78 luz zambrano food research international 2014 by Alan A. Rosales López

Food Research International 62 (2014) 974–983

Contents lists available at ScienceDirect

Food Research International journal homepage: www.elsevier.com/locate/foodres

Fresh-cut Red Delicious apples coating using tocopherol/mucilage nanoemulsion: Effect of coating on polyphenol oxidase and pectin methylesterase activities Maria L. Zambrano-Zaragoza a,⁎, Elsa Gutiérrez-Cortez a, Alicia Del Real c, Ricardo M. González-Reza a, Moises J. Galindo-Pérez a,b, David Quintanar-Guerrero b a Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Departamento de Ingeniería y Tecnología, Laboratorio de Procesos de Transformación y Tecnologías Emergentes en Alimentos, Km 2.5 Carretera Cuautitlán–Teoloyucan, San Sebastián Xhala, Cuautitlán Izcalli, Edo de México CP.54714, Mexico b Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Laboratorio de Posgrado en Tecnología Farmacéutica, Av. 1o de mayo s/n, Cuautitlán Izcalli C.P. 54745, Edo de México, Mexico c Departamento de Nanotecnología, Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro Qro. C.P. 76230, A.P. 1–1010, Mexico

a r t i c l e

i n f o

Article history: Received 29 January 2014 Accepted 3 May 2014 Available online 16 May 2014 Keywords: Nanoemulsions Nopal mucilage Pectin methylesterase Polyphenol oxidase Fresh-cut apples

a b s t r a c t The objective of this research was to determine the effect of α-tocopherol emulsion coatings with different particle sizes, with and without nopal mucilage (Opuntia ficus indica), applied to fresh-cut apples to evaluate pectin methylesterase (PME) and polyphenol oxidase (PPO) activity and changes associated with texture and the browning index at 4 °C for 21 days. The systems were: emulsion/mucilage; nanoemulsion; nanoemulsion/mucilage and solution all these with 2 g/L α-tocopherol; mucilage; and untreated apples. All treatments were applied by immersion. The particle size of the emulsions was 1281 nm with a zeta potential of −35 mV, while the nanoemulsions were b 200 nm with a zeta potential of b−40 mV, indicating stable systems. Respiration rate decreased to 35 mL O2/kg h when the apple was coated with nanoemulsions, compared to the control, α-tocopherol solution and mucilage (73, 63 and 61 mL O2/kg h). PME activity in the coated apples was lower in the submicron-size samples, helping maintain the firmness of the apples coated with nanoemulsion/mucilage (6.2 N) and nanoemulsion (5.1 N), compared to controls (2.7 N). At 21 days of storage, PPO activity decreased by 65% in the apples with nanoemulsion, as reflected in the lower browning indexes of 43.5 for nanoemulsions and 39.3 for nanoemulsion/mucilage. We conclude that the particle size of the emulsion droplets is a determining parameter in controlling texture and the browning index. The use of nopal mucilage helps control the browning index and is thus an additive with high potential for use in the preparation of nanocoatings. © 2014 Elsevier Ltd. All rights reserved.

Introduction Edible coatings are applied to protect fruits, maintain texture, assure ﬂavor retention, control volatile compounds, and decrease respiration, as well as to control release of nutraceuticals and antioxidant substances that can help increase the shelf life of fresh-cut fruits (Dhall, 2013; Pinheiro, Bourbon, Quintas, Coimbra, & Vicente, 2012). The latest generation of edible coatings proposes incorporating active compounds in submicron-size systems using nanoencapsulation techniques (Falguera, Quintero, Jiménez, Muñoz, & Ibarz, 2011). Nanotechnology has introduced innovations in food products on a nano-scale that affect the textural characteristics, color, sensory attributes, ⁎ Corresponding author. Tel.: +52 1525556232076; fax: +52 1525558175796. E-mail address: luz.zambrano@unam.mx (M.L. Zambrano-Zaragoza).

physicochemical stability, and controlled release of active agents, and may have the potential to affect shelf life (Ezhilarasi, Karthik, Chhanwal, & Anandharamakrishnan, 2013; Zambrano-Zaragoza et al., 2013). Nanoemulsions with particles ranging between 20 and 200 nm have applications in food preservation. They act through a protective effect on the fruit surface or as systems for the controlled release of lipophilic components such as antioxidants and other additives (Silva, Cerqueira, & Vicente, 2012). The principle problems with fresh-cut fruits concern changes in color and texture. Browning is caused by the action of polyphenol oxidase enzymes (PPO, EC 1.14.18.1) that interact with the phenols released during cutting to produce an undesirable brown color. Loss of ﬁrmness is due to pectin methylesterase activity (PME EC 3.1.1.11) (Barbagallo, Chisari, & Caputa, 2012). Recent studies report that high molecular weight polysaccharides can be used as encapsulants or carriers that support active agents applied to improve the functionality

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

of coatings used with fresh-cut fruits (Dhall, 2013; Rojas-Graü, SolivaFortuny, & Martín-Belloso, 2009). Nopal mucilage is a mixture of polysaccharides that, due to its emulsifying and water retention properties, plus its rheological behavior, is an interesting option for utilization as a carrier of active substances (Medina-Torres, Brito-De La Fuente, Torrestiana-Sánchez, & Katthain, 2000). Its use as an edible coating has been reported in strawberry preservation, where it achieved good results in increasing shelf life (DelValle, Hernández-Muñoz, Guarda, & Galotto, 2005), and improving optical properties and water vapor transport (Espino-Díaz et al., 2010). This mucilage has also been studied for its capacity for gallic acid encapsulation of bioactive compounds by spray-drying (Medina-Torres et al., 2013; Saénz, Tapia, Chávez, & Robert, 2009). Vitamin E, especially the active form of α-tocopherol, is widely used as an antioxidant in foods with high lipid content, and there are reports of its use as a nutraceutical effector in fresh-cut apples, where it showed the potential to modify nutritional properties to augment the potential consumer base beyond that of vitamin E, a dietary supplement in capsule form (Park, Zhao, Leonard, & Traber, 2005). Regarding potential applications of α-tocopherol as an antioxidant and nopal mucilage as a polysaccharide support in an edible coating formulated to enhance fruit preservation, this work proposed evaluating the effect of the particle size of emulsions with α-tocopherol/mucilage on PPO and PME activity in fresh-cut “Red Delicious” apples.

975

Preparation of the film-forming dispersions Mucilage dispersion The mucilage dispersion was prepared with 10 g/L of dry nopal mucilage and 10 g/L of glycerol in distilled water at 40 °C using a variable speed agitator (Eurostar Power Control-Visc IKA-WERKE®) at 500 rpm. Preparation of (o/w) emulsions The conventional emulsification method was used (Kulmyrzaev, Sivestre, & McClements, 2000). Briefly, 0.8 g/L of Span® 80 and 2 g/L of α-tocopherol were added to the dispersed phase. The continuous phase was composed of 10 g/L of glycerol, 4.2 g/L of Tween® 80 Considering HLB = 6 in the preparation. Formation of the emulsion was performed with a variable speed agitator (Eurostar Power Control Visc, IKA® WERKE) at 2000 rpm for 5 min. Then the nopal mucilage was added (10 g/L). Preparation of the nanoemulsions The nanoemulsions were obtained from an emulsion at 5 cycles of 5 min at 10,000 rpm of high agitation and rest intervals of 5 min between cycles, using a rotor/stator homogenizer (Ultra-Turrax IKA® T50 USA) and a dispersion tool (Model S 25 N-25 G, IKA®). Systems were prepared with and without nopal mucilage. The α-tocopherol solution was prepared with 2 g/L of TPGS in distilled water. Characterization of the film-forming dispersions

Materials and methods Materials The surfactants Span® 80 (Sorbitan monooleate, Mw = 428.6 g/mol, HLB = 4.3, μ ≈ 1000 cps at 25 °C) and Tween® (Polyoxyethylene (20) sorbitan monooleate, Mw = 1310 g/mol HLB = 15, μ ≈ 425 at 25 °C) were obtained from ICI Surfactants of Mexico. The antioxidant α-tocopherol acetate (98%) (HLB = 6, MW = 472 g/mol), α-tocopherol polyethylene glycol 1000 succinate (TPGS) and the glycerol plasticizer (ρ = 1.26 g/mL, MW = 92.09 g/m0l) were purchased from Sigma Aldrich®, USA. Distilled water was Milli-Q® quality. All other reagents were analytical grade.

Particle size (PS), poly-dispersion index (PDI) and zeta potential (ζ) The particle size (PS), poly-dispersion index (PDI), and zeta potential (ζ) of the colloidal systems were determined using a Z-sizer 4 (Malvern Instruments® ZEN NS 3600, Worcestershire, UK) ﬁtted with laser light scattering at a 90° angle, considering an refraction index of dispersant of 1.33 and of 1.59 for material. All dispersions were diluted in water. Tests were performed in triplicate for ζ and standardized with standard polystyrene dispersions to determine ζ (ζ = −55 mV), which measures the degree of repulsion between adjacent particles. The optimal ζ index is N|30|, considered a physically stable system (Bala et al., 2005). Coating formulations and application on fresh-cut apples

Biological material A batch of 40 kg of “Red Delicious” apples was purchased at a local fruit distribution center (Cuautitlán Izcalli, State of Mexico, Mexico). The apples were selected according to a subjective test of size, appearance and color, measurements of soluble solid content of 11-to-14° Brix, and ﬁrmness ≈ 7 N. All testing was performed randomly. The apples selected were stored for no more than 3 days at 4 ± 1 °C and 90–95% RH before use.

Nopal mucilage extraction Mucilage was obtained from cladodes of nopal (Opuntia ficus indica, var. esmeralda), with an average weight of 400 ± 10 g and ≈ 100 days of ripening. Nopal was collected at the Lords Ranch in the state of Guanajuato (México) during the 2010 spring–summer harvest, and was washed, disinfected and peeled following the methodology proposed by Contreras-Padilla et al. (2012). Mucilage extraction was performed by wet-milling in a disk centrifuge (DIDACTA Italy TAG 1/d) at 8000 rpm with screw gravity and a volumetric flow rate of 200 mL/min, to obtain the soluble fraction, which was then precipitated with ethanol (96%) to separate the precipitate by filtration under vacuum conditions (30 mm Hg), before final dehydration. Final moisture of the mucilage was 5 g water/g dry solid.

To evaluate the effect of nopal mucilage and particle size on the apple preservation. The batches considered in the study were: control, mucilage, emulsion/mucilage, nanoemulsion, nanoemulsion/mucilage and α-tocopherol solution. All coating systems contained 2 g/L of α-tocopherol. Controls were apples with no treatment. All apples were sanitized by immersion for 5 min in a chlorine solution at 70 ppm. To prevent contamination by microorganisms all cutting boards, utensils and recipients were disinfected. The sanitized fruits were manually peeled, cut the extremes with a knife and then cored and cut in eight wedges After cutting, the apple pieces were immersed in a 1% CaCl2 solution and then in the corresponding treatment. All samples were drained at 4 °C to remove excess coating. Once coated, the wedges were packed in 250 mL polypropylene containers with 90 g of fruit and stored at 4 ± 1 °C and 85% RH for 21 days. Morphological studies Morphological characterization of the emulsions was carried out by scanning electron microscopy (SEM) using the methodology proposed by Zambrano-Zaragoza, Mercado-Silva, Gutiérrez-Cortez, CastañoTostado, & Quintanar-Guerrero (2011). Samples were coated with gold using a JFC-1100 Sputter coater (JEOL, Tokyo, Japan) (~ 20 nm) and observed under a JSM 5600 LV-SEM ® LV microscope (JEOL, Tokyo, Japan) at a resolution of 5 nm and 28 kV and a pressure of 12-to-20 Pa in the chamber.

976

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

Morphological characterization of the apple surfaces with different treatments was performed by longitudinal tissue section and dipping in the corresponding system, followed by coating with gold as described above. The voltage used to take the micrographs was 20 kV as indicated in the methodology proposed by Zambrano-Zaragoza et al. (2014).

63 °C (Alandes et al., 2006) and evaluating the acid formed with a 0.025 N NaOH solution. The reaction mixture contained 500 μL of enzyme extract and 25 mL of a pectin solution (2 g/L) with 125 mM of NaCl. PME activity is proportional to the consumption rate of NaOH/ time unit. All measurements were performed in quintuplicate.

Respiration rate

Polyphenol oxidase activity (PPO)

The effect of coating on the O2 consumption rate was determined using the static method (Iqbal, Rodrigues, Mahajan, & Kerry, 2009; Wang, Duan, & Hu, 2009). Brieﬂy, 170 mL-glass bottles were used. Apple slices of known weight were placed inside and the bottles sealed. Samples of head space gas were drawn through a needle inserted into a septum placed in the top of container and connected to an oxygen analyzer (Quantek® Instruments model 905, USA) to obtain the oxygen concentration expressed as a fraction of the volume inside the container. Behavior was monitored for 20 days. The O2 consumption rate (RO2) was measured by the difference in O2 concentrations at different time intervals, expressed as:

PPO enzyme extraction was performed following the methodology proposed by Soysal (2009) with some modifications. Briefly, 30 g of coated surface was ground in a an Ultraturrax T18 IKA® (USA) with 20 mL of sodium phosphate buffer (0.2 M pH 7.0), 10 mL/L Triton® X-100 and polyvinylpyrrolidone (50 g/L) for 30 s at 4 °C. The homogenate was filtered to remove the solid particles and centrifuged at 10,000 rpm for 35 min. The supernatant consisted of the crude enzyme extract used to determine PPO activity. PPO activity was obtained by the method proposed by Zhou, Smith, and Lee (1993) using a mixture of 0.2 mL (0.050 M M citrate-phosphate buffer, pH 5) was added to 2.8 mL enzyme extract. Changes in absorbance at 420 nm for 15 min were measured in a Cintra 10 UV–vis spectrophotometer (GBC Scientific Equipment, Australia) and compared to a sample without enzymatic extract. Units of PPO activity were defined as a change of 0.001 Abs/min.

RO2 ¼

ðyi O2 −yO2 Þ V f ðt−t i Þ W

where: yiO2 and yO2 are, respectively, the O2 concentration in the gas mixture at time ti – any time except time zero expressed in hours – and RO2 is the respiration rate; W is the weight of the apples (kg); and Vf is the free volume inside the container. Physicochemical changes Weight loss of 8 apple pieces from each treatment group was monitored during storage. Percentage weight loss relative to initial weight was calculated by weighing samples every 3 days in triplicate. The juice from approximately 90 g of apple wedges was extracted from the polypropylene containers for each sample and used to measure total soluble solids (TSS) using an ABBE refractometer (Spectronic, Inc., USA). Titratable acidity was ascertained by titration with 0.1 N NaOH at pH 8.1 and expressed as malic acid (mg) (Zambrano-Zaragoza et al., 2013). pH was measured with a potentiometer (Philips Harris model E3039018G/K, Shenstone England) equipped with an electrode (model P43-120). All measurements were performed in triplicate.

Browning Index (BI) The effect of coatings on the color and browning index of fresh-cut apples was determined by measuring their surface color using a Minolta CR-300 colorimeter (Konica Minolta, Tokyo, Japan) every 3 days for 21 days The browning index was calculated as follows (ZambranoZaragoza et al., 2014). BI ¼

½100ðx−0:31Þ 0:172

Where: x¼

ða þ 1:75Þ : ð5:646L þ a þ 3:12bÞ

Statistical analysis Firmness measurement Firmness of the apple wedges was measured every other day up to day 21 of storage. In the tests, 5 wedges from each treatment batch were evaluated using an INSTRON 4411 with a load cell of 5 kN equipped with a fixed, 6-mm punch thickness (Instron, Mass., USA). Each sample wedge was fractured by a downward motion using a velocity of 150 mm min−1 to measure the maximum force (highest N value) required to penetrate 5 mm into the wedge. This measurement was taken to represent the firmness index (Varela, Salvador, & Fiszman, 2007; Zambrano-Zaragoza et al., 2013). All tests were performed in quintuplicate. Pectin methylesterase activity (PME) PME activity was determined using the methodology proposed by Alandes, Hernando, Quiles, Pérez-Munuera, and Lluch (2006) with some modifications. Briefly, 30 g of coated apple tissue surface were homogenized in an ultraturrax T18 (IKA®, USA) and the enzyme extracted using pH 8.0 buffer (tris 0.2 M) with 1 M NaCl for 2 h (1:2 w/v). The homogenate was filtered to remove solid particles and centrifuged at 10,000 rpm for 45 min. The supernatant consisted of the crude enzyme extract used to obtain PME activity, measured by determining the amount of acid released per time unit at pH 7.0 and

Results were analyzed using the statistical analysis software MINITAB® Release 14 to determine the effect of coating composition and average particle droplets on PPO and PME activity, the browning index, and ﬁrmness. Differences among treatments were a function of variation with respect to the mean performing an ANOVA and Tukey test (α = 0.05). Results and discussion Characterization of the ﬁlm-forming dispersions Fig. 1 shows the PS distribution as a function of the (%) intensity of the particles. The average PS obtained from the α-tocopherol acetate nanoemulsions was within the range reported for this system type less 1000 nm depending on the method of preparation (Weiss, Takhistov, & McClements, 2006) and particularly in nanoemulsions with less of 2% (w/w) of vitamin E obtained particles sizes between 130 and 150 nm (Saberi, Fang, & McClements, 2013). The nanoemulsion/mucilage showed a slightly higher PS than that obtained for nanoemulsions alone (Rao & McClements, 2011). However, both systems had monomodal behavior. The nanoemulsion had an average PS of 190 nm with a PDI of 0.119, while the nanoemulsion/mucilage had a PS of 247 nm and a PDI of 0.205. The difference in particle size in these submicron systems is

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

Intensity (%)

20 15 10 5 0 10

100

1000

10000

Particle size (nm) NE/M

E/M

Fig. 1. Particle size distribution of ﬁlm-forming coating systems. NEM: nanoemulsionmucilage; NE: nanoemulsion; EM: emulsion-mucilage; and E: emulsion.

attributable to the influence of the nopal mucilage, which could function as an encapsulant to envelop the nano-droplets of α-tocopherol. It has been reported that the use of this biopolymer also contributes to protecting the active agent (Medina-Torres et al., 2013). Both systems were in the submicron range and in the PS range mentioned in several earlier studies (Saberi et al., 2013; Weiss et al., 2006). The emulsion/ mucilage systems exhibited bimodal behavior. The first peak had an average PS of 220 nm (2.6%), whereas the average PS in the second was 1281 nm (14.1%) with a wider PS (339–3091 nm) and PDI of 0.46. A predictive parameter of the emulsion stability is ζ, which represents the surface charge of the particles, and may be related to the probability of coalescence due to the effect of net charge interaction (Choi, Kim, Cho, Hwang, & Kim, 2011). The ζ for the colloidal systems studied values were below −40 mV, considering a criteria of ζ ≥ | 30| to indicate excellent physical stability of dispersions due to the reduction of gravity, sedimentation, and the prevention of flocculation (ZambranoZaragoza et al., 2011). An important aspect of colloidal systems is their architecture. Fig. 2(a and b) shows the morphology of the emulsion and nanoemulsion obtained showing good homogeneity and integrity of the systems as well as a classical spherical shape of the droplets formed (Ushikubo & Cunha, 2014). Also, it was possible to distinguish α-tocopherol droplets of submicron size for nanoemulsion (Fig. 2a) and of 1–5 μm for the emulsion (Fig. 2b); findings that correlate with the particle sizes obtained by light scattering. Microstructural characterization of the apple coatings Scanning electron microscopy (SEM) provides a better understanding of the relationships of water vapor transmission, mechanical and optical properties with the films' structural characteristics (Jouki, Yazdi, Mortazavi, & Koocheki, 2014). Fig. 3(a–f) shows the surface appearance

of the mucilage and apple tissue after 24 h of the different treatments. Fig. 3(a′) presents the surface appearance of the mucilage before its application as a coating on the apple surface, while Fig. 3(a) shows the appearance of the control apples with surface characteristics of parenchymal tissue and some other cellular structures as mentioned previously Soliva-Fortuny, Lluch, Quiles, Grigelmo-Miguel, & MartínBelloso, 2003; Zambrano-Zaragoza et al., 2014. Fig. 3(b) shows a continuous surface coating because the solution of α-tocopherol has no resistance to the incorporation in the apple, but yet the water vapor permeability is greater for the miscibility of the components. In Fig. 3(c), meanwhile, one can observe the coated apples with mucilage and the micrograph which shows that the hydrocolloid forms a protective layer on the product surface. Mucilage is a hydrocolloid that has been reported to be a compound with good properties in terms of forming edible coatings (Del-Valle et al., 2005; Vargas, Pastor, Chiralt, McClements, & González-Martínez, 2008). The mucilage film displayed a compact, smooth and continuous microstructure with no irregularities (Jouki et al., 2014). Fig. 3(d) highlights the coating formed by the emulsion/mucilage, showing droplets that probably result from the accumulation of water on the surface that combines with the presence of stabilized α-tocopherol in a rich medium in soluble components. It is suggested that mucilage forms a thin film on the surface of the fruit that prevents the coalescence of droplets of α-tocopherol but allows infiltration into the cells inside the apple tissue. Fig. 3(e) illustrates the formation of a nano-coating with the α-tocopherol nanoemulsion. This coating adheres to the fruit surface with α-tocopherol nano-droplets and evidence of water retained from the fruit. Finally, Fig. 3(f) shows the coating formed by α-tocopherol nanoemulsion/mucilage with evidence of aggregates with different PS homogeneously distributed throughout the coating and exudate droplets on the external surface of the cell walls (Zambrano-Zaragoza et al., 2014). The coating formed exhibited compact, smooth, continuous film, without microstructure irregularities. The addition of α-tocopherol made a heterogeneous structure due to the oil droplets which were trapped by the polyssacaride on a continuous network showed in Fig. 3(c to f) (Jouki et al., 2014). Based on the surface aspect of the coating it was possible to establish that this dispersion formed a continuous thin coating over the entire surface. The appearance of this coating on the tissue surface is dependent on the particle size of the film-forming dispersion. Respiration rate, weight loss and physicochemical changes Fig. 4 shows the respiration rate and weight loss of the fresh-cut apples as a function of treatment with α-tocopherol dispersions, with and without nopal mucilage. The width of the bars represents the deviation of the results with respect to the average with α = 0.05. Fig. 4 highlights the changes in respiration rate of the control apples, revealing a significant increase in respiration rate during the first 9 days of storage, due to effects related to the tissue stress produced during preparation operations with the fresh-cut product (Chiumarelli, Ferrari, Sarantópoulos, & Hubinger, 2011), though this respiration rate then remained practically

Emulsion droplets

Nanoemulsion droplets

1 µm

977

15 kX

100 µm

Fig. 2. Morphological aspect of nanoemulsion and emulsion.

7 kX E

978

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

a’

20kV X1500

20kV

X1000

10µm

Control

20kV

X1000

10µm

Micropores

20kV

X1500

10µm

20kV

X1500

10µm

f NEM coating

20kV

X1500

10µm

20kV

X1500

10µm

NEM

Fig. 3. Apples coated with different ﬁlm-forming dispersions: a) control; a′) mucilage; b) S = α-tocopherol solution; c) M = apple mucilage; d) EM = emulsion-mucilage; e) NE = nanoemulsion; and, f) NEM = nanoemulsion-mucilage.

90 80

mL O2/kgh

70 60 50 40 30

0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21

NEM

0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21

Coating

0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21

Days

Fig. 4. Changes in respiration rate in fresh-cut apples at 4 °C, C = control, EM = emulsion-mucilage, M = Mucilage, NE = nanoemulsion, NEM = nanoemulsion-mucilage and S = α-tocopherol solution.

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

constant (73 mL O2/kg h) up to 15 days of storage. The fresh-cut apples coated with emulsion/mucilage showed no statistically significant changes (α = 0.05) in respiration rate with respect to the mucilage-coated apples; a finding attributed to the size and non-homogenous distribution of the α-tocopherol droplets. The fresh-cut apples treated with α-tocopherol solution increased the respiration rate to 63 mL O2/kg h. The apples coated with submicron size systems showed a greater reduction in respiration rate during the storage period, a behavior that favors preservation. According to Qi, Hu, Jiang, Tian, and Li (2011), the initial reduction of respiration rate is an important factor in increasing the shelf life of fresh-cut fruits. The use of submicron size systems helps improve control of the reduction of the respiration rate, attributable to the better distribution of the antioxidant in the coating formed on the apples. The effect of treatment on the weight loss associate with the juice exuded from apples during storage, an aspect that is a key quality factor in the preservation of fresh-cut (Mantilla, Castell-Perez, Gomes, & Moreira, 2013). However in this study the weight loss was less than 0.4%. Furthermore is important noted than the apples with α-tocopherol solution showed skin deep dehydration suggesting low protection and water loss by transpiration. The best results were achieved when nanoemulsions with and without mucilage were used as film-forming dispersions, as they achieved the lowest weight loss after 7 days at 4 °C. With regards to physicochemical changes are showed in Table 1, the soluble solids showed only a minimal variation in all the coated, freshcut apples. Initial Brix was between 12.8 and 13.5°, while at the end of storage it averaged 11.5°; a behavior that coincided with other reported results (Olivas, Mattinson, & Barbosa-Cánovas, 2007; Rocha, Brochado, & Morais, 1998). However, it is important to highlight that the samples treated with α-tocopherol solution showed a slight decrease in the °Brix due to the immersion treatment. The pH factor remained in a range of 3.5 to 3.8 for all coated samples, though a slight increase in pH is attributed to changes in initial respiratory activity due to the cutting and preparation that was controlled for each coating application (Albanese, Cinquanta, & Di Matteo, 2007; Cortez-Vega, Becerra-Prado, Soares, & Fonseca, 2008). No statistically significant changes (p ≤ 0.05) in acidity expressed as a function of malic acid were obtained during storage time with slight decrease to end the storage period. The changes were

979

associated with changes in respiration activity particularly in the fresh-cut apple with α-tocopherol solution this behavior was reported by Perera, Balchin, Baldwin, Stanley, and Tian (2003) in apples “Gala” The acidity was in ranged from 0.4 at 0.32% expressed as malic acid. Changes in firmness and PME activity The loss of firmness in the fresh-cut fruits was the most notable change. This is the result of the degradation of the cell wall that results from peeling, which accelerates metabolic activity and water loss in products (Olivas et al., 2007; Rojas-Graü, Tapia, & Martín-Belloso, 2008). Fig. 5 shows the comparative behavior between loss of firmness and PME activity. The height of the box indicates the deviation of results with respect to the average and it is marked with a point in the box center. Fig. 5(a) presents the evolution of the measures of firmness during storage up 21 days at 4 °C, showing that the use of coating helps to reduce firmness loss significantly (p b 0.05) by maintaining the product structure for a longer time. Fresh-cut apples with mucilage retained their texture longer than did the control. Similar results were reported by Benítez, Achaerandio, Sepulcre, and Pujolà (2013). Also, Del-Valle et al. (2005) reported that nopal mucilage helps retain the texture of strawberries during refrigerated storage. Fig. 5(a) shows that samples treated with nanoemulsion/mucilage maintained the texture best with respect to the initial firmness of the product and the control fresh-cut apples. This behavior is attributed to the interactions between Ca+ and the nopal mucilage, which apparently promote changes in the crosslinking of the polymer chains, resulting in lower water loss. Moreover, in the nanoemulsion/mucilage the α-tocopherol nano-droplets, attributed to the effect of particle size of the droplets of α-tocopherol which allowed a better protection. It has been reported that αtocopherol due to the hydrophobic nature contributed to decrease the water loss. This effect is achieved by synergy between the support polysaccharide and the nanoemulsion (Martins, Cerqueira, & Vicente, 2012). Finally the apple treated with α-tocopherol solution showed a decrease in the texture at the 9 days, increased later due to the dehydration of surface of apple wedge. Fig. 5(b), meanwhile, shows the activity of the PME enzymes, which may be beneficial or harmful in maintaining fruit texture, depending on many factors, such as composition, moisture, pectin content, etc. (Jolie, Duvetter,

Table 1 Effect of coating treatment on the physicochemical properties of fresh-cut apples stored at 4 °C for 21 days. Days 0

C pH Acidity °Bx pH Acidity °Bx pH Acidity °Bx pH Acidity °Bx pH Acidity °Bx pH Acidity °Bx pH Acidity °Bx pH Acidity °Bx

3.60 0.38 12.80 3.60 0.34 13.80 3.60 0.36 12.83 3.60 0.33 13.80 3.60 0.34 12.93 3.60 0.34 13.10 3.60 0.35 12.30 3.60 0.32 11.70

EM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.02a 0.20a 0.01a 0.01a 0.10a 0.01a 0.03a 0.40b 0.01a 0.04 0.50a 0.01 0.04a 0.50b 0.01a 0.02a 0.61a 0.01a 0.05a 0.51b 0.01a 0.01a 0.17c

3.70 0.35 13.53 3.60 0.35 13.40 3.70 0.33 13.33 3.70 0.33 13.70 3.70 0.29 13.50 3.60 0.28 12.43 3.60 0.30 12.30 3.66 0.29 11.73

M ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.04a 0.25a 0.00a 0.02a 0.34a 0.01a 0.02a 0.11a 0.01a 0.03a 0.52a 0.01a 0.02a 0.36a 0.01a 0.01b 0.51b 0.01a 0.01a 0.10b 0.06a 0.02b 0.47c

3.60 0.36 13.23 3.56 0.35 13.30 3.70 0.41 13.10 3.70 0.39 13.20 3.70 0.38 13.20 3.60 0.38 12.66 3.70 0.34 12.20 3.63 0.33 11.30

NE ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.03a 0.30a 0.06a 0.01a 0.10a 0.01a 0.02b 0.80a 0.01a 0.03b 0.80a 0.01a 0.03b 0.51a 0.01a 0.03b 0.30b 0.01a 0.03a 0.10b 0.06a 0.01a 0.50c

3.70 0.37 13.56 3.60 0.33 13.60 3.70 0.35 13.46 3.70 0.35 13.46 3.70 0.33 12.93 3.60 0.38 13.00 3.70 0.33 12.30 3.60 0.35 12.10

NEM ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.03a 0.05a 0.01a 0.03a 0.10a 0.01a 0.06a 0.64a 0.01a 0.04a 0.38a 0.01a 0.01a 0.46b 0.01a 0.05a 0.70a 0.01a 0.02a 0.10b 0.01 0.01a 0.64b

*Means at the same column and row with different letters are signiﬁcantly different (p b 0.05). C = control; EM = emulsion mucilage; M = mucilage; NE = nanoemulsion; NEM = Nanoemulsion mucilage; S = α-tocopherol solution.

3.66 0.36 13.36 3.66 0.35 13.4 3.70 0.36 13.97 3.70 0.38 13.56 3.70 0.36 13.63 3.63 0.36 13.20 3.63 0.34 12.86 3.66 0.33 12.00

S ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.02a 0.15a 0.05a 0.01a 0.46a 0.01a 0.02a 0.45a 0.01a 0.04a 0.35a 0.01a 0.03a 0.47a 0.05a 0.03a 0.80a 0.06a 0.05a 0.44b 0.06a 0.06a 0.45b

3.53 0.33 13.20 3.60 0.33 12.70 3.53 0.36 12.06 3.73 0.33 12.06 3.76 0.27 12.13 3.76 0.27 11.90 3.73 0.28 11.80 3.80 0.29 11.70

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.20a 0.04a 0.30a 0.20a 0.01a 0.45b 0.20a 0.03a 0.67b 0.06a 0.01a 0.70b 0.01a 0.01a 0.26b 0.05a 0.06a 0.70b 0.10a 0.02a 0.60b 0.01a 0.02a 0.69b

980

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

Firmeness (N)

NEM

Coating

0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21

Days

PME (UEA/g)

NEM

Coating

0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21

Days

Fig. 5. Relation between changes in ﬁrmness and pectin methylesterase activity in fresh-cut apples at 4 °C: a) ﬁrmness; b) pectin methylesterase, C = control, EM = emulsion-mucilage, M = Mucilage, NE = nanoemulsion, NEM = nanoemulsion-mucilage and S = α-tocopherol solution.

Van Loey, & Hendrickx, 2010). Fig. 5(b) shows that the control apples exhibited an increase in PME activity during the first 9 days, followed by a reduction directly proportional to the amount of residual substrate in the apple associated with firmness loss. The apples coated with mucilage showed a greater increase in PME activity during the first 6 days due to the pectin present in the mucilage, although the PME activity was lower than control. The samples treated with emulsion/mucilage shown a higher PME activity at initial time; however this decreased during storage time. Otherwise, the fresh-cut apples treated with αtocopherol solution showed an increase in PME activity to maximum at 15 days of storage at 4 °C, probably due to that the solution permeates into the fruit, decreasing the protective effect, because non limiting the gas exchanger, which is associated with the behavior in the respiration rate, which leads us to infer that the use of α-tocopherol modifies the respiration rate and acts as an antioxidant agent by limiting oxygen absorption. The effect of PS is a critical factor in maintaining the firmness observed in the apples coated with nanoemulsion/mucilage

because PME activity in these samples increased after 9 days of refrigerated storage. This increase could be attributed to the calcium pectate that forms from the mucilage pectins and contributes to maintenance and uniform texture (Jolie et al., 2010). Changes in the browning index and polyphenol oxidase activity (PPO) Fig. 6 shows the effect of applying coatings with α-tocopherol on the browning index (BI) and PPO activity in fresh-cut apples during refrigerated storage (4 °C), revealing that the coated samples had a statistically signiﬁcant effect (α = 0.05) regardless of protection level in relation to control apples in terms of decreasing the browning index and PPO enzyme activity. This BI was used to assess changes associated with the development of browning, deﬁned as coffee color purity, which is one of the main biochemical indicators of enzymatic browning in fresh-cut fruits (Pathare, Opara, & Al-Said, 2013). Fig. 6(a) shows the evolution of the BI, which reveals that the control apples had the fastest

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

981

Browning Index

140

0 3 6 129 1 15 218 0 3 6 9 12 1 15 218

0 3 6 129 1 15 218

Coating

0 3 6 9 12 15 18 21 0 3 6 19 152 1 281

Days

0 3 6 129 1 15 281

PPO activity (U/min mL)

120 100

80 60 40 20

NEM

Coating

0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21

Days

Fig. 6. Effect of polyphenol oxidase activity on the browning index in fresh-cut apples at 4 °C: a) browning index; and, b) polyphenol oxidase activity. C = control, EM = emulsion-mucilage, M = Mucilage, NE = nanoemulsion, NEM = nanoemulsion-mucilage and S = α-tocopherol solution.

browning development from the beginning of storage, the highest rate of browning in the first 9 days, and evidence of browning after just 24 h of refrigeration. Although the apples coated with nopal mucilage showed some control of browning, the development of brown colors occurred slowly and clearly up to 12 days. The coating affect was associated with the decrease in oxygen uptake (Fig. 4). It has been reported that the use of high molecular weight polysaccharides contributes to slowing respiration and controlling enzymatic activity, thereby reducing the browning rate (Chiumarelli et al., 2011). The apples treated with αtocopherol solution showed control of browning to 9 days slightly increasing and until 15 days the BI was increased due to the solution is infiltrated into the tissue and loss antioxidant activity. With respect to the apples treated with the α-tocopherol emulsion/ mucilage, it was possible to demonstrate that the antioxidant helps decrease browning. It is important to point out that the apples coated with the α-tocopherol nanoemulsion and nanoemulsion/mucilage had a slower rate of browning but no statistically significant differences between the two treatments (p ≤ 0.05) up to day 12, after this time and between the 15 and 21 days probably the α-tocopherol was loss causing

an increase in the BI. However, browning development in the apples with nanoemulsions was 30% less than in controls, in contrast to the result obtained by Li et al. (2011), who tested a coating on Fuji apples with nano-ZnO but found an equal development of browning as in controls after 12 days of storage. Fig. 6(a) shows that adding nopal mucilage as an ingredient in the ﬁlm-forming dispersion of the nanoemulsions helped reduce the browning index better than in the apples tested with the nanoemulsion alone, since it seems that the activity of α-tocopherol as an antioxidant agent and modiﬁer of the barrier properties is enhanced by the controlled release of the agent, which forms a double barrier with the nopal mucilage that increases the contact area with the fruit surface (Mei & Zhao, 2003). Clearly, the effects observed can be attributed to the ability of the coating to function as an oxygen barrier. The α-tocopherol contributes to modifying the transport properties by limiting enzyme action (Park et al., 2005), while PS impedes a more homogenous distribution and effect on the tissue surface. The most important changes associated with the cutting and fracturing of tissues are caused by the release of enzymes, particularly PPO

982

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983

activity, which causes browning upon contact with the phenolic substrate (Olivas et al., 2007; Rojas-Graü et al., 2009). Brown pigments develop due to the abiotic stress of cutting, peeling, etc. (Rocha & Morais, 2001). Fig. 6(b) shows the changes in PPO activity during refrigerated storage at 4 °C. The control fresh-cut apples increased PPO activity to a maximum of 126 U/mL min after 8 days; an activity measured according to the browning index. This response was related to depletion of the substrate and the formation of quinones on the apple surface as a response to the oxidation of phenolic compounds that results in the quinones interacting with the proteins generated through covalent condensation with the groups \SH and \NH2 of the amino acids, which are susceptible to alkylation by quinones. Thus, the bonds formed between quinones and proteins promote changes in the protein structure and functional characteristics that result in the inhibition of the synthesis of the “novo” of phenols, thereby reducing PPO activity due to the lack of a suitable substrate (Wojdyło, Oszmiański, & Laskowski, 2008). The batch with mucilage showed no statistically significant difference (p ≤ 0.05) with respect to the control samples in terms of PPO activity after 12 days, though it did present a reduction in the browning index (Fig. 5(a)), probably because the mucilage creates a modified atmosphere capable of capturing part of the exudates, including phenolic compounds, that limit substrate availability, thus generating a lower browning rate at the start of storage. Fig. 6(b) shows that the emulsion/ mucilage had a maximum of ≈120 U/mL min, which is attributable to the use of α-tocopherol as an antioxidant, which is also associated with a lower browning index. The PPO activity was practically constant during the first 6 days, with a maximum of 72 U/mL min at 12 days. This comportment was related to slow browning that had the TPGS for the first 9 days. However, the α-tocopherol solution does not protect as coating because there was an infiltrating into the apples reason that can not oxygen restriction. The apples coated with submicron-sized film-forming dispersions had the best performance with a clear decrease in PPO activity compared to all other coatings. The nanoemulsion provided a protective effect since the maximum PPO activity (25.5 U/mL min) was not achieved until day 13 of storage. The nanoemulsion/mucilage showed a similar trend to that of the emulsion/mucilage, though it also clearly evidenced that the smaller particle size helped decrease PPO activity, which was 62% less than in the case of the emulsion/mucilage. On the basis of these findings it can be concluded that the decrease in particle size in the film-forming dispersions helps increase the interaction probability of α-tocopherol, thus limiting the presence of oxygen and forming a homogeneous modified atmosphere with beneficial effects for the preservation of fresh-cut apples. Conclusions The coatings formed with nanoemulsion had a significant effect on PME and PPO activity in the fresh-cut apples. The protective effect of nanoemulsion is potentiated by the addition of nopal mucilage, which acts as an encapsulant that enhances the action of α-tocopherol. The submicron-size systems helped maintain the characteristics of color and firmness while also controlling weight loss, indicating that particle size has important implications for the control of the changes that can affect product quality during refrigerated storage. Acknowledgments The authors acknowledge the financial support for this work from PAPIIT (Ref. IT200814) DGAPA-UNAM. References Alandes, L., Hernando, I., Quiles, A., Pérez-Munuera, I., & Lluch, M.A. (2006). Cell wall stability of fresh-cut fuji apples treated with calcium lactate. Journal of Food Science, 71(9), S615–S620.

Albanese, D., Cinquanta, L., & Di Matteo, M. (2007). Effects of an innovative dipping treatment on the cold storage of minimally processed annurca apples. Food Chemistry, 105(3), 1054–1060. Bala, I., Bhardwaj, V., Hariharan, S., Sitterberg, J., Bakowsky, U., & Ravi Kumar, M. N. V. (2005). Design of biodegradable nanoparticles: A novel approach to encapsulating poorly soluble phytochemical ellagic acid. Nanotechnology, 16(12), 2819–2822. Barbagallo, R. N., Chisari, M., & Caputa, G. (2012). Effects of calcium citrate and ascorbate as inhibitors of browning and softening in minimally processed ‘birgah’ eggplants. Postharvest Biology and Technology, 73, 107–114. Benítez, S., Achaerandio, I., Sepulcre, F., & Pujolà, M. (2013). Aloe vera-based edible coatings improve the quality of minimally processed ‘hayward’ kiwifruit. Postharvest Biology and Technology, 81, 29–36. Chiumarelli, M., Ferrari, C. C., Sarantópoulos, C. I. G. L., & Hubinger, M.D. (2011). Fresh-cut tommy atkins mango pre-treated with citric acid and coated with cassava (manihot esculenta crantz) starch or sodium alginate. Innovative Food Science and Emerging Technologies, 12(3), 381–387. Choi, A., Kim, C., Cho, Y., Hwang, J., & Kim, C. (2011). Characterization of capsaicin-loaded nanoemulsions stabilized with alginate and chitosan by self-assembly. Food and Bioprocess Technology, 4(6), 1119–1126. Contreras-Padilla, M., Gutiérrez-Cortez, E., Valderrama-Bravo, M. d. C., Rojas-Molina, I., Espinosa-Arbeláez, D.G., Suárez-Vargas, R., et al. (2012). Effects of drying process on the physicochemical properties of nopal cladodes at different maturity stages. Plant Foods for Human Nutrition, 67(1), 44–49. Cortez-Vega, W. R., Becerra-Prado, A.M., Soares, J. M., & Fonseca, G. G. (2008). Effect of L-ascorbic acid and sodium metabisulfite in the inhibition of the enzymatic browning of minimally processed apple. International Journal of Agricultural Research, 3(3), 196–201. Del-Valle, V., Hernández-Muñoz, P., Guarda, A., & Galotto, M. J. (2005). Development of a cactus-mucilage edible coating (Opuntia ficus indica) and its application to extend strawberry (Fragaria ananassa) shelf-life. Food Chemistry, 91(4), 751–756. Dhall, R. K. (2013). Advances in edible coatings for fresh fruits and vegetables: A review. Critical Reviews in Food Science and Nutrition, 53(5), 435–450. Espino-Díaz, M., De Jesús Ornelas-Paz, J., Martínez-Téllez, M.A., Santillán, C., BarbosaCánovas, G. V., Zamudio-Flores, P. B., et al. (2010). Development and characterization of edible films based on mucilage of Opuntia ficus-indica (L.). Journal of Food Science, 75(6), E347–E352. Ezhilarasi, P. N., Karthik, P., Chhanwal, N., & Anandharamakrishnan, C. (2013). Nanoencapsulation techniques for food bioactive components: A review. Food and Bioprocess Technology, 6(3), 628–647. Falguera, V., Quintero, J. P., Jiménez, A., Muñoz, J. A., & Ibarz, A. (2011). Edible films and coatings: Structures, active functions, and trends in their use. Trends in Food Science & Technology, 22(6), 292–303. Iqbal, T., Rodrigues, F. A. S., Mahajan, P. V., & Kerry, J. P. (2009). Mathematical modeling of the influence of temperature and gas composition on the respiration rate of shredded carrots. Journal of Food Engineering, 91(2), 325–332. Jolie, R. P., Duvetter, T., Van Loey, A.M., & Hendrickx, M. E. (2010). Pectin methylesterase and its proteinaceous inhibitor: A review. Carbohydrate Research, 345(18), 2583–2595. Jouki, M., Yazdi, F. T., Mortazavi, S. A., & Koocheki, A. (2014). Quince seed mucilage films incorporated with oregano essential oil: Physical, thermal, barrier, antioxidant and antibacterial properties. Food Hydrocolloids, 36, 9–19. Kulmyrzaev, A., Sivestre, M. C., & McClements, D. J. (2000). Rheology and stability of whey protein stabilized emulsions with high CaCl2 concentrations. Food Research International, 33(1), 21–25. Li, X., Li, W., Jiang, Y., Ding, Y., Yun, J., Tang, Y., et al. (2011). Effect of nano-ZnO-coated active packaging on quality of fresh-cut ‘fuji’ apple. International Journal of Food Science and Technology, 46(9), 1947–1955. Mantilla, N., Castell-Perez, M. E., Gomes, C., & Moreira, R. G. (2013). Multilayered antimicrobial edible coating and its effect on quality and shelf-life of fresh-cut pineapple (ananas comosus). LWT - Food Science and Technology, 51(1), 37–43. Martins, J. T., Cerqueira, M. A., & Vicente, A. A. (2012). Influence of α-tocopherol on physicochemical properties of chitosan-based films. Food Hydrocolloids, 27(1), 220–227. Medina-Torres, L., Brito-De La Fuente, E., Torrestiana-Sánchez, B., & Katthain, R. (2000). Rheological properties of the mucilage gum (Opuntia ficus indica). Food Hydrocolloids, 14(5), 417–424. Medina-Torres, L., García-Cruz, E. E., Calderas, F., González Laredo, R. F., Sánchez-Olivares, G., Gallegos-Infante, J. A., et al. (2013). Microencapsulation by spray drying of gallic acid with nopal mucilage (Opuntia ficus indica). LWT — Food Science and Technology, 50(2), 642–650. Mei, Y., & Zhao, Y. (2003). Barrier and mechanical properties of milk protein-based edible films containing nutraceuticals. Journal of Agricultural and Food Chemistry, 51(7), 1914–1918. Olivas, G. I., Mattinson, D. S., & Barbosa-Cánovas, G. V. (2007). Alginate coatings for preservation of minimally processed ‘gala’ apples. Postharvest Biology and Technology, 45(1), 89–96. Park, S., Zhao, Y., Leonard, S. W., & Traber, M. G. (2005). Vitamin E and mineral fortification in fresh-cut apples (fuji) using vacuum impregnation. Nutrition and Food Science, 35(6), 393–402. Pathare, P. B., Opara, U. L., & Al-Said, F. A. (2013). Colour measurement and analysis in fresh and processed foods: A review. Food and Bioprocess Technology, 6(1), 36–60. Perera, C. O., Balchin, L., Baldwin, E., Stanley, R., & Tian, M. (2003). Effect of 1methylcyclopropene on the quality of fresh-cut apple slices. Journal of Food Science, 68(6), 1910–1914. Pinheiro, A.C., Bourbon, A. I., Quintas, M.A.C., Coimbra, M.A., & Vicente, A. A. (2012). Κ-carrageenan/chitosan nanolayered coating for controlled release of a model bioactive compound. Innovative Food Science and Emerging Technologies, 16, 227–232.

M.L. Zambrano-Zaragoza et al. / Food Research International 62 (2014) 974–983 Qi, H., Hu, W., Jiang, A., Tian, M., & Li, Y. (2011). Extending shelf-life of fresh-cut ‘fuji’ apples with chitosan-coatings. Innovative Food Science and Emerging Technologies, 12(1), 62–66. Rao, J., & McClements, D. J. (2011). Food-grade microemulsions, nanoemulsions and emulsions: Fabrication from sucrose monopalmitate & lemon oil. Food Hydrocolloids, 25(6), 1413–1423. Rocha, A.M. C. N., Brochado, C. M., & Morais, A.M. M. B. (1998). Influence of chemical treatment on quality of cut apple (cv. jonagored). Journal of Food Quality, 21(1), 13–28. Rocha, A.M. C. N., & Morais, A.M. M. B. (2001). Characterization of polyphenoloxidase (PPO) extracted from ‘jonagored’ apple. Food Control, 12(2), 85–90. Rojas-Graü, M.A., Soliva-Fortuny, R., & Martín-Belloso, O. (2009). Edible coatings to incorporate active ingredients to fresh-cut fruits: A review. Trends in Food Science and Technology, 20(10), 438–447. Rojas-Graü, M.A., Tapia, M. S., & Martín-Belloso, O. (2008). Using polysaccharide-based edible coatings to maintain quality of fresh-cut fuji apples. LWT — Food Science and Technology, 41(1), 139–147. Saberi, A. H., Fang, Y., & McClements, D. J. (2013). Fabrication of vitamin E-enriched nanoemulsions: Factors affecting particle size using spontaneous emulsification. Journal of Colloid and Interface Science, 391(1), 95–102. Saénz, C., Tapia, S., Chávez, J., & Robert, P. (2009). Microencapsulation by spray drying of bioactive compounds from cactus pear (Opuntia ficus-indica). Food Chemistry, 114(2), 616–622. Silva, H. D., Cerqueira, M.A., & Vicente, A. A. (2012). Nanoemulsions for food applications: Development and characterization. Food and Bioprocess Technology, 5(3), 854–867. Soliva-Fortuny, R. C., Lluch, M.A., Quiles, A., Grigelmo-Miguel, N., & Martín-Belloso, O. (2003). Evaluation of textural properties and microstructure during storage of minimally processed apples. Journal of Food Science, 68(1), 312–317. Soysal, C. (2009). Effects of green tea extract on “Golden Delicious” apple polyphenoloxidase and its browning. Journal of Food Biochemistry, 33(1), 134–148. Ushikubo, F. Y., & Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34, 145–153.

983

Varela, P., Salvador, A., & Fiszman, S. (2007). Changes in apple tissue with storage time: Rheological, textural and microstructural analyses. Journal of Food Engineering, 78(2), 622–629. Vargas, M., Pastor, C., Chiralt, A., McClements, D. J., & González-Martínez, C. (2008). Recent advances in edible coatings for fresh and minimally processed fruits. Critical Reviews in Food Science and Nutrition, 48(6), 496–511. Wang, Z., Duan, H., & Hu, C. (2009). Modelling the respiration rate of guava (Psidium guajava L.) fruit using enzyme kinetics, chemical kinetics and artiﬁcial neural network. European Food Research and Technology, 229(3), 495–503. Weiss, J., Takhistov, P., & McClements, D. J. (2006). Functional materials in food nanotechnology. Journal of Food Science, 71(9), R107–R116. Wojdyło, A., Oszmiański, J., & Laskowski, P. (2008). Polyphenolic compounds and antioxidant activity of new and old apple varieties. Journal of Agricultural and Food Chemistry, 56(15), 6520–6530. Zambrano-Zaragoza, M. L., Mercado-Silva, E., Del Real, A. L., Gutiérrez-Cortez, E., CornejoVillegas, M.A., & Quintanar-Guerrero, D. (2014). The effect of nano-coatings with αtocopherol and xanthan gum on shelf-life and browning index of fresh-cut “Red Delicious” apples. Innovative Food Science and Emerging Technologies, 22, 188–196. Zambrano-Zaragoza, M. L., Mercado-Silva, E., Gutiérrez-Cortez, E., Castaño-Tostado, E., & Quintanar-Guerrero, D. (2011). Optimization of nanocapsule preparation by the emulsion–diffusion method for food applications. LWT — Food Science and Technology, 44(6), 1362–1368. Zambrano-Zaragoza, M. L., Mercado-Silva, E., Ramírez-Zamorano, P., Cornejo-Villegas, M.A., Gutiérrez-Cortez, E., & Quintanar-Guerrero, D. (2013). Use of solid lipid nanoparticles (SLNs) in edible coatings to increase guava (Psidium guajava L.) shelf-life. Food Research International, 51(2), 946–953. Zhou, P., Smith, N. L., & Lee, G. Y. (1993). Potential puriﬁcation and some properties of monroe apple peel polyphenol oxidase. Journal of Agricultural and Food Chemistry, 41(4), 532–536.