Enrichment of Apple Slices with Bioactive Compounds from Pomegranate Cryoconcentrated Juice as an Osmodehydration Agent

Pomegranate juice is an important source of bioactive compounds, and cryoconcentrated juice is an interesting osmodehydration agent to enrich a vegetal matrix. is investigation aimed to incorporate bioactive compounds from pomegranate cryoconcentrated juice into apple slices using osmodehydration (OD) assisted by pulse vacuum (PV) and ohmic heating (OH). e apple slices (3× 4× 0.5 cm) were osmodehydrated using a 47 °Brix pomegranate cryoconcentrated juice at 30, 40, or 50°C for 180min with an electric eld of 6.66V/cm (50V) and a 5min pulsed vacuum. Over the time, all of the treatments applied to the apple slices increased the soluble solids and bioactive compounds compared with the fresh sample. PVOD/OH at 50°C had the highest content of total anthocyanin during processing, and the best results for the total avonoids were obtained with OD/OH at 50°C and 40°C. e osmodehydration assisted by pulse vacuum and ohmic heating using a cryoconcentrated juice is a useful combined technique to acquire enriched vegetal samples with bioactive compounds.


Introduction
As consequences of changing lifestyle and eating habits, diseases such as obesity, cardiovascular problems, diabetes type II, hypertension, and cancer among others have increased in the last decades [1,2].ese adverse e ects have developed an interest in healthy food and the consumption of functional food [3].Functional foods include bioactive compounds (BAC) that have health bene ts and nutritional value.Vitamins, polyphenols, and minerals are examples of BAC [3].
Pomegranate (Punica granatum L.) recently has gained interest in the past few years because of its nutritional and antioxidant activities [4].Pomegranate juice is a potential source of anthocyanins, avonoids, organic acids, and ellagic acid [5][6][7].Additionally, recently, pomegranate juice has been used as a functional ingredient, for example, in fermented milk and smoothies [8,9].erefore, its health bene ts are associated with those chemical characteristics.
e process of enriching vegetables to create functional products with BAC has been achieved through the combination of emerging technologies such as cryoconcentration with ohmic heating (OH) and assisted techniques such as pulsed vacuum (PV) and osmodehydration (OD).In this context, the food industry must link consumer demands and these available technologies [10].
Cryoconcentration, also called freeze concentration, as mentioned above, is an emerging technology, which consists of entirely or partially freezing the liquid food solution for later separation of the ice fraction from the liquid [11].Vitamins, as well as polyphenols, are thermolabile compounds, and by using freeze concentration, the high nutritional value and organoleptic characteristics can be protected [12].erefore, the BAC present in pomegranate juice could be protected using this technology.
OH is a thermal process, in which energy is generated by the passage of an alternating electrical current that diffuses through the food [1].PV has been widely used to obtain a rapid penetration of compounds in vegetable tissues and enriches fruits and vegetables with antioxidants, vitamins, and minerals, among others [13].
Osmotic dehydration is a process in which water is partially removed from the cellular material or product when exposed directly to a concentrated solution of solutes or hypertonic medium [14,15].It has the advantage of higher nutritional content compared with drying methods because it has a minimal effect on the components of the food matrix [15].e type of osmotic agent used and its molecular weight or ionic behavior affect the kinetics of water elimination and the gain of solids [15].e OD mass transfer occurs through a semipermeable cell membrane, which makes substantial changes to the tissue structure [16].
e OD applied to vegetables is commonly performed using sucrose or other osmotic agents such as salt; however, recently, Lech et al. [17] reported the use of a fruit juice as an alternative OD solution (commercial chokeberry juice), obtaining interesting results in the final vegetable product in terms of bioactive compounds and antioxidant activity.In addition, Cano-Lamadrid et al. [18] reported a similar use of fruit juices such as OD solutions in this case was used to improve the sensorial and antioxidant capacity of pomegranate cultivar Mollar de Elche, a popular Spanish pomegranate.
is investigation aimed to determine the incorporation of bioactive compounds from frozen concentrated pomegranate juice into osmodehydrated apple slices using pulsed vacuum and ohmic heating treatments and to evaluate the mechanical and optical properties of the enriched product.e pomegranates were cut in half, and the seeds were removed manually for juice extraction and filtered to eliminate any residue from the fruit and be ready for the experimental procedure.Apples were cut into sizes of 3 × 4 × 0.5 cm and submerged in a solution of 2% citric acid and 1% ascorbic acid for 3 min to prevent oxidation.e pomegranate (cv.Wonderful) has a slightly acid sweet flavor, a flavor that is compatible with the apple used as a vegetable matrix to be osmodehydrated.

Osmodehydration
Agent.Cryoconcentrated pomegranate juice (osmodehydration agent) was obtained as described by Orellana-Palma et al. [19] and Petzold et al. [20,21] with modifications.Samples were frozen at −20 °C for 12 h and placed in a refrigerated centrifuge (Eppendorf AG, model 5430R, Hamburg, Germany) operated at 15 °C for 15 min for the first cycle and 10 min for the second and third cycles at a speed of 1878 RCF, until the juice reached 48 °Brix (value obtained at the end of cryoconcentration cycles).e final cryoconcentrated juice had the following phenolic content: total polyphenols of 4953.64 ± 95.48 mg gallic acid equivalents/L, total anthocyanin of 117.64 ± 1.30 mg cyaniding-3-glucoside/L, and total flavonoids of 368.84 ± 4.62 mg catechin/L.For the optical parameters (CIE L * a * b * coordinates), L * 1.07 ± 0.41, a * 4.45 ± 0.24, and b * 1.05 ± 0.04.

Osmodehydration Treatments Assisted by Pulsed Vacuum and Ohmic
Heating.Osmodehydration with atmospheric pressure (OD) or pulsed vacuum (PVOD) and conventional and ohmic heating (OH) at 30, 40, and 50 °C were conducted using a thermoregulated bath, as described by Moreno et al. [22,23].Osmotic treatments were carried out in a stainless steel tank made with two concentric cylindrical electrodes (3.7 and 19 cm in diameter) and a nonconducting bottom, and the distances between them were 7.50 cm [24].Pomegranate juice was exposed to an alternating current at 60 Hz and 50 V, generating an electric field E of 6.66 V/cm, given by the following equation: e temperature during the ohmic heating treatments was controlled through a refrigeration system.

Total Phenolic Content (TPC).
e total phenolic content was determined through a colorimetric method by Folin-Ciocalteu (FC) reagent [25] using gallic acid as the standard.Treated sample extracts (100 µL) were mixed with 7900 µL of distilled water, 500 µL of FC reagent, and 1500 µL of sodium carbonate.e samples were incubated for 120 min before measurement at 760 nm in a spectrophotometer (Shimadzu Scientific 1600 UV/VIS, USA).e results were expressed as mg of gallic acid equivalents in 100 g of dry matter (mg GAE/100 g d.m.).

Total Anthocyanin Content (TAC).
Total anthocyanin content was determined spectrophotometrically by a pH differential method using two buffer solutions, potassium chloride 0.025 M at pH 1 and sodium acetate 0.4 M at pH 4.5, to treat the sample extracts following the methodology of Lee [26] with modifications.One part of the extract was mixed with one part of the buffer, and measurements were made at 510 and 700 nm in a spectrophotometer (Shimadzu Scientific 1600 UV/VIS, USA).e results were expressed as mg of cyanidin-3-glucoside in 100 g of dry matter (mg cyanidin-3glucoside/100 g d.m.).

Total Flavonoid Content (TFC).
Total flavonoid content was determined using a spectrophotometric method, using (+)-catechin as the standard.e assay was carried out as described by Dewanto [27].An aliquot (0.25 mL) of the extract was mixed with distilled water (1.25 mL), and 75 µL of sodium nitrite 5% solution was added.After 6 min, 150 µL of 10% solution of hydrated sixth aluminum chloride was added and the mixture was allowed to stand for 5 more min.Afterwards, 0.5 mL of 1 M sodium hydroxide was incorporated.Distilled water was added until it reaches a volume of 2.5 mL.Spectrophotometric measurements were made at 510 nm (Shimadzu Scientific 1600 UV/VIS, USA).e results were expressed as mg of catechin/100 g of dry matter (mg of catechin/100 g d.m.).

Sample Compositional Analysis.
Soluble solids (X ss ) and moisture content (X w ) were determined during the osmodehydration processing time, and the effects on water loss and mass, solid gain, and water activity (a w ) were examined.Moisture content was determined according to the method defined by the AOAC (Association of Official Analytical Chemist, 2000).Samples, fresh and treated, were dried at 60 °C in a vacuum oven (Lab Line Instruments Inc.) at 10 kPa.
e solute gain was determined as described by Moreno et al. [23], taking 3 g of sample (fresh or treated) and 25 mL of distilled water and grinding with Ultra-Turrax (Ika-Werke, model T25 basic, USA).
e solid content was measured using a digital refractometer (Leica Mark II, Buffalo, NY, USA).
e X ss and X w contents were determined for both fresh and treated samples, and the compositional changes were calculated through the following equations: where W 0 and W t represent the sample weights at time 0 and t, respectively.W is the weight of the distilled water and A stands for the sample °Brix.e net changes of the soluble solid and moisture contents (ΔM ss t and ΔM w t ) were calculated with the following equations: where M 0 0 and M t 0 stand for the sample weight at time 0 and t, respectively.X ss 0 , X w 0 , X ss t , and X w t are soluble solids (ss) and water (w) mass fractions at time 0 and t, correspondingly.
e water activity (aw) of samples was determined using a dewpoint hygrometer (Aqua Lab Model 4TE, Pullman, USA).

Mechanical Properties.
e firmness of the samples was evaluated with a Texture Analyzer TA-XT (Stable Microsystems, Haslemere, UK), using a slice shear blade.e mechanical parameter was considered as the maximum peak force and reported in N.

Optical Properties.
e color of apple slices, fresh and treated, was measured with a spectrocolorimeter (Konica Minolta CM -500).e CIE L * a * b * coordinates used a D 65 illuminant and 10 °observer as a system reference.e chroma (C * ab), hue (h * ab), and the color differential (ΔE) were calculated according to the following equations: 2.10.Statistical Analysis.e experiment was carried out using a randomized factorial design (4 × 3) considering the osmodehydration treatments and temperatures as the primary sources of variance (Figure 1).e results were subjected to analysis of variance (ANOVA) and least significant difference test (LSD) using Statgraphics Centurion XVI software with p ≤ 0.05.Measurements were done in triplicate, except for the mechanical properties, which were tested six times.

Total Phenolic Content (TPC).
Figure 2 shows the TPC of the apples treated over the osmodehydration time.e PVOD/OH treatment at 30 °C had a higher TPC (Figure 2(a)) over time (180 min), increasing to twice the initial value, followed by PVOD/OH at 50 °C (Figure 2(b)). is behavior is connected with that of the bioactive compounds from the cryoconcentrated pomegranate juice used as a osmodehydration agent; similar results and explanation were reported using chokeberry juice in the OD processing of carrot and zucchini [17].e samples with the lowest phenolic retention were PVOD at 40 °C and OD/OH at 50 °C (Figures 2(b)-2(c)).Furthermore, the TPC decreased for all samples after 30 min, except for PVOD/OH at 30 °C (Figure 2(a)); this decrease was attributed to the treatment combination, temperature, and time.Additionally, after 180 min, most of the treatments retained at least the initial value of TPC, except for PVOD at 30 °C and 40 °C.Such findings agree with those reported by Moreno et al. [23], in which for these osmodehydrated treatments, the initial content of polyphenols was reduced significantly during processing time compared with that of the fresh sample.

Total Anthocyanin Content (TAC). As mentioned by
Mahmoud et al. [28], anthocyanin pigments are responsible for the pomegranate red color and for the color of the osmodehydrated apples.Figure 3 shows the total anthocyanin content during processing time.As observed, PVOD/OH at 50 °C had the highest anthocyanin content, reaching a value near 117 mg of cyanidin-3-glucoside/L, very close to the anthocyanin content of the cryoconcentrated pomegranate juice (Figure 3 4 Journal of Food Quality pulsed vacuum, and electroporation, which enables the osmodehydration agent (cryoconcentrated pomegranate juice) to enter the apple matrix pores [29,30].Reports showed that anthocyanins are less a ected at 40 °C than at 30 °C and 50 °C; this is due to a combination of temperature, time, pH, enzymes, stability, and physicochemical properties of these individual pigments [31,32].In the same study conducted by Kechinski et al. [33], it was demonstrated that anthocyanins were less likely to su er from thermal degradation; the susceptibility to heat might be caused by the di erent forms of the pigments and the interactions of other components in the fruit.For the rest of the treatments, they present a decrease in the TAC after 180 min of treatment.).e avonoids had a drop at 90 min in most of the treatments.e loss of macromolecules such as avonoid during heat treatment might be caused by temperature and time [34].Wet thermal treatments may a ect the cell's structure compromising the integrity and causing the migration of components, leading to damages by leakage or breakdown by several chemical reactions [35].For the 30 °C treatment (Figure 4(a)), the OD presented better retention of avonoids during the processing time. 1 shows the compositional changes between the fresh sample and di erent treatments of osmodehydration after 180 min of processing.According to previous ndings, osmotic dehydration with concentrated juice is more signi cant compared with osmotic dehydration with sucrose [36]. is behavior is in agreement with a recent study of Lech et al. [37], who conclude that the osmodehydration process is a ected by the solution applied and the particular chemical properties of this solution.

Compositional Changes and Water Activity. Table
e combination of PVOD/OH at 40 °C and 50 °C had the highest solute gain, and PVOD at 30 °C had more signi cant water loss and solute gain due to the di usion and convection forces, both of which facilitate the mass transfer process.Application of pulsed vacuum eases the osmotic agent into the matrix pores and facilitates the water loss and has a bene cial e ect on the kinetics, reaching equilibrium easily [23,38].Additionally, the combination of OD/OH encouraged more substantial concentrated levels in the samples than OD.In this case, the mass transfer in PVOD and PVOD/OH in comparison to OD is due to the unsteadiness of particles in the juice.Likewise, inadequate dissemination of the components may occur when gradient pressure in the vegetable tissue provokes the irregular ow of the osmotic agent through the structure and consequently accumulates bioactive components in some areas [39], making the mass transfer harder in OD.  6 Journal of Food Quality e most significant water activity reduction was in PVOD/OH at 50 °C, followed by OD/OH at 50 °C in contrast to the fresh sample.
is difference is attributed to temperature, pH, and electroporation, which promotes the gain from cryoconcentrated pomegranate juice and water loss [40,41].Other treatments also presented a significant difference compared with the fresh one.
On the other hand, it is important to mention the potential use of ultrasound as a technology that in general intensifies the mass exchanges and reduces drying times.In the case of ultrasound application in combination with the apple osmotic dehydration, Ciurzyńska et al. [42] demonstrated that it is an effective technology to accelerate the solid gain when using sucrose as an osmotic solution; however, when using chokeberry juice, it does not change.

Changes in Optical and Mechanical Properties.
Table 2 shows the values obtained for the fresh and treated apple for the optical parameters: L * , a * , b * , the hue angle (h * ab), chrome (C * ab), and change in color (ΔE).Previous studies have demonstrated that L * (luminosity) increases with the osmotic treatment [40].However, samples with a combination of PVOD and PVOD/OH have reduced L * due to the concentration of anthocyanins from the pomegranate juice. is same effect was reported by Petzold et al. [21], in which the treated sample had a lower L * than the fresh one.Temperature also contributed to this effect.Samples treated at 40 °C and 50 °C have the lowest value, and ΔE was significantly higher in the treatments with lower L.
is is due to the anthocyanin pigments being present in the pomegranate cryoconcentrated juice, especially for PVOD/OH at 40 °C and 50 °C (Section 3.2).On the other hand, we observed no significant difference in C * ab of PVOD/OH treatments.For h * ab, there was a significant difference between the fresh sample and the osmotically treated samples.
In general, the sample optical changes after the osmodehydration process could be connected with the plant material used (i.e., cryoconcentrate pomegranate juice), agreeing with the previous results that used chockeberry juice as an osmodehydration solution [17,18,37].
e firmness values acquired from the mechanical test are shown in Table 2. e sample of PVOD at 30 °C had the highest firmness value, which indicates a higher resistance [43].e fresh sample differs significantly from the samples treated at 50 °C.However, the values of treated samples at 40 °C had no difference with the fresh one, as well as with that of OD/OH and PVOD at 30 °C.

Conclusion
e combination of pulsed vacuum and ohmic heating in the osmodehydration of apple slices at 30 °C affected the retention of total phenolic content positively during processing time (180 min).
e highest preservation of anthocyanin pigments was observed with PVOD/OH at 50 °C, followed by the other two temperatures with the same type of treatment.On the other hand, the best results of flavonoids were obtained with OD/OH at 50 °C and 40 °C.Osmotic dehydration combined with pulsed vacuum and ohmic heating at 50 °C and 40 °C intensified the solid gain and water loss due to the diffusion and convection forces which accelerated the mass transfer process.PVOD/OH and OD/OH at 50 °C had the most significant water activity mainly caused by the electroporation and temperature.e changes in color were significant in C * ab (chrome) and L * (lightness), for apple slices treated with PVOD and PVOD/OH, due to the retention of anthocyanins from the pomegranate cryoconcentrated juice.e highest firmness was observed in the sample of PVOD at 30 °C.erefore, our results suggest that the PVOD/OH process at 50 °C for 120 min is the optimal treatment for osmodehydrated apples with pomegranate cryoconcentrated juice to obtain enriched apple slices.Finally, a possible future work is to study the proposed technology using other pomegranate cultivars such as the Mollar cultivars of Elche or Valenciana.

Figure 4
displays the total avonoid content kinetics.Similar to the TPC and TAC, avonoids were best retained by OD/OH at 50 (Figure 4(c)) and 40 °C (Figure 4(b)
Wonderful) and apples (cv.Granny Smith) were acquired in the local market (Chillán, Chile) during the same harvesting season (March 2017) and stored under refrigeration (≈5 °C) until processing.

Table 1 :
Composition parameters of fresh and processed samples (processing time: 180 min), water mass fraction (X w ), soluble solids mass fraction (X ss ), water loss (ΔM w Di erent letters in each column indicate signi cant di erences at p ≤ 0.05, according to a LSD test.OD: osmotic dehydration at atmospheric pressure; PVOD: pulsed vacuum, both with conventional heating; OH: ohmic heating (OD/OH and PVOD/OH), electric eld intensity at 6.66 V/cm (50 V).

Table 2 :
Firmness and color determination in fresh and treated apples with three temperatures (30 °C, 40 °C, and 50 °C).