RRMSE); the latter was evaluated according to Eq. (5): RRMSE(%) = √√√√ 1 N N∑ n=1 ( yobs − ycalc ycalc )2 · 100 (5) where yobs represents the observed value, ycalc the calculated value and N the number of observations. 3. Results and discussion 3.1. Osmotic dehydration kinetics The Peleg’s equation (Eq. (4)) was used to model the kinet- ics of water loss and solute uptake during OD. Constants of the Peleg’s equation (k1 and k2) and their inverse and equi- librium concentrations are reported in Table 1. The predictive capability of the Peleg’s model can be observed in Fig. 1, which compares the observed and calculated values of the mass frac- tion, which is expressed as a function of the process time, for water (a), sucrose (b), calcium (c) and ascorbic acid (d). In general, the model showed a good fit with the exper- imental data, as high R2 values and low RRMSE were found (Table 1), confirming its suitability for describing mass transfer phenomena in OD, as reported by Palou et al. (1994). The same model was also successfully applied by other researchers, such as Sacchetti et al. (2001). Regarding Table 1, it can be observed that the initial rate of dehydration was increased by the presence of Ca2+ and AA in the osmotic solution, as higher 1/k1 values were found for Suc-CaLac, Suc-AA and Suc-CaLac-AA treatments compared to the one with only sucrose in the solution. As reported in Fig. 1a, the presence of calcium in both Suc-CaLac and Suc- CaLac-AA solutions promoted a higher reduction in the water content than the treatments without Ca2+. Contrary to Ca2+, the presence of AA in the sucrose solution (Suc-AA treatment) caused only a slight depletion in the water content. In addi- tion, the equilibrium water contents, calculated on the basis of the parameter k2, were more affected by Ca2+, showing lower values than the other treatments (Table 1). It should be noted that when the osmotic solution contained both Ca2+ and AA solutes, the equilibrium water content was reduced to the lowest value (0.6428 g g−1, Table 1), which corresponds to the lowest water activity of all solutions. The water activities were 0.962 ± 0.002 for Suc, 0.953 ± 0.001 for Suc-CaLac, 0.954 ± 0.001 for Suc-AA and 0.944 ± 0.004 for Suc-CaLac-AA osmotic solu- tions. Conversely, despite the differences between the initial rates of water transfer found for the Suc and Suc-AA treat- ments (Table 1) as well as between the water activities of these two solutions, their equilibrium water levels were quite sim- food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 5 Table 1 – Kinetic model of water, sucrose, calcium and ascorbic acid transfer in each osmotic solution according to Peleg’s model (Eq. (4)) and equilibrium content (g g−1 total mass). Solution k1 (s) SD p-Level 1/k1 × 103 (s−1) k2 (g total mass g−1) SD p-Level 1/k2 × 103 (g total mass g−1)−1 R2 RRMSE (%) wk,eq (g g−1 total mass) Water Suc 11.18 0.78 <0.001 89.45 6.10 0.33 <0.001 163.90 0.997 3.8 0.6802 Suc-CaLac 9.12 0.64 <0.001 109.70 5.68 0.28 <0.001 176.09 0.997 3.1 0.6708 Suc-AA 8.93 0.46 <0.001 112.00 5.98 0.21 <0.001 167.26 0.998 3.1 0.6832 Suc-CaLac-AA 9.10 1.07 <0.01 109.92 4.83 0.45 <0.01 206.88 0.992 5.3 0.6428 Sucrose Suc 14.51 1.32 <0.01 68.92 6.92 0.54 <0.001 144.54 0.998 3.8 0.1967 Suc-CaLac 14.59 1.53 <0.001 68.53 8.08 0.64 <0.001 123.79 0.998 3.2 0.1759 Suc-AA 11.49 2.18 0.01 87.05 8.13 1.01 <0.01 123.03 0.980 8.60 0.1752 Suc-CaLac-AA 17.36 3.17 0.01 57.62 6.25 1.19 0.01 160.02 0.979 12.0 0.2122 Calcium Suc-CaLac 392.99 7.40 <0.001 2.54 523.45 4.31 <0.001 1.91 1.000 0.7 0.0019 Suc-CaLac-AA 524.34 83.76 <0.01 1.91 408.00 40.04 <0.01 2.45 0.986 7.0 0.0025 Ascorbic acid Suc-AA 85.44 14.08 <0.01 11.70 81.48 7.24 <0.01 12.27 0.986 5.9 0.0123 i w w t a t a ( t m s r a w a M a C i i t S f S b a 2 c e 8 t c b m t b e w t t a Suc-CaLac-AA 104.72 10.56 <0.01 9.55 68.83 lar. These discrepancies are related to the presence of AA, hich can affect the cellular structure and thus influence the ater and solutes transfer, as well as the equilibrium con- ents, as further discussed below. Effects promoted by Ca2+ nd AA, however, can be better observed when also assessing he sucrose transfer. The initial rates of sucrose mass transfer (1/k1) found in ll treatments were lower than the ones calculated for water Table 1). This behaviour is expected in plant tissue because he cell wall porosity and selective permeability of the cellular embranes reduce the transport of larger molecules, such as ucrose, through the cell tissue. While the cell membranes emain intact, intracellular spaces occupied by protoplasts nd vacuoles are unavailable to sucrose transport. Conversely, ater can diffuse throughout the cell walls and membranes nd occupy all liquid phases of the plant cell (Mauro and enegalli, 2003). The addition of Ca2+ and AA had a variable nd unexpected influence on sucrose transport. When only a2+ was added, the treatment promoted an increase in the nitial rate of the water transfer, but did not cause any change n the initial rateof the sucrose transfer, in comparison with he Suc treatment (Table 1). Moreover, it can be seen that the uc-CaLac treatment led to the lowest sucrose content after our hours of processing (Fig. 1b). Conversely, the highest value of 1/k1 corresponded to the uc-AA treatment. However, the addition of AA affected the ehaviour of the sucrose content with time in both Suc-AA nd Suc-CaLac-AA treatments, which sharply increased after h of processing, as shown in Fig. 1b. Moreover, because of this hange in the trend of these curves, the fitting efficiency wors- ned, as confirmed by the highest RRMSE values in Table 1, .6% for Suc-AA and 12% for Suc-CaLac-AA. The worst fit- ings were related to the presence of AA that would have aused damage to the structure of cell walls and cellular mem- ranes, changing transport during OD. The effects of AA on the icrostructure of apple tissue and water distribution within he different cellular compartments changed the cell mem- ranes permeability, as reported in a previous study (Mauro t al., 2016). In fact, AA can affect cellular tissue in different ays. Several studies have evaluated the role of AA in plant issues that undergo stress; however, little is known regarding he mechanisms that explain its effects when plant tissues re not exposed to stress, as reported by Qian et al. (2014), 4.76 <0.001 15.53 0.994 4.8 0.0145 who observed severe damage in the cellular structure of Ara- bidopsis thaliana seedlings that were exposed to exogenous AA. Additionally, an increase in the cell wall porosity is also expected because of the medium acidification (Zemke-White et al., 2000). In addition, discrepancies were more visible in the Suc- CaLac-AA treatment, which presented the lowest initial rate 1/k1 and the highest equilibrium value of 0.2122 g g−1 (Table 1). The poor fittings related to the presence of AA in osmotic solutions added some uncertainty to the equilibrium content calculated for the Suc-AA and Suc-CaLac-AA treatments. During OD, damages in the tissue promoted by the solu- tion components and/or by the dehydration could release enzymes and cause depolymerisation, solubilisation and de- methylation of pectins. Pectin, an important component of the primary cell wall, is mainly formed by homogalacturonan blocks. In the presence of divalent cations, such as Ca2+ and, depending on the degree of methylesterification and the distri- bution of the methyl-substituent groups, homogalacturonan can dimerise, reinforcing the cell adhesion and controlling the wall porosity (Bonnin and Lahaye, 2013). The calcium effect on firmness has been observed in various fruit tissues that undergo OD in the presence of calcium salts, and it has been attributed to reduction in the cell wall porosity and to the for- mation of calcium pectate (Mavroudis et al., 2012; Pereira et al., 2006; Silva et al., 2014a,b). Consequently, Ca2+ limited sucrose impregnation because of these interactions with pectin. Good impregnation levels of both Ca2+ and AA were obtained, as shown in Fig. 1c and d. The Ca2+ contents in both Suc-AA and Suc-CaLac-AA were very close until two hours of processing; afterwards, the impregnation quickly increased in those samples treated in Suc-CaLac-AA solution (Fig. 1c). Regarding the AA levels in Fig. 1d, a similar behaviour was observed, where, after two hours of processing, the AA impreg- nation tended to increase in both,= Suc-AA and Suc-CaLac-AA. The equilibrium concentrations of both Ca2+ and AA were also higher when samples were treated in Suc-CaLac-AA solution (Table 1). However, when Ca2+ was combined with AA, only after 2 h of OD was an increase in solute impregnation noticed, which is probably observed because the damage caused by AA surpassed the restraining effects of the cell structure impreg- nated with Ca2+ to the solute transport. 6 food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 Fig. 2 – Total heat production (J/g) of fresh and osmotically dehydrated samples during 16 h at 10 ◦C. Different letters indicate significant differences by the Tukey test at p < 0.05. 3.2. Metabolic profiles The results of the total metabolic heat produced during 16 h at 10 ◦C, measured through isothermal calorimetry after osmotic treatments, were performed for 0, 30, 60, 120 and 240 min and are given in Fig. 2. Provided that the concentration of O2 and CO2 can also give useful information about tissue metabolism, after calorimetric analysis, the composition of the headspace of the vials was evaluated. The measured respiration rates (RRO2 and RRCO2) are presented in Fig. 3. As a consequence of Suc treatment, there was a slightly decreasing trend in metabolic heat production (Fig. 2), which was proportional to treatment time until two hours of pro- cessing, as well as a lower respiration rate compared to the fresh samples, both in terms of the CO2 produced and O2 con- sumed (Fig. 3). A partial loss of cell viability could be expected after OD treatment, even if the osmotic solution concentration was very low (Panarese et al., 2012). In a previous experiment (Mauro et al., 2016), cell viability was found to be preserved in apple tissue subjected to OD treatment with 40% sucrose solu- tion, as observed by an FDA staining technique that allows for determination of the plasma membrane integrity. Conversely, neutral red staining revealed the incidence of plasmolysis that could help explain the decrease in the metabolic heat pro- duced by the tissue. Salvatori and Alzamora (2000) found that a 25% w/w sucrose solution can cause vesciculation and rupture of the cell membranes in apple tissue. According to Mavroudis et al. (2004), only few layers of cells on the surface are expected to die upon osmotic treatment, while plasmolysis and shrink- age occur in the remaining tissue. The presence of calcium in the osmotic solution caused a further decrease in the metabolic heat production. This result is in accordance with previous literature reports (Castelló et al., 2010; Luna-Guzmán et al., 1999), and it confirms the ability of calcium to slow down tissue metabolic activity and thus to enhance the stability of minimally processed fruit. In various fruits, both whole and cut, an effect of calcium on respiration has been observed together with a reduction of ethylene production and general slowing of ripening and senescence (Lester, 1996; Saftner et al., 1999). In particular, different explanations have been put forward for the reduction of the respiration rate: a protective osmotic effect due to the high salt concentration (Ferguson, 1984); an indirect effect on substrate transport from the alteration of the membrane permeability (Bangerth et al., 1972); the forma- tion of a transient barrier between fruit and atmosphere that hinders gas exchange (Saftner et al., 1999); inhibition of plant aquaporins that regulate membrane permeability, causing an increase in the cytoplasmic ATP concentration that remains available for other biochemical routes (Kinoshita et al., 1995); and delay of senescence-related changes (Lester, 1996). At the same time, an excess of calcium has been related to a hastening of senescence because of damages to the plasma membrane structure and functionality. Nevertheless, the effect of calcium on respiration has not been fully clarified to date. In the present experiment, the res- piration rate values were similar to those of samples that were only dehydrated with sucrose, and they were only slightly lower after 30 and 240 min, indicating that the reduction of heat produced could be related more to other biochemical phenomena than to the reduction of respiratory activity. Conversely, the presence of AA in the osmotic solution pro- moted a drastic increase in the metabolic heat production as the treatment time increased up until 50% compared to the fresh sample. This increase can probably be attributed to the physiological stress caused on the tissue, as already observed for sliced potatoes (Limbo and Piergiovanni, 2007; Rocculi et al., 2005). The damage to cellular structures, which is promoted by osmotic AA solution, can mainly be caused food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 7 Fig. 3 – Respiration rates, expressed as the oxygen consumed (RRO2) and carbon dioxide produced (RRCO2), for treatment times of 30 min (yellow), 60 min (green), 120 min (blue) and 240 min (red). Error bars indicate standard deviation. Different letters in the auxiliary tables indicate significant differences according to the Tukey test at p < 0.05. (For interpretation of the r d to b t H u r d u s o m c w b a o a w p r t d a t S d w R a t t 1 u a f eferences to color in this figure legend, the reader is referre y its lower pH. At low pH, plasma membrane ATPases in the issue increase active H+ pumping to address the excess of + uptake, increasing the demand for respiratory energy. An lterior pH decrease can also cause a decline in the respiration ates. The combination of AA with Ca initially promoted a ecrease in the heat production to a level that was lower than ntreated samples; however, after 2 h of OD, the metabolism harply rose. This behaviour suggests that, during the first part f the treatment, calcium acted as stabilizer and reduced the etabolic activity of the tissue; however, as the treatment pro- eeded, a progressive damage to cellular structures occurred, hich was probably related to the AA intake. Conversely, for samples dehydrated in the presence of AA, oth alone or in combination with CaLac, there was a notice- ble change in the respiratory pathway, particularly in terms f the increased oxygen consumption compared with Suc nd Suc-Ca samples (Fig. 3). The highest standard deviations ere observed for RRO2 measurements in some AA sam- les. Although high variability was found in the respiration ates, reflecting the large natural variability of the raw and the reated material, useful indications were obtained. CO2 pro- uction was constant for all treatment times in AA samples, nd there was a reduction of approximately 20% compared to he fresh tissue. The production was higher compared to in uc and Suc-CaLac samples. Conversely, a noticeable RRCO2 ecrease was verified in the Suc-CaLac-AA condition, which as proportional to the treatment time. The respiratory quotient (RQ), calculated as the ratio RO2/RRCO2, is an indicator of the respiration pathway dopted by tissues. The complete oxidation of glucose through he aerobic pathway produces an equal CO2 level compared o the O2 consumed; as a result, the respiratory quotient is . Variations in the RQ may depend on a different substrate sed for respiration, such as malate or long chain fatty acids, lthough an increase in RQ generally indicates the onset of ermentative routes (Taiz and Zeiger, 1998). However, accord- the web version of this article.) ing to Makino (2013), an RQ in the range of 0.7–1.3 could be considered an indicator of aerobic respiration. Approximately following this indication, it was possible to identify the sam- ples that were characterized by aerobic metabolism in Fig. 3 that shows values of RRO2 and RRCO2 of samples at different OD times. In our experiment, fresh samples had an RQ value of 1.24, while in Suc and Suc-CaLac, RQ values were lower and closer to 1, showing negligible anaerobic metabolism. Anaerobic metabolism can be prompted by either low oxy- gen or high carbon dioxide concentrations in the environment that are, respectively, lower than 2%–5% and higher than 4%–5% (Cortellino et al., 2015; Iversen et al., 1989). Although these values were never exceeded, in some samples, and in particular in the fresh ones, the CO2 content was very close to this limit after 20 h, which may have caused the develop- ment of some fermentative pathways, leading to an imbalance between CO2 production and O2 consumption in the tissue. This, in turn, increased the RQ. As a result, only samples treated with Suc-AA and Suc-CaLac-AA solutions seemed to have a non-aerobic response to the treatment. As a consequence of OD, an increase in the RQ was observed by Torres et al. (2008) and by Castelló et al. (2010) on mango and strawberry tissues. Anaerobic metabolism is often found in plant tissue as a physiological response to stress conditions, such as dehydration, and as an optional metabolic pathway (Torres et al., 2008). While oxygen diffusion through the tissue decreases because of structural alteration in the cells as treat- ment proceeds, an increase of CO2 production has generally been observed by these authors. The oxygen consumed was attributed to the effort of some enzymatic systems to react to the stress caused by osmotic treatment (Lewicki et al., 2001). Conversely, Moraga et al. (2009) did not find changes from the presence of calcium lactate in the RQ of osmo-dehydrated grapefruit, although the respiration rate generally decreased. In samples that were dehydrated in the presence of AA, a lower RQ was calculated and was found to decrease slightly by increasing the treatment time between 0.72 and 0.64 (data not 8 food and bioproducts processing 1 0 3 ( 2 0 1 7 ) 1–9 shown). The combination of sucrose and ascorbic acid caused cellular damage to the tissue, and the effect on the plas- malemma and tonoplast was different and unclear, but there was a strong influence on tissue functionality (Mauro et al., 2016). When both AA and CaLac were used, the RQ decreased sharply, from 0.59 to 0.09, as dehydration proceeded. This decrease is mainly due to the higher oxygen consumption observed compared to CO2 production. It is important to note that the variation of the gas com- position in the sample headspace could be from both the respiratory metabolism of the tissue and the presence of other enzymatic reactions. According to Igual et al. (2008), this O2 consumption can be considered as the “apparent” respiration rate. Because molecular oxygen can be used as substrate by many enzymes in plant tissue, it can contribute to the “apparent” respiration rate if measured in terms of oxy- gen consumption and not in terms CO2 production (Taiz and Zeiger, 1998). Concerning this particular issue, the effect of sugar, calcium and ascorbic acid on the complexity of fresh tissue enzymatic activity must be considered. 4. Conclusions The investigated osmotic dehydration treatments showed dif- ferent effects on the product in terms of both mass transfer phenomena during processing and metabolic activity of the apple tissue. The presence of calcium lactate (CaLac) and ascorbic acid (AA) affected the water and solute transport, which was attributed to changes in the cellular structure. Sig- nificant impregnation of solutes promoted by AA was related to severe damage caused to the cell structure, increasing spaces viable for solute transport. Ca2+ contributed to the improvement of dehydration and to limit the sucrose impreg- nation; however, in combination with AA, its capacity to restrain solute transport was diminished after 2 h of OD, which was probably because of the excessive AA gain. Metabolic heat production in samples treated in sucrose solutions was slightly lower than in untreated samples, and it was further reduced with calcium lactate (CaLac) addi- tion. However, samples impregnated with ascorbic acid (AA) showed higher heat production because there was a metabolic response of the apple tissue to AA treatment. When combined with Ca2+, heat production decreased sharply to a level that was lower than untreated samples, except for those treated for 240 min (higher solid gain), which showed the highest heat production values. These results confirm previous findings, suggesting that AA solution can promote a stress response on specific fresh-cut vegetable tissues and an increase of their endogenous metabolic activity, which was further confirmed by a higher O2 consumption that was observed by head space gas determination. 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