82 luz zambrano innovative food science & emerging technologies 2014

Innovative Food Science and Emerging Technologies 22 (2014) 188–196

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The effect of nano-coatings with α-tocopherol and xanthan gum on shelf-life and browning index of fresh-cut “Red Delicious” apples M.L. Zambrano-Zaragoza a,b, E. Mercado-Silva b, A. Del Real L. c, E. Gutiérrez-Cortez a, M.A. Cornejo-Villegas a, D. Quintanar-Guerrero d,⁎ a Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Departamento de Ingeniería y Tecnología, Laboratorio de Procesos de Transformación de Alimentos y Tecnologías Emergentes, Km 2.5 Carretera Cuautitlán-Teoloyucan, San Sebastián Xhala, Cuautitlán Izcalli, Edo de México CP. 54714, Mexico b Laboratorio de Fisiología y Bioquímica Poscosecha de Frutas y Hortalizas, Departamento de Investigación y Posgrado en Alimentos, Facultad de Química, Universidad Autónoma de Querétaro, C.U. Cerro de las Campanas S/N, Santiago de Querétaro, Qro. C.¨P. 76010, Mexico c Departamento de Nanotecnología, Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, Juriquilla, Querétaro C.P. 76230, Mexico d Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Laboratorio de Posgrado en Tecnología Farmacéutica, Av. 1ro de mayo s/n Cuautitlán Izcalli, C.P. 54745, Edo de México, Mexico

a r t i c l e

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Article history: Received 8 August 2012 Accepted 25 September 2013 Available online 8 October 2013 Editor Proof Receive Date 2 January 2014 Keywords: Nanocapsules Nanoemulsion Nanospheres DL-α-tocopherol Xanthan gum Browning index

a b s t r a c t The objective of this study was to prepare nanoparticles and nanocapsules using the emulsification–diffusion method, and then evaluate the effectiveness of several systems made with DL-α-tocopherol on the browning index and firmness in fresh-cut apples. Poly-ε-caprolactone was used as a biopolymer to form the membrane of nanocapsules and the matrix of nanospheres. To provide greater functionality to the coating, xanthan gum was added to some of the systems tested. Changes in the treated fruit were monitored during 18 days of cold storage.

The micrographs obtained give evidence of the presence of capsular entities. Particle size was 174 to 240 nm, and the zeta potential was −44 to −56 mV, which indicates that the systems were stable. With respect to the browning index, nanocapsules proved to be the most effective system, followed by nanospheres. Firmness changes were reduced by applying nanocapsules and nanospheres, both of which limited variations in firmness to below 15% (6.1 and 6.3 N, respectively). These results confirm that the use of nanoparticle systems does indeed help maintain the quality of fresh-cut apples.

Industrial relevance: This study shows the advantages of using edible coatings containing nanosystems to preserve fresh cut fruits in particular the apple. The results show clearly that nanotechnological coatings decrease the browning index and preserve the firmness by longer times compared with the xanthan gum and control. Nanocapsules containing DL-α-tocopherol were the best system followed by nanoemulsions and nanospheres. The systems were prepared by the emulsion–diffusion method from acceptable food materials. This process is efficient, versatile and of simple implementation to industrial level. On the other hand, the nanosystems can be easily applied by dipping or spraying as conventional coatings in production lines, and they do not require special equipment. Apparently the effect of nanosystems is attributed to their high superficial area and the modification of membrane permeability due to their lipophilic nature. Furthermore, the food industry is continuously growing, so that the application of antioxidants in a nanocapsule form is a choice of easy application, in accordance with current systems of conservation of fresh-cut fruit, helping to increase the shelf life of apples and other products of high marked demand, thereby reducing product losses. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction The demand for fresh-cut fruits has increased considerably due to their content of vitamins, phenols and other antioxidants related to the prevention of various cancers and degenerative diseases (Son, Moon, & Lee, 2001). However, fresh-cut processing causes quality deterioration associated with tissue breakdown that results in metabolic, physicochemical and textural changes (Cortez-Vega, ⁎ Corresponding author. Tel.: +52 5556232065; fax: +52 5558175796.

E-mail address: quintana@unam.mx (D. Quintanar-Guerrero).

1466-8564/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ifset.2013.09.008

Becerra-Prado, Soares, & Fonseca, 2008; Perez-Gago, Serra, Alonso, Mateos, & Del Río, 2005). One of the main challenges for the freshcut fruit industry is the browning effect that develops as a result of the polyphenol oxidase activity that occurs after peeling and cutting.

This problem must be treated with a browning inhibitor that impedes the development of brown discoloration (Alvarez-Parrilla et al., 2007).

Browning-inhibitor formulations generally contain reducing agents such as organic acid, cysteine, honey, CaCl2 and polyphosphates among others (Son et al., 2001; Thongsook & Tivaboonchai, 2011).

Edible coatings are one option for reducing the deterioration caused by even minimal processing of fresh-cut fruits, and for extending

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189

shelf life, because they form a semipermeable barrier consisting of O2, CO2, moisture and solute movement that serves to reduce respiration index, weight loss, and oxidation reaction rates (Perez-Gago, Serra, Alonso, Mateos, & Del Río, 2003; Qi, Hu, Jiang, Tian, & Li, 2011). These coatings function by creating a modified atmosphere on the product surface, and have the additional ability to release additives, antioxidants, and other protective substances. Polysaccharide-based coatings are commonly used to extend the shelf life of fresh-cut fruits and vegetables (Lin & Zhao, 2007; Mei & Zhao, 2003; Park, Zhao, Leonard, & Traber, 2005; Tapia et al., 2007). Xanthan gum is a polysaccharide with a primary structure consisting of 1,4-linked β-D-glucose residues, and a trisaccharide side chain attached to alternate D-glucose residues. The side chain is made up of β-D-mannose-1,4-β-D-glucuronic acid-1,2-α-D-mannose.

However, the use of xanthan gum as an edible coating on fresh fruits has been little explored despite its mechanical properties and ability to retain the aroma and flavor of the fruit, which make it an interesting option to be used as a coating, or as a support for other particles (Chen & Nussinovitch, 2000; García-Ochoa, Santos, Casas, & Gómez, 2000).

Nanotechnology has a great potential to change the way food products are preserved (Weiss, Takhistov, & McClements, 2006), so it is necessary to explore its possibilities for controlling brown discoloration and extending the shelf life of fruits. Recent applications of nanotechnology in food systems have focused mainly on functional products, packaging materials and microbial control (Mcclements, Decker, Park, & Weiss, 2009; Weiss et al., 2006), so little information is currently available on their functionality for preserving fruits and vegetables. Nanoparticles are defined as solid submicron-sized colloidal particles, and include nanospheres and nanocapsules. Nanospheres are solid structures in which the active substance of interest is dispersed in a polymer matrix or lipid matrix. Nanocapsules, in contrast, are membrane-type systems in which the active substance is usually enveloped in an oily core surrounded by a membrane (Mora-Huertas, Fessi, & Elaissari, 2010; Quintanar-Guerrero, Allémann, Doelker, & Fessi, 1998).

A wide variety of polymeric particulate carriers have been developed to protect active molecules. Poly-ε-caprolactone (PCL) is an aliphatic polyester that can be degraded by hydrolysis, is non-toxic, and is compatible. It is classified as ‘Generally Recognized as Safe’ (GRAS) by U.S. Food and Drug Administration (FDA) (Das, Rao, Wilson, & Chandy, 2000). Other advantages of PCL are its in vitro stability and low cost (Kim, Kim, Kang, Pyo, & Jeong, 2010; Nakagawa, Surassmo, Min, & Choi, 2011). In recent years, some studies have used nanocapsules in food applications with PCL as the carrier system for the active food ingredients (Nakagawa et al., 2011; Surassmo, Min, Bejrapha, & Choi, 2010; Zambrano-Zaragoza, Mercado-Silva, Gutiérrez-Cortez, Castaño-Tostado, & Quintanar-Guerrero, 2011).

However, the use of PCL has not yet been explored in direct applications: thus it is important to consider its potential use with fresh-cut fruits. The objective of this study, therefore, was to prepare nanodispersions: nanocapsules and nanospheres by the emulsification–diffusion method and nanoemulsion by high shear homogenization in order to evaluate their effectiveness as coatings for the conservation of fresh-cut apple.

stabilizers Span® 80 and Tween® 80, which were mixed to obtain a hydrophilic–lipophilic balance (6) (ICI Surfactants, Mexico). The distilled water was of Milli-Q® (Millipore, USA, Bedford, MD).

Xanthan gum was purchased from Sigma-Aldrich® (Germany) and was used as a continuous matrix for the coatings and the propylene glycol as a plasticizer (99%) (Sigma-Aldrich, USA). Density markers and colloidal silica (Percoll®) were provided by Pharmacia LKB (Sweden). All other reagents were of analytical grade and were used without any purification.

Red Delicious apples (Malus domestica Borkh) were purchased at local wholesale markets in Mexico City. The apples were selected based on maturity, color, size and uniformity of shape. Damaged or defective apples were discarded. The selected fruits were randomly divided into 10 kg batches and kept in refrigerated conditions at 4 °C for 24 h prior to use.

2. Materials and methods

2.3. Coating formulations and application on fresh-cut apples

2.1. Materials

All whole apples were sanitized by immersion in chlorine solution at 70 ppm for 5 min. Cutting boards, utensils and containers were also sanitized to minimize contamination due to microorganisms.

After cutting, the apple pieces were first placed in a 1% CaCl2 solution and subsequently immersed in the corresponding nanodispersion coating. Nanodispersions were prepared with 3 g/L of xanthan gum.

The batches tested in the study were the following: nanocapsules, nanospheres and a nanoemulsion with, and without, the addition of xanthan gum as a film-forming hydrocolloid. The control samples used in the experiment were cut apples with no coating of any kind.

All samples were drained at 4 °C to remove any excess coating. Once

The poly-ε-caprolactone (PCL) Mw ≈ 80,000 (ρ = 1.147 g/cm3 at 25 °C) was used as biopolymer to form the membrane (nanocapsules) and the matrix (nanospheres) was obtained from Sigma Aldrich® (USA). Pluronic F-127 (PF-127) (BASF, Mexico) was used as stabilizer (nonionic) and DL-α-tocopherol acetate (98%) (Sigma Aldrich®, USA) was the oily core for nanocapsules. The partially water-miscible solvent (analytical grade) was ethyl acetate (Fermont, Mexico). This solvent was selected for the preparation of the nanocapsules due to its low toxicity (ICH class: 3). Nanoemulsion was achieved using the

2.2. Preparation of the nanosystems Nanocapsules and nanospheres were prepared using the emulsification–diffusion method proposed by Quintanar-Guerrero et al. (1998), and considering the methodology optimization approach proposed by Zambrano-Zaragoza et al. (2011). An aqueous phase saturated with ethyl acetate (20 mL) containing pluronic-127 (50 g/L) and an oily phase saturated with water (40 mL) containing PCL (~250 mg) and DLα-tocopherol acetate (2 g/L) were used.

To evaluate the effect of biopolymer on nanocapsules and nanospheres, a nanoemulsion was prepared from an emulsion made with dispersed DL-α-tocopherol acetate (2 g/L), Span® 80 (0.8 g/L) and Tween® 80 (4.2 g/L). To rule out any effect on the functionality of the coating due to the biopolymer and active agent, a coating in the form of a nanoemulsion that contained DL-α-tocopherol was tested.

This emulsion was prepared using a variable speed agitator (Caframo RZR-1®, Ontario, Canada; propeller: PR 31, Heidolph®, Schwabach, Germany) at 2000 rpm/10 min. The nanoemulsion was obtained from the emulsion by high shear homogenization using a rotor/stator device (Ultra turrax T50, IKA USA). In order to obtain a stable nanoemulsion with the required submicronic particle size it was necessary to homogenize it with 5 cycles of 10,000 rpm/5 min with a 5-min rest interval between cycles.

2.2.1. Nanodispersions characterization The particle size, polydispersion index (PDI) and zeta potential (ζ) of the nanocapsules, nanospheres and nanoemulsion were determined using a Z-sizer 4 (Malvern Instruments® ZEN NS 3600, Worcestershire, UK) fitted with laser light scattering at an angle of 273°. Each suspension was diluted in water. Tests were performed in triplicate for ζ and were standardized with polystyrene dispersions (ζ = −55 mV).

The density of the nanodispersions was evaluated as a function of polymer concentration by isopycnic centrifugation in colloidal silica gradients (Percoll®) with specific gravity markers (Phamacia, LKB), following the methodology proposed by Zambrano-Zaragoza et al. (2011).

M.L. Zambrano-Zaragoza et al. / Innovative Food Science and Emerging Technologies 22 (2014) 188–196

coated, the apple pieces were packed in 250 mL polypropylene containers with 90 g of fruit and stored at 4 ± 1 °C and 85% RH for 18 days.

2.4. Morphological studies The morphological characterization of nanocapsules was carried out by scanning electron microscopy (SEM) using the methodology proposed

by Zambrano-Zaragoza et al. (2011). The 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 chamber pressure of 12 to 20 Pa. The morphological characterization of the apple surface with the different coatings was performed by longitudinal tissue section after dipping into the corresponding coating system, followed by coating with gold, as described above. The voltage used in taking the micrographs was 20 kV. 2.5. Color evaluation The effect of nanosystem coatings on the color and browning index of fresh-cut apples was determined by measuring their surface color with a Minolta CR-300 colorimeter (Konica Minolta, Tokyo, Japan) every 3 days during the 18 days of storage time. Readings were obtained on a CIELAB scale (L*, a*, b*). Three wedges from each treatment were taken and 6 color measurements were made at 2 locations for each sample. The browning index (BI) was calculated and was used as an indicator of the intensity of brown discoloration. BI was calculated as follows (Olivas, Mattinson, & Barbosa-Cánovas, 2007; Palou, López-Malo, Barbosa-Cánovas, Welti-Chanes, & Swanson, 1999):

BI ¼

½100ðx−0:31Þ 0:172

where:

x¼

ða þ1:75Þ : ð5:646L þ a −3:012b Þ

2.6. Firmness measurement The firmness of the apple wedges was measured every other day up to the 18th day of storage. For each test, 5 wedges from each treatment batch were evaluated using an INSTRON 4411 with load cell of 5 kN equipped with 5 mm puncture thickness fix (Instron, Mass., USA). Each sample wedge was fractured by downward motion (150 mm·s− 1). The maximum force (highest N value) applied to break the wedge was taken as the index of firmness (HernándezMuñoz, Almenar, Valle, Velez, & Gavara, 2008). All tests were performed in quintuplicate.

2.7. Physicochemical changes The weight loss of 8 apple pieces from each treatment group was monitored during storage. The percentage of 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. This juice was used to measure total soluble solids (TSS) using an ABBE refractometer (Spectronic, Inc., USA), and titratable acidity which was determined by titration with 0.1 N NaOH to pH 8.1 and expressed as malic acid (mg) (Lin, Leonard, Lederer, Traber, & Zhao, 2006; Olivas et al., 2007). All measurements were performed by triplicate.

2.8. Statistical analysis The results were analyzed using the Statistical analysis software MINITAB® Release 14. The evaluation of effectiveness nanosystems versus time was performed by a two-way analysis of variance. Mean separation was determined by the Tukey's test (α = 0.05).

3. Results and discussion 3.1. Nanosystem characterization The densities of nanosystem were as follows: nanocapsules, 1.022 g/mL; nanospheres, 1.098 g/mL; and the nanoemulsion 0.962 g/mL. Fig. 1 shows particle size distributions by % volume.

All the systems tested showed a monomodal behavior. These results correlate with those obtained by Relkin, Yung, Kalnin, and Ollivon (2008), who reported a monomodal behavior for α-tocopherol nanoemulsions prepared by the homogenization method with particle sizes below 200 nm. Similarly, Yuan, Gao, Mao, and Zhao (2008) found a monomodal behavior in the preparation of β-carotene nanoemulsions.

Yin et al. (2009) reported a bimodal behavior when they used Tween® 20 as a surfactant and a β-carotene nanoemulsion following the solvent displacement method. In nanocapsules prepared by the emulsification– diffusion method, the following particle sizes have been reported: ranging from 200 to 500 nm with different oil materials (QuintanarGuerrero et al., 1998); from 300 to 500 nm when PCL was used as core biopolymer (Choi, Soottitantawat, Nuchuchua, Min, & Ruktanonchai, 2009); and from 183 to 206 nm when fish oil was encapsulated (Choi, Soottitantawat, Nuchuchua, Min, & Ruktanonchai, 2009). The exact dispersion behavior depends largely on the active substance–biopolymer association, the method used and the preparation conditions (Sinha, Bansal, Kaushik, Kumria, & Trehan, 2004). All the systems obtained in the present study were submicronic in size, suggesting that the conditions employed in the previous optimization study can be applied when preparing these types of systems (Zambrano-Zaragoza et al., 2011).

Table 1 shows the results of particle size, PDI and ζ in nanospheres, nanocapsules and nanoemulsion with, and without the addition of xanthan gum. The smallest particle size was found in the nanospheres and nanoemulsion. The larger particle size in the nanocapsules is due to the DL-α-tocopherol that was added as an antioxidant, since it has been shown that active content affects the particle size (Choi, Ruktanonchai, Soottitantawat, & Min, 2009). Also, the nanocapsules and nanospheres had the lowest PDI at values below 0.2, indicative of narrower particle size distribution. Moreover, there was no influence on particle size and PDI after the addition of xanthan gum, as the PDI always remained below 0.25. According to Lemarchand, Couvreur, Vauthier, Costantini, and Gref (2003), narrow PDI values (b0.3) are indicative of a homogeneous particle distribution. Zigoneanu, Astete, and Sabliov (2008) reported PDI values of approximately 0.1 for DL-αtocopherol-containing nanoparticles, using polyvinyl alcohol as a

40

Volume (%)

190

30 20 10 0 10

100

1000

Particle size (nm) Fig. 1. Particle size distribution of the nanosystems. ( ) nanocapsules. nanoemulsion, (

) Nanospheres, (

)

M.L. Zambrano-Zaragoza et al. / Innovative Food Science and Emerging Technologies 22 (2014) 188–196 Table 1 Particle size, PDI, and ζ in the nanoparticle systems.

System

Particle size (nm)

PDI (polydispersion index)

Zeta potential (ζ) mV

Nanospheres Nanocapsules Nanoemulsion Nanospheres/xanthan gum Nanocapsules/xanthan gum Nanoemulsion/xanthan gum

175 ± 35a 239 ± 3b 197 ± 4c 187 ± 7a 220 ± 11b 197 ± 36c

0.123 ± 0.01a 0.086 ± 0.04b 0.123 ± 0.03a 0.140 ± 0.04c 0.131 ± 0.09c 0.165 ± 0.07d

−45.3 ± 1.1a −44.7 ± 0.8a −53.0 ± 1.1b −47.7 ± 3.6c −52.7 ± 0.7b −56.6 ± 1.5b

Significance level α = 0.05.

Means followed by the same letter in the columns did not differ by Tukey's test.

stabilizer. On the other hand, Bala et al. (2005) found that when ellagic acid is used to prepare nanoparticles, PDI varies from 0.075 to 0.099. The ζ interval was between −44.7 and −56.6mV indicating little likelihood of dispersion aggregation, since an absolute value less than or greater than 25 mV is indicative of flocculated and deflocculated emulsions, respectively (Mirhosseini, Tan, Hamid, & Yusof, 2008). The variations obtained in nanosystems that contained xanthan gum can be attributed to the negative charge of this substance, which is composed of β-1,4 glucose bound to branched mannose and glucoronic acid (Katzbauer, 1998). The ζ values obtained suggest that the dispersions are stable (Tewa-Tagne, Briançon, & Fessi, 2007). Mirhosseini et al. (2008) mentioned that absolute values of ζ N |25| mV reduce the likelihood of dispersion aggregation. In the present experiment, all system had ζ below −44 mV. 3.2. Morphological characterization Fig. 2 presents the nanocapsule dispersion micrographs that show the capsular entities consisting of a nucleus surrounded by a membrane, presumably PCL. Fig. 2(a) shows only the nanocapsules with DL-α-tocopherol. Arrows indicate the nanocapsules with capsular structure; circles enclose the complete nanocapsules and confirm that they are of submicronic size (Quintanar-Guerrero et al., 1998).

Fig. 2(b) shows the nanocapsules with xanthan gum to demonstrate that although there is a slight increase in particle size of the polysaccharide used, it does not affect the integrity of the nanocapsules.

Fig. 3 shows the micrographs of the surface of the apple tissues according to the different treatments. Fig. 3(a) presents the control apple surface, which shows parenchymal organization with superficial modifications. Fig. 3(b) illustrates the characteristics of the apple

a)

surface after infiltration by the nanocapsules, which are homogeneously distributed in the tissue to achieve the lower browning that is evident on the surface of the apple wedges in this treatment group. The possible cause is that the DL-α-tocopherol is released and allowed to exert its antioxidant effect, thus preventing oxygen availability through the oxidative development reaction. In addition, it is assumed that the lipophilic character of the active agent modifies the permeability of the cell membrane, thereby decreasing the availability of phenols (Toivonen & y DeEll, 2002). In Fig. 3(c) one can see that morphology of nanospheres was consistently spherical, and that they remain on the surface of the tissue due to the presence of PCL, a key characteristic of biodegradation.

With respect to the rate of ester cleavage, this is limited by the access of water to the ester bond (Jenkins & Harrison, 2006).

Myllymäki et al. (1998) reported that the moisture sorption of PCL was negligible and that its presence decreased the moisture sorption of starch/PCL in proportion to the amount of PCL in the film. However, the homogeneous distribution of nanoparticles helps provide a protective effect by limiting oxygen diffusion and thus preventing the browning of the apple's surface. Fig. 3(d) shows the formation of thin film with different particle sizes of DL-α-tocopherol droplets on the tissue surface. According to Sapers et al. (1989) the use of surfactants species on Tween® and Span® affects the cellular membrane, causing the release of phenols, enzyme and the substrate together, in a process that leads to an increase in the browning index.

Fig. 4(a) to (d) illustrates the behavior of apple surface tissue coated initially with xanthan gum under the different systems studied. These figures show an evident modification of apple's surface that correlates with the lower shelf life observed in these apples. Fig. 4(a) reveals structures formed by xanthan gum (Veiga-Santos, Suzuki, Cereda, & Scamparini, 2005) with a homogeneous distribution over the tissue surface with no apparent superficial changes and no empty spaces. Fig. 4(b) shows the behavior of the apples coated with nanocapsules/xanthan gum. This coating caused changes that seem to be a transpiration process that occurred after the coating was applied and led to the formation of large droplets. It also shows droplets smaller than 1 nm, which are presumably DL-α-tocopherol nanocapsules involved in xanthan gum.

Fig. 4(c) and (d) shows aggregate formations that presumably were occupied by water produced by transpiration. This correlates with the faster changes in the browning index relative to the apples not treated with the coating that contain xanthan gum. According to SolivaFortuny, Lluch, Quiles, Grigelmo-Miguel, and Martín-Belloso (2003), the large hemispherical space that can be identified as drops on the outer surface of the cells suggests changes due to transpiration that may contribute to an increase in the rate of tissue deterioration.

b)

Nanocapsules Nanocapsules/ xanthan gum

Nanocapsules aggregates

20 kV

X10,000

1 µm

NC T

191

20 kV

X25,000 1 µm

NCX

Fig. 2. Nanocapsule micrographics, a) DL-α-tocopherol nanocapsules at 10,000×, b) DL-α-tocopherol nanocapsules with xanthan gum at 25,000×.

192

M.L. Zambrano-Zaragoza et al. / Innovative Food Science and Emerging Technologies 22 (2014) 188–196

b)

a)

Nanocapsules signs

d)

c) Nanospheres

Nanoemulsion droplets

Fig. 3. Apple coating with different treatments (a) control; (b) nanocapsules; (c) nanospheres; (d) nanoemulsion.

3.3. Color evolution Fig. 5 shows the variation in lightness L* and a* value of the fresh-cut apples as a function of storage time. Fig. 5(a) shows that lightness decreased considerably in the fresh-cut control apples during the first five storage days, at a rate of 4.15 day−1 (R2 = 0.87), suggesting that it is indeed important to apply coating materials. Of the coated samples, those treated with xanthan gum had the highest rate of change in L*

at 0.67day−1 (R2 = 0.93), while the lowest rate was seen in those treated with nanocapsules at just 0.18 day−1 (R2 = 0.89). Usually, changes in L* are associated with tissue surface modifications caused by the activity polyphenol oxidases, so this reduction is associated with the barrier formed by the coating on the product's surface (Soliva-Fortuny et al., 2003). These results show that the use of submicron-sized systems changed the appearance of the fruit surface after treatment onset, as it showed an increase in L* early during storage, which was attributed to

Fig. 4. Apple coating with xanthan/gum (a) xanthan gum; (b) nanocapsules/xanthan gum; (c) nanospheres/xanthan gum and (d) nanoemulsion/xanthan gum.

M.L. Zambrano-Zaragoza et al. / Innovative Food Science and Emerging Technologies 22 (2014) 188–196

85

193

90

a)

a)

80

Browning Index

80

L*

75

70

65

70 60 50 40 30

60 0

5

10

15

20

20 0

4

b)

3

10

15

9

2 1 0 -1 -2 -3

20

b)

8

Firmness (N)

a* value

5

7 6 5 4

-4 0

5

10

15

20

Storage time (days)

3 2 0

Fig. 5. L* and a* value changes for the different systems of fresh-cut apples stored at 4 °C. (a) L* value evolution during storage; (b) a* value variation during storage. ( ) Control, ) xanthan gum, ( ) nanoemulsion, ( ) nanoemulsion/xanthan gum, ( ) ( nanocapsules, ( ) nanocapsules/xanthan gum, ( ) nanospheres, ( ) nanospheres/xanthan gum. Vertical bars represent standard deviation.

the ability of nanosystems to reflect light. A similar phenomenon has been observed with submicron materials used as sunscreens. Regarding L* changes, several authors (Mchugh & Senesi, 2000; Perez-Gago et al., 2005; Soliva-Fortuny, Ricart-Coll, & Martín-Belloso, 2005; Rojas-Graü, Tapia, & Martín-Belloso, 2008) have pointed out that a decrease in this parameter is related to both an increase in pigment concentration and the application of a coating, since these act as an oxygen barrier that could limit the action of polyphenol oxidases. Finally, it was possible to conclude that fresh-cut apples treated with nanospheres and those treated with nanoemulsion with, and without xanthan gum, were the ones that had the lowest changes in L* values. Lee, Park, Lee, and Choi (2003) reported initial L* values of 79 for fresh-cut Fuji apples treated with ascorbic acid/carrageen that decreased to 76 during storage.

Thus, it is clear that the submicron-sized systems used in the present study achieved a better conservation of L* when applied to fresh-cut Red Delicious apples.

Fig. 5(b) shows the effects of nanosystems coatings on a* values of the apple wedges. In general an increase in a* implies a higher browning index (Rojas-Graü, Sobrino-López, Tapia, & Martín-Belloso, 2006). The control apples exhibit drastic increase in their a* values that averaged between −2.6 and +2.2 after 5 days of storage; a finding associated with the changes in the browning index. After day 5, however, these values remained virtually unchanged for the rest of the storage period.

The samples treated with xanthan gum and nanoemulsion/xanthan gum showed no statistically significant difference (p N 0.05), as they were less effective in preserving a* values, with variations from −2.6 to +1.5. The coatings that best preserved the a* values (−2.6) were nanocapsules and nanospheres, with changes below 29% (0.76) after 18 days of storage. According to Lee et al. (2003), a decrease in the L* value coupled with an increase in the a* value is a measure of the degree of browning that develops in fresh-cut fruits. In addition Rojas-Graü et al. (2008) mention that a higher a* value is indicative of increasing

10

20

Storage time (days) Fig. 6. Browning index and firmness progress as a function of the treatment of fresh-cut apples during storage. (a) Browning index; and (b) firmness changes. ( ) Control, ) xanthan gum, ( ) nanoemulsion, ( ) nanoemulsion/xanthan gum, ( ) ( ) nanocapsules/xanthan gum, ( ) nanospheres, ( ) nanocapsules, ( nanospheres/xanthan gum. Vertical bars represent standard deviation.

browning index in apples during storage, then a* value is an effective parameter to monitor enzymatic browning. All apple wedges coating had minimal variations of the b* value during storage (between 22 and 25). Although depending on the type of fruit or vegetable the b* value as a yellowing that potentiates the brown discoloration of the sample (Xing et al., 2010). The present results suggest that nanosystems without xanthan gum allow infiltration and coat the surface of fresh-cut apples better, thus modifying the rate of change and helping to reduce the changes associated with the browning. This effect is most likely attributable to a decrease in oxygen availability.

3.4. Browning index (BI) and firmness changes The browning index measures the purity of the brown color and is considered a necessary parameter in this process, where both enzymatic and non-enzymatic browning may take place. Fig. 6(a) shows the effect of the coated and control samples on the BI during storage at 4 °C. The control samples had the highest browning change, at 4.7 BI/day (R2 = 0.83) after 5 days of storage, though these samples showed only slight variations in the subsequent days. The samples coated with xanthan gum, nanoemulsion and nanoemulsion/xanthan gum showed no statistical difference (p N 0.05), as the BI presented a mean value of 47 compared to an initial value of 28. The lower effectiveness of the nanoemulsion was attributed mainly to its rapid absorption into the fresh-cut apple and the formation of droplets on the surface of apple wedges. This correlates with the micrographs shown in Fig. 4(d). No statistical difference with respect to BI emerged between the nanospheres and the nanospheres/xanthan, with an increase in browning of 0.84 unit of BI/day (R2 = 0.85), while the nanocapsules/xanthan gum had a slightly lower browning increase of 0.75 BI/day (R2 = 0.85). This

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a 28% decrease in firmness at the end of the storage period compared to base values. In and of itself, xanthan gum was not effective in preventing browning, and is associated with firmness loss due to the action of pectin methylesterase. Varela, Salvador, and Fiszman (2007) studied texture in two apple varieties: Granny Smith and Golden Delicious.

They performed puncture tests using up to 10 mm of surface from apple cubes for 14 days of storage, and found that the initial firmness of the Golden Delicious apples was 12.5 N, but this decreased by 48% at 7 days, and by 65.5% at 14 days. In our case, firmness reduction in the nanoparticle systems reached a maximum just 37% after 21days of storage, while a firmness reduction of only 15% was seen when nanocapsules were used.

These findings confirm the hypothesis that submicron-sized systems, and in particular those containing DL-α-tocopherol, effectively contribute in maintaining the firmness of the nanocapsule coating of Red Delicious apples, and that this is attributable to an improved system distribution on the surface and to the antioxidant effect over a large surface area.

occurred because, as was shown in Fig. 4(b), the droplet formationmost likely due to the interaction between the xanthan gum and the water produced by transpiration in the fresh-cut apple decreased the efficiency of the DL-α-tocopherol nanocapsules enveloped, in this case, in xanthan gum. Finally, the nanocapsules alone were the most effective coating as they had the lowest browning rate: 0.53 BI/day (R2 = 0.92).

This behavior is consistent with that observed in Fig. 3(b), which shows a homogeneous distribution of the nanocapsules over the surface of the fresh-cut apples, with no formation of micrometric spheres. It is possible that nanocapsules containing DL-α-tocopherol acetate have the ability to reduce BI, probably by modifying oxygen availability and triggering changes in cellular membrane that modify transport properties and polyphenol oxidase activity (Park et al., 2005; Perez-Gago, Serra, & Río, 2006). Treatment of fresh-cut Red Delicious apples with nanocapsules containing DL-α-tocopherol with a PCL biopolymer membrane significantly reduce the browning index due to the increased surface area covered. This may allow better oxygen reduction through an antioxidant effect.

Fig. 6(b) shows the changes in firmness as a function of storage time in both fresh-cut apples coated with the different systems and control apples. The two-way ANOVA (α = 0.05) showed a statistical difference related to coating composition and storage time. The greatest decrease in firmness occurred in the control samples, with a 60% loss of firmness compared to the initial state. The nanoemulsion was the least effective submicron-sized system, with a 37% firmness loss, followed by the nanoemulsion/xanthan and nanospheres/xanthan treatment, both of which had a firmness reduction of 28% during total storage time. The most effective fresh-cut apple treatments were the nanocapsules and nanospheres, with an average decrease of only 15% compared to the initial state. This finding reflects the functionality of the nanosystem, which correlated as well with a lower browning rate.

Immersion in the CaCl2 solution prior to previous coating helps to preserve the firmness (Lee et al., 2003; Manurakchinakorn, Chamnan, & Mahakarnchanakul, 2012); however, the use of active substances as the DL-α-tocopherol is a factor in reducing firmness loss. Texture conservation depends largely on the precise composition. Xanthan gum is a polysaccharide that exerts a protective effect on texture (Valencia-Chamorro, Pérez-Gago, Del Río, & Palou, 2008), as shown by

3.5. Physicochemical changes Red Delicious apple samples used in the experiment had a confidence interval characterized by minimum–mean–maximum with limits of: TSS of 12.4 b 13.1 N 14.2, a pH of 3.4 b 3.6 N 3.9, an acidity of 254 b 248 N 235 mg of malic acid/100 mL of juice, and an initial firmness between 6.9 b 6.5 N 6.1 N. There were no significant differences (p N 0.05) in fresh weight loss among treatments. However, during storage time, there was a steady decrease in weight for all treatments that was always less than 0.5%.

This behavior is attributed to the container, which limits the exchange of water vapor (Conforti & Totty, 2007).

Table 2 shows the behavior or the pH, acidity, and TSS in apples stored for 18 days. The control samples had a 27% decrease in TSS with respect to their initial days, followed by the apples coated with xanthan gum and nanoemulsion/xanthan gum, with a decrease of 22 and 18%, respectively. Changes were smaller in the samples coated with nanocapsules/xanthan gum and nanospheres/xanthan gum, a result that can be attributed to their chemical make-up and to the interaction of the xanthan gum with the surface tissue. The apple pieces coated with nanospheres/xanthan gum and nanocapsules/xanthan

Table 2 Physicochemical properties on Red Delicious fresh-cut apples treated with the different coatings during storage at 4 °C. Storage time (days) Control

Xanthan gum

Nanospheres

Nanospheres/xanthan gum

Nanocapsules

Nanocapsules/xanthan gum

Nanoemulsion

Nanoemulsion/xanthan gum

Acidity pH SST Acidity pH SST Acidity pH SST Acidity pH SST Acidity pH SST Acidity pH SST Acidity pH SST Acidity pH SST

0

5

9

15

18

248 ± 4aA 3.8 ± 0.1aA 13.9 ± 0.7aA 257 ± 12aA 3.7 ± 0.2aA 13.5 ± 0.4aA 249 ± 0.6aA 3.7 ± 0.3aA 13.2 ± 0.4aA 255 ± 10aA 3.7 ± 0.1aA 13.4 ± 0.5aA 248 ± 15aA 3.7 ± 0.2aA 13.3 ± 0.8aA 254 ± 8aA 3.6 ± 0.2aA 13.5 ± 0.4aA 254 ± 15aA 3.6 ± 0.5ªA 13.7 ± 0.4ªA 249 ± 19ªA 3.6 ± 0.1ªA 13.2 ± 0.5ªA

185 ± 5bA 4.2 ± 0.2bA 13 ± 0.1bA 213 ± 6bB 3.9 ± 0.2aB 12.7 ± 0.2bB 252 ± 0.7aC 3.7 ± 0.1aB 12.9 ± 0.4bA 252 ± 7aC 3.6 ± 0.2aB 13.1 ± 0.4aA 244 ± 6aD 3.7 ± 0.1aB 13.4 ±1.1aA 238 ±14bd 3.7 ± 0.1aB 12.9 ± 0.7bC 258 ± 7aC 3.7 ± 0.1aB 13.1 ± 0.9bA 230 ± 30bC 3.7 ± 0.2aB 12.9 ± 0.4bA

179 ± 8cA 4.2 ± 0.7bA 12.1 ±0.2cA 208 ± 12cB 3.9 ± 0.1aB 10.9 ± 0.7cB 238 ± 13bC 3.8 ± 0.2aB 11.9 ± 0.5cA 237 ± 7bC 3.7 ± 0.2aB 12.7 ± 0.5bC 245 ± 10aD 3.8 ± 0.3aB 12.2 ± 0.9bA 239 ± 20bc 3.7 ± 0.1aB 12.7 ± 1.1bC 248 ± 10b 3.6 ± 0.2aD 12.2 ±0.4cA 227 ± 11bB 3.7 ± 0.1aB 11.6 ± 0.4cE

183 ± 15bA 4.3 ± 0.2bA 11.7 ± 0.2dA 154 ± 10dB 4.1 ± 0.1bA 10.3 ± 0.5dB 221 ± 16cC 3.9 ± 0.1aB 11.7 ± 0.8cA 222 ± 5cC 3.7 ± 0.1aB 12.5 ± 0.9bC 228 ± 10bC 3.8 ± 0.2aB 12.2 ± 0.4bC 216 ± 9cC 3.9 ± 0.1aB 12.6 ± 0.8bC 232 ± 14c 3.9 ± 0.1aB 11.6 ± 0.4dA 223 ± 10bC 3.8 ± 0.1aB 11.6 ± 0.4cA

163 ± 7dA 4.3 ± 0.1bA 10.2 ± 0.7eA 195 ± 3eB 4.1 ± 0.2bA 10.5 ± 0.3eA 210 ± 8dC 3.9 ± 0.1aB 12.1 ± 1.3cB 220 ± 11cC 3.8 ± 0.2aB 12.6 ± 0.5bC 221 ± 13bC 3.9 ± 0.5aB 12.3 ± 0.5bC 215 ± 13cC 3.9 ± 0.2aB 12.9 ± 0.9bB 219 ± 17d 4.1 ± 0.2bC 11.4 ± 1.3dC 215 ± 12cC 4.0 ± 0.2bA 10.9 ± 1.6dC

SST = total soluble solids.

Acidity (mg of malic acid/100 mL of apple juice).

Significance level α = 0.05.

Means followed by the same letter lowercase in the columns and capital letter in row did not differ by Tukey's test.

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Fig. 7. Appearance of fresh-cut apples with different treatments at the end of storage time.

gum apparently interacted with the tissue such that the xanthan gum formed aggregates that altered the interaction with the surface of the fresh-cut apples to constitute a barrier against the modified environment. In this regard, McHugh and Senesi (2000) and Conforti and Totty (2007) reported variations in soluble solids according to the composition of polysaccharide-based coatings. On the other hand, Rößle, Auty, Brunton, Gormley, and Butler (2010) reported that the immersion of apple wedges on probiotic bacteria does not change the TSS content during the storage. However, Pizato, Cortez-Vega, de Souza, Prentice-Hernández, and Borges (2013) reported reduction of TSS content attributable to the fact that the immersion of fruits in xanthan gum solutions may have leached the TSS of fruit. Similar results were observed for the apple wedges treated with xanthan gum and systems with this component. This behavior confirms that the use of nanocapsules and nanospheres without xanthan gum contributes better to stabilize the fresh-cut apples during storage time.

Acidity is a very important factor that affects the flavor of fruits.

Table 2 shows the variations in malic acid content. The control apples and xanthan gum-coated apples had a greater decrease in acidity.

According to Olivas et al. (2007), the acidity of fresh-cut Golden Delicious apples tends to decrease during storage, a fact associated with metabolic changes and fruit maturation that varies with the composition of the coating. Also, Cortez-Vega et al. (2008) noted that treatment, variety and storage conditions are all important determining factors of the degree of change in acidity. In the present study, the submicron-sized systems with and without xanthan gum showed no statistically significant differences (p N 0.05) in acidity during the first 9 days of storage. This was attributed to the way in which these coatings interact with the surface of the fresh-cut apple. Finally, pH showed an inverse behavior in relation to acidity and in general had an increase on the pH in all fresh-cut apples which is expected when calcium chloride is added, as this compound is salt chlorinate of basic nature providing buffer capacity; similar effect was shown when xanthan gum is used for fresh-cut peach (Pizato et al., 2013).

Finally, Fig. 7 summarizes the visual changes of fresh-cut apples (18th day of storage); it is possible to observe that fresh-cut apples coated with nanocapsules and nanospheres had better appearance than those coated with nanosystems/xanthan gum. In brief, the browning and physicochemical data showed that the use of submicron size systems contribute to the increase of the shelf life of product compared with the control and xanthan gum coating.

4. Conclusions The nanocapsules containing DL-α-tocopherol acetate used as coating for fresh-cut apples proved to be the most effective system in reducing the browning index, thus helping increase the shelf life. Apparently, nanocapsules are able to remain on the tissue surface where they reduce the loss of quality in fresh-cut apples in refrigerated storage due to the increased homogeneity distribution on surface area covered, while also impeding oxygen supply due to polyphenol oxidase activity. In addition, it was possible to establish that submicronic systems increase the storage time of fresh-cut Red Delicious apples and help reduce the loss of firmness.

This is attributable to the antioxidant capacity of DL-α-tocopherol acetate, which prevents the action of pectin methylesterase through a homogeneous distribution of nanocapsule coating. In this case, the use of xanthan gum was not as effective as nanocapsules, despite the fact that it functions as good coating formation.

Acknowledgments The authors acknowledge the financial support for this work from PAPIIT (Ref. IT231511).

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