The Effects of Tocopherol Nanocapsules/ Xanthan Gum Coatings on the Preservation of Fresh-Cut Apples: Evaluation of Phenol Metabolism M. J. Galindo-Pérez, D. QuintanarGuerrero, E. Mercado-Silva, S. A. RealSandoval & M. L. Zambrano-Zaragoza Food and Bioprocess Technology An International Journal ISSN 1935-5130 Volume 8 Number 8 Food Bioprocess Technol (2015) 8:1791-1799 DOI 10.1007/s11947-015-1523-y
1 23
Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com�.
1 23
Author's personal copy Food Bioprocess Technol (2015) 8:1791–1799 DOI 10.1007/s11947-015-1523-y
ORIGINAL PAPER
The Effects of Tocopherol Nanocapsules/Xanthan Gum Coatings on the Preservation of Fresh-Cut Apples: Evaluation of Phenol Metabolism M. J. Galindo-Pérez 1,2 & D. Quintanar-Guerrero 2 & E. Mercado-Silva 3 & S. A. Real-Sandoval 1 & M. L. Zambrano-Zaragoza 1
Received: 6 January 2015 / Accepted: 6 April 2015 / Published online: 24 May 2015 # Springer Science+Business Media New York 2015
Abstract The aim of this study was to evaluate the effect of different xanthan gum coatings with nanoparticles (nanocapsules and nanospheres) on the production and oxidation of phenolic compounds generated by enzymatic activity (phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO)) on fresh-cut Red Delicious apples stored for 21 days at 4 °C. The nanoparticles were prepared using the emulsification-diffusion method. The film systems formed had particle sizes (Ps) of 190 to 260 nm, the polydispersity index (Pdi) was<0.3+, and the zeta potential was (ζ)>|35|mV parameters that suggest good physical stability. While all the nanoparticulate coatings proved to be effective, the best coating was the nanocapsules/xanthan gum combination because it decreased the initial respiration rate by 63 % compared to controls. The effect on PPO and PAL activity was associated with the protection of phenolic compounds, whose concentrations remained unchanged. Also, lower variations in total color differences were found.
* M. L. Zambrano-Zaragoza luz.zambrano@unam.mx 1
Laboratorio de Procesos de Transformación y Tecnologías Emergentes en Alimentos, Departamento de Ingeniería y Tecnología, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Km 2.5 Carretera Cuautitlán–Teoloyucan, San Sebastián Xhala, Cuautitlán Izcalli 54714, Estado de México, México
2
Laboratorio de Posgrado en Tecnología Farmacéutica, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Av. 1o de mayo s/n, Cuautitlán Izcalli 54745, Estado de México, México
3
Departamento de Investigación y Posgrado en Alimentos, Facultad de Química, Universidad Autónoma de Querétaro, Cerro de las Campanas s/n, 76010 Querétaro, México
Keywords Nanoparticles . Polyphenol oxidase . Phenylalanine ammonia lyase . Total phenols . Fresh-cut apples
Introduction In recent decades, fresh-cut fruits have enjoyed growing interest because they are convenient and provide a series of phytonutrients that are beneficial for health (Qi et al. 2011). Among the most abundant bioactive compounds are the antioxidants, especially phenolic compounds with physiological effects in vivo and in vitro (e.g., allergic activity; anti-caries properties; enzymatic inhibitors; and the capacity to reduce the risk of cardiovascular disease, some cancers, asthma, diabetes, etc.) (Hyson 2011). Apples are known to be an important source of phenolic compounds such as hydroxycinnamic acid, anthocyanins, flavonols, dihydrochalcones, and procyanidins (Tsao et al. 2003; Wojdyło et al. 2008). In phenol biosynthesis, the enzyme phenylalanine ammonia lyase (PAL; EC 4.3.1.24) has a central effect between the shikimic acid and phenylpropanoid pathways, as it catalyzes the conversion of L-phenylalanine into trans-cinnamic acid, a precursor of a wide variety of phenolic compounds (Choi et al. 2005). PAL activity increases significantly in fresh-cut fruits, inducing the synthesis and accumulation of phenols in the damaged tissue (Treutter 2001). When oxidized by polyphenol oxidase (PPO) enzymes (EC 1.10.3.1), these synthesized compounds produce the browning elements that affect the functionality, sensory characteristics, and marketing value of fresh-cut fruits (Soysal 2008). Edible coatings have been used to reduce enzymatic browning, limit the exchange of O2 and CO2, delay the oxidation of phenolic compounds, and increase product shelf life (Dhall 2013). Coatings can also be used as carriers for other
Author's personal copy 1792
additives, such as antimicrobials, antioxidants, and nutraceuticals (Lin and Zaho 2007). Xanthan gum is a polysaccharide product of the metabolism of Xanthomonas campestris that has been used as a coating and carrier of αtocopherol and calcium in baby carrots (Mei et al. 2002) and as an anti-browning agent with Gala apples that preserves the fruit’s physicochemical characteristics (Freitas et al. 2013). A second generation of coatings incorporates additives that use microencapsulation or nanoencapsulation processes to obtain the controlled release of the active compounds (Falguera et al. 2011). Nanotechnology has had a great impact on different areas of science, and food processing is no exception, as it has generated new techniques and materials that increase the shelf life of food products. Nanoparticles are defined as colloidal or solid particles, including nanospheres and nanocapsules, whose principal characteristic is their size: from 1 to 500 nm (Vert et al. 2012). Nanocapsules consist of a liquid center surrounded by a polymer membrane, while nanospheres are composed of a dense polymer matrix (Quintanar-Guerrero et al. 1998; Mora-Huertas et al. 2010). In the case of food applications, submicron systems should be made with generally recognized as safe (GRAS) substances, including biodegradable polymers like poly-ε-caprolactone (ZambranoZaragoza et al. 2011). Recently, our group explored the use of solid lipid nanoparticle/xanthan gum coatings to increase the shelf life of guavas (Zambrano-Zaragoza et al. 2013). Also, we tested nanocapsules containing α-tocopherol as a coating to improve preservation of fresh-cut Red Delicious apples. That work showed that submicron systems have the potential to reduce the browning index and texture changes while maintaining product quality (Zambrano-Zaragoza et al. 2014a); however, the effect of nanosystems on PAL and PPO enzymatic activity in fresh-cut fruit has not been explored, despite the importance of these parameters for functional changes. For these reasons, the goal of this study was to evaluate the effect of nanoparticles (nanocapsules with αtocopherol and nanospheres) with xanthan gum coatings on PPO and PAL activity and correlate findings with the synthesis and degradation of phenolic compounds in fresh-cut Red Delicious apples.
Food Bioprocess Technol (2015) 8:1791–1799
nanocapsules. The partially water-miscible solvent (analytical grade) used was ethyl acetate (Fermont, Mexico). The distilled water was of Milli-Q® quality (Millipore, USA). Xanthan gum was purchased from Sigma-Aldrich® (Germany) and used as a continuous matrix for the coatings. Propylene glycol was utilized as the plasticizer (99 %) (Sigma-Aldrich, USA). All other reagents were of analytical grade and were used without purification. Biological Material Red Delicious apples (Malus domestica Borkh) were purchased at local wholesale markets in Cuautitlan Izcalli, Mexico, selected on the basis of maturity, color, size, and uniformity of shape, considering a soluble solid content of 11–14°Bx and firmness≈7 N, as tested by puncture. The fruits selected were randomly divided into batches and kept in refrigerated conditions at 4 °C for 24 h prior to use. Nanoparticle Preparation In order to evaluate the effect of encapsulated α-tocopherol on PAL and PPO enzyme activity, the concentration of total phenols, and the total color differences of fresh-cut apples, nanocapsules containing α-tocopherol and nanospheres without this additive were prepared using the emulsificationdiffusion method described in detail elsewhere (QuintanarGuerrero et al. 1998) and considering the methodology optimization approach proposed by Zambrano-Zaragoza et al. (2011) for food applications. Briefly, ethyl acetate and water were mixed to equilibrium. PCL (~250 mg) and dl-αtocopherol acetate (2 g/L) were dissolved in the organic phase, while Pluronic® F-127 (50 g/L) was dissolved in the aqueous phase. The two solutions were then mixed and emulsified at 4000 rpm for 10 min (Ultra-Turrax® T18, IKA, Germany), followed by the addition of excess water to promote diffusion of the solvent in the aqueous phase and nanocapsules formation. In the preparation of the nanospheres, tocopherol was omitted. Finally, the excess solvent was removed under reduced pressure at 70 mmHg and 30 °C (RV10, IKA® Labortechnik, Germany).
Materials and Methods
Characterization of Nanoparticles
Materials
The particle size (Ps), polydispersion index (Pdi), and zeta potential (ζ) of the nanocapsules and nanospheres with, and without, xanthan gum 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 Milli-Q®-grade water. Tests were performed in triplicate for ζ and standardized using standard polystyrene dispersions (ζ=−55 mV).
Poly-ε-caprolactone (PCL) Mw≈80,000 (ρ=1.147 g/cm3 at 25 °C) from Sigma-Aldrich® (USA) was used as a biopolymer to form the membrane (nanocapsules) or matrix (nanospheres). Pluronic® F-127 (BASF, Mexico) was the (nonionic) stabilizer, and dl-α-tocopherol acetate (98 %) (SigmaAldrich®, USA) was used to form the oily core of the
Author's personal copy Food Bioprocess Technol (2015) 8:1791–1799
Coating Applications on Fresh-Cut Apples The whole apples were sanitized by immersion in a chlorine solution at 70 ppm for 5 min. Cutting boards, utensils, and containers were also sanitized to minimize contamination by microorganisms. After cutting, the apple pieces were first placed in a CaCl2 solution (10 g/L) and subsequently dipped in one of the prepared coatings. To determine the influence of the polysaccharide matrix, one group of apples was coated with xanthan gum (3 g/L) and 5 g/L propylene glycol. The following treatments were applied: control, nanocapsules, nanocapsules/xanthan gum, nanospheres, and nanospheres/ xanthan gum. The latter two batches were included to determine the influence of the polymer wall on the behavior of the coatings applied on the fresh-cut apples. After the fresh-cut apple pieces were dipped into the dispersion, they were drained for 2 min at 10 °C. Once coated, they 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 Characterization of Apple Coatings The morphological characterization of the apple surfaces with the different coatings was performed by scanning electron microscopy following the method proposed by ZambranoZaragoza et al. (2014a). The surfaces were then observed under a high-vacuum scanning electron microscope LV-SEM JSM 5600 LV (Jeol, Japan; resolution=5 nm) at a voltage of 20 kV and 12- to 20-Pa pressure in the chamber. Initial Respiration Rate The effect of the coatings on respiration rates was determined by the static method as reported by Wang et al. (2009) and Iqbal et al. (2008), using 170-mL glass bottles in which the apple pieces were sealed. Gas readings were determined by measuring headspace using a needle inserted through a septum placed in the center of the bottle cap and connected to a O2/CO2 analyzer (Quantek Instruments model 905, USA) to obtain the CO2 volume fraction expressed inside the container. The production rate of CO2 (RCO2) was calculated by the difference in CO2 concentrations at different time intervals, expressed as follows: RCO2 ¼
ðyCO2 −yi CO2 Þ V f * W ðt−t i Þ
where yi CO2 is the initial concentration of gas in the mixture, y CO2 is the gas concentration at any other time, t is anytime other than time 0 (t =0) expressed in hours, RCO2 is the CO2 production rate, W is the product weight (kg), and Vf is the volume (mL) inside the container (Iqbal et al. 2008). The determination of
1793
initial CO2 production was performed in triplicate for each treatment. Determination of Total Color Difference (ΔE*) The CIELab colorimetric parameters L*, a*, and b* were obtained for the cut apple surfaces with a Minolta CR-300 colorimeter (Konica Minolta DP-301 console, Japan). Total color differences (ΔE*) were determined to associate browning development with phenolic content and PAL and PPO enzyme activities. ΔE* indicates the color difference between two tones according to the following scale: ΔE*=0–0.5: trace level difference, ΔE*=0.5–1.5: slight difference, ΔE*=1.5– 3.0: noticeable difference, ΔE*=3.0–6.0: appreciable difference, ΔE*=6.0–12.0: large difference, and ΔE*>12.0: very obvious difference (Goyeneche et al. 2014). Three apple pieces were taken from each package, and two measurements were made per sample to record the parameters during the storage period. ΔE* was calculated using the following relationship: qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi * * 2 2 2 ΔE * ¼ L −L0 þ a* −a*0 þ b* −b*0 where L*, a*, and b* are the colorimetric measurements of the sample at time t, while L*0, a*0, and b*0 are the colorimetric parameters at time 0 (Goyeneche et al. 2014). Determination of Total Phenols Extraction of phenolic compounds from the coated apple surfaces was performed according to the method described by Supapvanich et al. (2011). Folin-Ciocalteu’s colorimetric method (Waterhouse 2005) was used to determine total phenols. Briefly, 20 μL of phenolic extract was mixed with 1.58 mL of distilled water and 100 mL of Folin-Ciocalteu reagent (1:10). After 4 min, 300 μL of sodium carbonate solution (200 g/L) was added. The mixture was stirred slightly at 25 °C for 2 h. Absorbance was measured at 765 nm (UV-Vis Cintra 10e, GBC Scientific Equipment Ltd. Dandenong Victoria, Australia). Results were expressed as milligrams of gallic acid equivalents (mg GAE) per 100 g of fresh weight. All determinations were performed in triplicate. Determination of PAL Activity Evaluation of PAL activity was performed according to the method proposed by Ke and Saltveit (1986). Four grams of tissue was homogenized with 0.4 g of polyvinylpyrrolidone, 16 mL of buffer borate (50 mM, pH 8.5), and 14 μL of 2mercaptoethanol. After homogenization in an Ultra-Turrax® T18 (IKA, Germany), the sample was centrifuged at 10, 000 rpm for 40 min at 4 °C and finally filtered. PAL activity was determined in the supernatant at 40 °C using 300 μL of L-
Author's personal copy 1794
Food Bioprocess Technol (2015) 8:1791–1799
phenylalanine as a substrate and measuring absorbance at λ= 290 nm in a UV-Vis spectrophotometer (UV-Vis Cintra 10e, GBC Scientific Equipment Ltd., Australia). Activity was expressed as the amount of PAL that produced 1 μmol of cinnamic acid per gram of sample in 1 h. All measurements were performed in triplicate.
Determination of PPO Activity PPO enzyme extraction was performed following the methodology modified by Zambrano-Zaragoza et al. (2014b). Thirty grams of coated tissue was ground and homogenized in an Ultra-Turrax® T18 (IKA, Germany) at 5000 rpm for 2 min with 20 mL of sodium phosphate buffer (0.2 M; pH 7.0) containing 50 g/L polyvinylpyrrolidone, using an external ice bath. The homogenate was filtered through Whatman® 42 paper and centrifuged at 10,000 rpm for 30 min. The supernatant obtained was from the enzyme extract used to measure PPO activity. PPO activity was determined using a mixture of 0.2 mL of enzymatic extract and 2.8 mL of catechol (50 mM in 0.2 M citrate-phosphate buffer at pH 6.5). Changes in relative absorbance compared to the white without enzymatic extract were measured at 420 nm (UV-Vis Cintra 10e, GBC Scientific Equipment Ltd., Australia). One unit of PPO activity was defined as a change of 0.001 Abs/min from the initial slope. All determinations of PPO activity were performed in triplicate.
Statistical Analysis Results were analyzed using the statistical analytical software Minitab 16 (Minitab®, USA), ANOVA, and comparison of means of treatments (α = 0.05) to establish the effect of the different coating compositions on PPO and PAL activity, phenolic content, and color changes during storage of freshcut apples.
Results and Discussion Characterization and Stability of Nanoparticles Table 1 shows the Ps, Pdi, and ζ of the nanoparticles used to prepare the coatings. All systems were submicron size in a range <300 nm. Ps depends mainly on the preparation method, the polymer membrane, and the amount and type of active oil encapsulated (Weiss et al. 2006; Mora-Huertas et al. 2010). The Ps reported for the nanocapsules prepared using the emulsification-diffusion method ranged from 200 to 500 nm for the different oils, using PCL as the biopolymer membrane (Quintanar-Guerrero et al. 1998; Zambrano-Zaragoza et al. 2011). The reduction in the Ps of the colloidal systems helps reduce the gravitational force and sedimentation, while also preventing flocculation (McClements and Rao 2011). The Pdi obtained was <0.3, suggesting a narrow distribution of Ps, which implies a higher probability of achieving the largest number of particles with the average trend (Lemarchand et al. 2003). All systems had ζ below −40 mV, which implies that these systems with, or without, xanthan gum had good stability and likelihood of flocculation and coalescence. It has been shown that colloidal systems with ζ > |25| mV are deflocculated, since higher absolute values of ζ have greater between-particle electrical repulsion and better dispersion (Chee-Teck 2003).
Microstructural Characterization of Coatings on Fresh-Cut Apples Figure 1a–f shows the microstructural characterization of the apple surface coatings compared to the control apples after 24 h of treatment with the different coatings. Figure 1a represents the control apple surface with the parenchymal tissue and other cellular structures of normal apples (Hye-Yeon et al. 2013; Zambrano-Zaragoza et al. 2014b). Figure 1b shows the xanthan gum coating on the fresh-cut apple surfaces, which formed a well-distributed uniform coating whose
Table 1 Average values of Ps, Pdi, and ζ of nanoparticles prepared by emulsification-diffusion method with and without xanthan gum and initial respiration rate of fresh-cut apple System
Ps (nm)
Pdi
ζ (mV)
Initial respiration rate (mL CO2 kg−1 h−1)
Nanocapsules Nanocapsules/xanthan Nanospheres Nanospheres/xanthan
243 a±8 259 a±2 190 b±26 196 b±18
0.11 a±0.02 0.09 a±0.03 0.17 b±0.01 0.26 b±0.04
−43.6 a±1.33 −43.57 a±1.32 −45.36 a±1.4 −48.6 b±2.93
4.10 a±0.3 2.85 b±0.2 3.22 c±0.3 3.48 c±0.4
Xanthan gum Control
– –
– –
– –
5.52 d±0.3 7.75 e±0.8
Each value is the mean±standard deviation of the mean of three replicates. Different letters indicate differences (p<0.05) between treatments Ps particle size, Pdi polydispersion index, ζ zeta potential
Author's personal copy Food Bioprocess Technol (2015) 8:1791â&#x20AC;&#x201C;1799
1795
Fig. 1 Micrographs of apple tissue coated with the different systems: a control, b xanthan gum, c nanocapsules, d nanocapsule/xanthan, e nanospheres, and f nanospheres/ xanthan
appearance is characteristic of xanthan gum applications (Veiga-Santos et al. 2005; Zambrano-Zaragoza et al. 2014b). Figure 1c shows the fresh-cut apple surface coated with nanocapsules; note that these are dispersed over the entire tissue surface. The arrows in the micrographs indicate the nanocapsules found in the coated apple. Also visible are signs left by the capsular entities as previously described by Zambrano-Zaragoza et al. (2014a). Figure 1d shows that in the apple pieces coated with nanocapsules/xanthan gum, the distribution of nanocapsules occurs preferentially on the surface, embedded in a matrix apparently formed by the xanthan gum that completely covers the apple tissue. In this image, clear differences with respect to the nanocapsules without xanthan gum are observed, showing that nanocapsules mixed with the polysaccharide present some larger particles, presumably a result of transpiration processes that occur after minimal processing of the fruit (Soliva-Fortuny et al. 2003). According to Zambrano-Zaragoza et al. (2014a), the water released by the fruit is trapped by the polysaccharide and forms the globules seen in the micrograph. Figure 1e presents the distribution of the nanospheres, which were abundant on the surface of the fresh-cut apple pieces. Arrows indicate some entities composed of the nanospheres that were deposited on the fruit
tissue, showing similar characteristics to those found on the apples coated with nanocapsules. Finally, Fig. 1f shows the distribution of the nanospheres/xanthan gum coating with a structure similar to the samples seen in Fig. 1d. Although this system forms a continuous coating, some aggregates do appear in the film, caused by the water trapped by the xanthan gum during fruit transpiration. This behavior has also been observed when apples are coated with Îą-tocopherol nanoemulsions using nopal mucilage as the matrix polysaccharide (Zambrano-Zaragoza et al. 2014b). Initial Respiration Rates of Fresh-Cut Apples with Different Coatings Table 1 shows the changes in the initial respiration rates of apples after coating with the different treatments. All the freshcut apple coatings decreased respiration rates with respect to controls. The xanthan gum coating had the least effect on decreasing initial respiratory activity with a reduction of 29 % relative to controls, followed by nanocapsules (47 %), nanospheres/xanthan gum (55 %), and nanospheres (58 %). The most effective treatment proved to be nanocapsules/ xanthan gum, which decreased the initial respiration rate by
Author's personal copy 1796
Food Bioprocess Technol (2015) 8:1791–1799
63 %. Lee et al. (2003) found decreases of 5 and 20 % in the respiration of fresh-cut Fuji apples coated with carrageenan and whey protein, respectively, suggesting that the insolubility of the coating helps decrease the respiration rate. Qi et al. (2011) reported a 20 % decrease in the initial respiration rate of Fuji apples coated with 2 % ascorbic acid+0.5 % CaCl2 +1 % chitosan. Thus, it is possible to show that applying nanosystems has a favorable effect on decreasing initial respiratory rates and makes it possible to retard the deterioration of fresh-cut fruit.
to day 12. This behavior is attributable to the polymer PCL used in preparing nanoparticles by the emulsificationdiffusion method, since it forms a barrier that helps decrease gas exchange (Myllymäki et al. 1998), thus reducing initial respiration rates and browning reactions in fresh-cut apples. Freitas et al. (2013) reported that a xanthan gum coating without CaCl2 had no significant effect on decreasing the loss of luminosity or increasing a* values in Gala apples; however, xanthan gum has effectively served as a carrier and support for other additives, such as nanocapsules as a means of maintaining the quality of fresh-cut fruits.
Changes in Total Color Difference (ΔE*) Total Phenols The ΔE* parameter has been used to monitor the enzymatic browning of many processed fruits and vegetables, including fresh-cut apples (Soliva-Fortuny et al. 2001; Limbo and Piergiovanni 2006; Goyeneche et al. 2014). Table 2 shows the changes in ΔE* values. The samples coated with the nanosystems had lower ΔE* compared to control samples. The difference was very obvious (ΔE*> 12) at day 6, indicating a delay in browning. The xanthan gum coatings had smaller changes in ΔE* with a change rate of 0.92 ΔE*/day. However, very obvious differences at 15 days were observed, suggesting a protective effect of the polysaccharide that was associated with a reduction of the initial respiration rate. The apples coated with the nanodispersions had the best control and prevention of browning development as they presented the largest differences in coloration (ΔE*=6.0–12) at the end of storage. Treatment with nanospheres/xanthan gum was the least effective of the nanosystems with appreciable differences (ΔE*=3.0–6.0) at day 6 and a rate change of 0.47 ΔE*/day. In contrast, both the samples coated with nanospheres and those treated with nanocapsules had the same change rate of 0.37 ΔE*/day. Meanwhile, the nanocapsules/xanthan gum coatings had a rate of 0.34 ΔE*/day and showed significant color differences up
Table 2 Days
3 6 9 12 15 21
Figure 2 shows the variations in total phenols of the apple coatings during 21 days of storage at 4 °C. The amplitude of the boxes represents the variability of results from the same batch and treatment. All samples showed a mean of 440 to 450 mg GAE/100 g of apple at the start of storage, values that are consistent with those reported by Tsao et al. (2003) for Red Delicious apples. The phenolic compound content of the control samples declined during storage to a final concentration of 390 mg GAE/100 g of fruit, which correlates with the increase of ΔE*(R = −0.88), whereas the samples treated with nanoparticle coatings had minimal changes in terms of their phenolic content. That small change was associated with lower development of browning as a function of ΔE* values. The samples treated with the xanthan gum or nanoparticle/xanthan gum coatings increased their phenolic content at different times. The apples coated with xanthan gum had the highest phenolic content, but concentrations decreased up to day 6 in association with a rapid rise in ΔE* (R= −0.81). Later, an accumulation of phenols occurred. The samples treated with nanocapsules and nanospheres showed little variation in total phenolic content, probably
Changes in ΔE* during storage of fresh-cut apple coating with xanthan gum and different nanoparticle systems ΔE*changes C
NCS
NCS/XG
NSP
NSP/XG
XG
6.48±0.18 aa 16.98±0.82 ba 17.97±0.46 ba 19.22±0.72 ca 20.31±1.27 ca 18.58±0.71 ca
1.63±0.44 ab 2.68±1.14 bb 3.51±0.25 cb 4.33±0.33 db 5.49±0.07 eb 6.58±0.33 fb
1.65±0.67 ab 2.31±0.98 ab 2.01±0.57 ac 3.43±0.38 bc 4.96±0.55 cc 6.41±0.17 db
1.78±0.33 ab 2.96±0.74 bb 3.93±0.22 cb 4.28±0.55 db 5.62±0.54 dc 8.04±0.25 ec
1.69±0.16 ab 3.63±0.57 bc 6.16±0.78 dd 6.54±0.72 de 6.80±0.49 dd 8.92±1.03 ec
4.49±0.42 ac 6.39±0.63 bd 8.72±0.66 de 10.37±0.75 ef 12.91±1.07 fg 13.21±1.43 fe
Each value is the mean±standard deviation of the mean of three replicates. First different letters indicate differences (p<0.05) between storage times, and second different letters indicate significant difference (p<0.05) between treatments C control, NCS nanocapsules, NCS/XG nanocapsules/xanthan gum, NSP nanospheres, NSP/XG nanospheres/xanthan gum, XG xanthan gum
Author's personal copy Food Bioprocess Technol (2015) 8:1791–1799
1797
PAL Activity
Fig. 2 Total phenols in fresh-cut apples under different treatments. C control, NCS nanocapsules, NCS/XG nanocapsules/xanthan gum, NSP nanospheres, NSP/XG nanospheres/xanthan, XG xanthan gum
due to the functionality of their submicron size and the polymer used, as well as the fact that the nanocapsules contained α-tocopherol, which impeded oxygen penetration into the fruit (Myllymäki et al. 1998), thus reducing the oxidation of phenolic compounds. It is important to note that the nanocapsules/xanthan gum coating had a higher phenolic content during the test period, likely because it allowed an accumulation of phenols in the xanthan gum matrix, though the presence of nanoencapsulated α-tocopherol and the PCL polymer allowed only limited oxygen absorption, which impeded development of browning. Therefore, it was possible to demonstrate that these special submicron-sized coatings have a great potential for maintaining the concentration of phenolic compounds almost unchanged during 21 days of storage, thus preserving the functional characteristics of fresh-cut Red Delicious apples.
Fig. 3 Changes in phenylalanine ammonia lyase (PAL) activity as a function of treatment. NCS nanocapsules, NCS/XG nanocapsules/ xanthan gum, NSP Nanoespheres, NSP/XG nanospheres/xanthan gum, XG xanthangum
PAL activity increases significantly in fruits that are injured during the fresh-cutting process, so inhibiting browning is one of the most important control factors for increasing the shelf life of those products (Choi et al. 2005). Figure 3 shows the changes associated with PAL activity as a function of the composition of different coatings. All coatings had some degree of effectiveness in terms of controlling PAL activity, with a mean reduction of 75 %. PAL activity showed a parabolic behavior for controls and xanthan gum, as reported in other studies. After reaching a peak, PAL activity decreased due to inactivation caused by phenol accumulation (Ortega-Garcia and Peragon 2009). The control apples increased their PAL activity (2.33 mol/g/dia), suggesting that there was no protection against the injuries produced during cutting, which allowed an increase of browning attributed to PAL activity and subsequent oxidation due to enzymatic PPO activity (Choi et al. 2005). Xanthan gum did have a protective effect, as it reduced the availability of oxygen, leading to lower rates of enzymatic activity. Figure 3 also shows that the nanocapsule and nanocapsules/xanthan gum coatings presented the smallest increase in PAL activity, associated with the induction of scarring on apple surfaces. According to Choi et al. (2005), the phenols generated by PAL activity are retained in the vacuoles and participate in browning reactions only when membrane alteration allows the substrates to mix with enzymes. Here, the Ps of the coating and the homogeneity of its distribution on the surface are key factors. The nanospheres coating also had a protective effect, but its results are attributable to the terminal carboxyl groups that impede PAL activity by modifying pH, which prevents the development of browning by altering the surface’s
Fig. 4 Changes in polyphenol oxidase activity (PPO) during storage of fresh-cut apples treated with different coatings. C control, NCS nanocapsules, NCS/XG nanocapsules/xanthan gum, NSP nanospheres, NSP/XG nanospheres/xanthan, XG xanthan gum
Author's personal copy 1798
Food Bioprocess Technol (2015) 8:1791–1799
permeability to oxygen in a way that limits exchange with the environment (Myllymäki et al. 1998).
decreased the production and oxidation of phenolic compounds on the cut apple surfaces.
PPO Activity
Acknowledgments Galindo-Pérez thanks to Consejo Nacional de Ciencia y Tecnología (CONACyT) for the financial grant received (support number:392051). The authors acknowledge the financial support for this work provided by PAPIIT IT231511 and PAPIIT IT200814 from DGAPA-UNAM and the technical support for acquisition of micrographics to M en I. Alicia del Real López.
Evaluations of the effectiveness of treatment relate directly to the decrease in PPO activity during storage (Rojas-Graü et al. 2006). Figure 4 shows the behavior of PPO activity as a function of coating and time. All apple coatings showed that PPO activity reached a maximum before declining. Qi et al. (2011) reported similar results on PPO activity using chitosan as the coating and release platform for anti-browning substances in fresh-cut Fuji apples. In the control samples, an increase in PPO activity of 85 U/mL/day was observed up to day 6, when it reached 1388 U/mL. These changes in activity correlated with an increase in the ΔE* parameter (R=0.96) and a reduction of phenolic compounds (R=−0.95), all of which indicate that activation of the PAL enzyme causes synthesis of phenols that are oxidized by PPO, thus promoting the formation of browning compounds. The apple coating with nanospheres/ xanthan gum showed a similar behavior with a maximum activity at 1384 U/mL on day 6 associated with an increase in ΔE*(R=0.98) and a decrease in the phenolic content (R= −0.80). The nanospheres and nanocapsules coatings reached their maximum PPO activity on day 9 at 1428 U/mL and day 12 at 1327 U/mL, respectively, which was reflected mainly in an increase in ΔE* values in both cases (R=0.82), though their phenolic concentrations remained constant. The nanocapsules/xanthan gum coating proved to be the best treatment for controlling PPO activity, with a maximum of 1327 U/mL at day 15. Compared to the other treatments after 10 days of storage, clear differences were observed that reflected lower catalytic activity, a behavior more often associated with slight variations in ΔE* values (R=0.77). The coatings containing nanoparticles succeeded in retarding PPO activity because they are capable of limiting O 2 absorption.
Conclusions This study shows that polymeric nanoparticles containing αtocopherol and incorporated into a polymeric matrix (xanthan gum) have a great potential for preserving the physicochemical characteristics of fresh-cut Red Delicious apples during 21 days of storage. It is important to note that polymeric nanoparticles contribute significantly to reducing initial respiration rates by over 50 % with respect to controls. With respect to the activity of the PAL and PPO enzymes, we observed that the polymeric nanoparticles decreased this catalytic activity significantly compared to controls. Particularly important in this regard were the nanocapsules and nanocapsules/xanthan gum systems, which showed the lowest catalytic activity and
References Chee-Teck, T. (2003). Beverage emulsions. In S. E. Friberg, K. Larsson, & J. Sjöblom (Eds.), Food emulsions (pp. 485–525). New York: Marcel Dekker, Inc. Choi, Y., Tomás-Barberán, A., & Saltveit, M. E. (2005). Wound-induced phenolic accumulation and browning in lettuce (Lactuca sativa L.) leaf tissue is reduced by exposure to n-alcohols. Postharvest Biology and Technology, 37(1), 47–55. 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. 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 and Technology, 22(6), 292–303. Freitas, I. R., Cortez-Vega, W. R., Pizato, S., Prentice-Hernández, C., & Borges, C. D. (2013). Xanthan gum as a carrier of preservative agents and calcium chloride applied on fresh-cut apple. Journal of Food Safety, 33(3), 229–238. Goyeneche, R., Agüero, M. V., Roura, S., & Di Scala, K. (2014). Application of citric acid and mild heat shock to minimally processed sliced radish: color evaluation. Postharvest Biology and Technology, 93, 106–113. Hye-Yeon, S., Wan-Shin, J., Nak-Bum, S., Sea, C. M., & Kyung, B. S. (2013). Quality change of apple slices coated with Aloe vera gel during storage. Journal of Food Science, 78(6), C817–C822. Hyson, D. A. (2011). A comprehensive review of apples and apple components and their relationship to human health. Advances in Nutrition: An International Review Journal, 2(5), 408–420. Iqbal, T., Rodrigues, F. A., Mahajan, P. V., Kerry, J. P., Gil, L., Manso, M., et al. (2008). Effect of minimal processing conditions on respiration rate of carrots. Journal of Food Science, 73(8), E396–E402. Ke, D., & Saltveit, M. E. (1986). Effects of calcium and auxin on russet and phenylalanine ammonia-lyase activity in iceberg lettuce. HortScience, 21, 1169–1171. Lee, J. Y., Park, H. J., Lee, C. Y., & Choi, W. Y. (2003). Extending shelflife of minimally processed apples with edible coatings and antibrowning agents. LWT–Food Science and Technology, 36(3), 323–329. Lemarchand, C., Couvreur, P., Vauthier, C., Constantini, D., & Gref, R. (2003). Study of emulsion stabilization by graft copolymers using the optical analyzer Turbiscan. International Journal of Pharmaceutics, 254(1), 77–82. Limbo, S., & Piergiovanni, L. (2006). Shelf life of minimally processed potatoes: part 1. Effects of high oxygen partial pressures in combination with ascorbic and citric acids on enzymatic browning. Postharvest Biology and Technology, 39(3), 254–264. Lin, D., & Zaho, Y. (2007). Innovations in the development and application of edible coating for fresh minimally processed fruits and vegetables. Comprehensive Reviews in Food Science and Food Safety, 6(3), 60–75.
Author's personal copy Food Bioprocess Technol (2015) 8:1791–1799 McClements, D. J., & Rao, J. (2011). Food-grade nanoemulsions: formulation, fabrication, properties, performance, biological fate, and potential toxicity. Critical Reviews in Food Science and Nutrition, 51(4), 285–330. Mei, Y., Zhao, Y., Yang, J., & Furr, H. C. (2002). Using edible coatings to enhance nutritional and sensory qualities of baby carrots. Journal of Food Science, 67(5), 1964–1968. Mora-Huertas, C. E., Fessi, H., & Elaissari, A. (2010). Polymer-based nanocapsules for drug delivery. International Journal of Pharmaceutics, 385(1), 113–142. Myllymäki, O., Myllärinen, P., Forssell, P., Suortti, T., Lähteenkorva, K., Ahvenainen, R., et al. (1998). Mechanical and permeability properties of biodegradable extruded starch/polycaprolactone films. Packaging Technology and Science, 11(6), 265–274. Ortega-García, F., & Peragon, J. (2009). The response of phenylalanine ammonia-lyase, polyphenoloxidase and phenols to cold stress in the olive tree (Olea europaea L. cv. Picual). Journal of the Science of Food and Agriculture, 89(9), 1565–1573. 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. Quintanar-Guerrero, D., Allémann, E., Fessi, H., & Doelker, E. (1998). Preparation techniques and mechanisms of formation of biodegradable nanoparticles from preformed polymers. Drug Development and Industrial Pharmacy, 24(12), 1113–1128. Rojas-Graü, M. A., Sobrino-López, A., Tapia, M. S., & Martín-Belloso, O. (2006). Browning inhibition in fresh-cut ‘Fuji’ apple slices by natural antibrowning agents. Journal of Food Science, 71(1), S59– S65. Soliva-Fortuny, R. C., Grigelmo-Miguel, N., Odriozola-Serrano, I., Gorinstein, S., & Martín-Belloso, O. (2001). Browning evaluation of ready-to-eat apples as affected by modified atmosphere packaging. Journal of Agricultural and Food Chemistry, 49(8), 3685– 3690. 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, Ç. (2008). Kinetics and thermal activation/inactivation of Starking apple polyphenol oxidase. Journal of Food Processing and Preservation, 32(6), 1034–1046. Supapvanich, S., Pimsaga, J., & Srisujan, P. (2011). Physicochemical changes in fresh-cut wax apple (Syzygium samarangenese [Blume] Merrill & L.M. Perry) during storage. Food Chemistry, 127(3), 912– 917. Treutter, D. (2001). Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regulation, 34(1), 71–89. Tsao, R., Yang, R., Young, C., & Zhu, H. (2003). Polyphenolic profiles in eight apple cultivar using high-performance liquid chromatography
1799 (HPLC). Journal of Agricultural and Food Chemistry, 51(21), 6347–6353. Veiga-Santos, P., Oliveira, L. M., Cereda, M. P., Alves, A. J., & Scamparini, A. R. P. (2005). Mechanical properties, hydrophilicity and water activity of starch-gum films: effect of additives and deacetylated xanthan gum. Food Hydrocolloids, 19(2), 341–349. Vert, M., Hellwich, K. H., Hess, M., Hodge, P., Kubisa, P., Rinaudo, M., et al. (2012). Terminology for biorelated polymers and applications (IUPAC Recommendations 2012). Pure and Applied Chemistry, 84(2), 377–410. Wang, Z., Duan, H., & Hu, C. (2009). Modelling respiration rate of guava (Psidium guajava L.) fruit using enzyme kinetics, chemical kinetics and artificial neutral network. European Food Research and Technology, 229(3), 495–503. Waterhouse, A. L. (2005). Determination of total phenolics. In R. E. Wrolstad, T. E. Acree, E. A. Decker, M. H. Penner, D. S. Reid, S. J. Schwartz, C. F. Shoemaker, D. Smith, & P. Sporns (Eds.), Handbook of food analytical chemistry: pigments, colorants, flavors, texture, and bioactive food components (pp. 463–470). New York: Wiley. 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 antioxidants activity of new and old apple varieties. Journal of Agricultural and Food Chemistry, 56(15), 6520–6530. Zambrano-Zaragoza, M. L., Mercado-Silva, E., Gutiérrez-Cortez, E., Castaño-Tostado, E., & Quintanar-Guerrero, D. (2011). Optimization of nanocapsules preparation by the emulsiondiffusion method for food applications. LWT–Food Science and Technology, 44(6), 1362–1368. Zambrano-Zaragoza, M. L., Mercado-Silva, E., Ramirez-Zamorano, P., Cornejo-Villegas, M. A., Gutiérrez-Cortez, E., & QuintanarGuerrero, 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. Zambrano-Zaragoza, M. L., Mercado-Silva, E., Del Real, L. A., Gutiérrez-Cortez, E., Cornejo-Villegas, M. A., & QuintanarGuerrero, D. (2014a). The effect of nano-coatings with αtocopherol and xanthan gum on shelf-life and browning index of fresh-cut BRed Delicious^ apples. Innovative Food Science and Emerging Technologies, 22, 188–196. Zambrano-Zaragoza, M. L., Gutiérrez-Cortez, E., Del Real, A., González-Reza, R. M., Galindo-Pérez, M. J., & QuintanarGuerrero, D. (2014b). Fresh-cut Red Delicious apples coating using tocopherol/mucilage nanoemulsion: effect of coating on polyphenol oxidase and pectin methylesterase activities. Food Research International, 62, 974–983.