Accepted Manuscript Analytical methods Performance of an active paper based on cinnamon essential oil in mushrooms quality Y. Echegoyen, C. Nerín PII: DOI: Reference:
S0308-8146(14)01247-3 http://dx.doi.org/10.1016/j.foodchem.2014.08.032 FOCH 16255
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 April 2013 1 July 2014 10 August 2014
Please cite this article as: Echegoyen, Y., Nerín, C., Performance of an active paper based on cinnamon essential oil in mushrooms quality, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.08.032
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PERFORMANCE OF AN
ACTIVE
PAPER
BASED
ON
CINNAMON
ESSENTIAL OIL IN MUSHROOMS QUALITY. Y. Echegoyen, C. Nerín∗ I3A, Department of Analytical Chemistry, University of Zaragoza, C/ María de Luna 3, 50018, Zaragoza, Spain Abstract The antioxidant capacity of two active papers (based on solid and emulsion paraffin) with cinnamon essential oil was studied. Mushroom samples were introduced in macroperforated PET trays covered with the active papers, and weight loss and browning monitored for nine days. The antioxidant capacity of the different papers was evaluated based on scavenging 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and tyrosinase inhibition kinetics, and the release of aromatic volatile oils was determined by HSPMEGC-MS. Differences in performance were observed: the active papers were more efficient at avoiding weight loss and mushroom browning when compared to the nonactive paraffin-based papers. The efficiency increased when the bottom and walls of the trays were covered rather than the bottom alone. Better results were observed when cinnamon was incorporated as emulsion paraffin instead of a solid. Keywords: mushroom quality, active packaging, cinnamon essential oil, antioxidant capacity. 1. Introduction Half of the world’s fruit and vegetable crops are lost due to postharvest deterioration. Browning disorders in agricultural products are normally induced by dehydration, mechanical injury or wound deterioration (Whitaker & Lee, 1995). Polyphenol oxidase
∗
To whom correspondence should be addressed: [email protected]; Tlf: +34976761873; Fax: +34976762388; I3A, Department of Analytical Chemistry, University of Zaragoza, C/ María de Luna 3, 50018, Zaragoza, Spain.
1
(PPO) is the major enzyme involved in browning reactions. PPO catalyzes the oxidation of phenolic compounds to o-quinones, resulting in brown pigmentation (Chang & Quimio 1984). Most research on browning of white button mushroom (Agaricus bisporus) has associated browning with PPO or related enzymes, specifically tyrosinase, an enzyme belonging to the PPO family (Nerya, Ben-Arie, Luzzatto, Musa, Khativ & Vaya, 2006; Qiu et al., 2009). Cool storage conditions slow the rate of deterioration (Escriche-Roberto, Galotto-López, Gómez-Ariza & Serra-Belenguer, 2001), but temperatures that are too low may cause chilling injury. The most important factors determining the rate of enzymatic browning of fruit and vegetables are the concentrations of active PPO and phenolic compounds, pH, temperature and oxygen availability of the tissue (Martínez & Whitaker, 1995). Extending the shelf life of mushrooms is difficult for a number of reasons: it is a delicate fresh product and the handling can seriously damage the tissue, facilitating the appearance of black or brown spots, which decrease their value. Mushrooms have a high respiration rate and produce a large amount of water vapor, which increases the decay of mushrooms in closed packaging systems. Mushrooms are fungi and, therefore, naturally have a short shelf life. The initial problem is the browning, more evident in the gills or lamella than in the cap. As fresh products, mushrooms cannot be treated with antioxidants to protect them against oxidation, active packaging is a smart solution to extend their shelf life. The use of essential oils to extend the shelf life is becoming popular since consumers have become more conscious about potential health problems associated with synthetic preservatives (Gómez-Estaca, Lacey, López-Caballero & Gómez-Guillén, 2010; Holley & Patel, 2005). Synthetic preservatives, added to food items as antimicrobials and/or antioxidants, are generally-recognized-as-safe and, usually, have a beneficial effect on
2
food preservation. However, there have been problems concerning the safety of some chemicals, including potential allergens (e.g. benzoic acid and sulphites) and the formation of carcinogenic compounds such as nitrosamines from nitrites, and the possible
rodent
carcinogenicity
of
butylated
hydroxyanisole
and
butylated
hydroxytoluene (Parke & Lewis, 1992). Previous studies with essential oils as food additives have revealed them to be advantageous, as observed by an increase in food shelf life (Chouliara, Karatapanis, Savvaidis & Kontominas, 2007; Jiang, Feng & Zheng, 2012). The amount of essential oils, however, determined the acceptability of products since strong aromas might be imparted to the product. One alternative to the direct incorporation of essential oils in foodstuffs is active packaging where volatile compounds in the essential oil create a protective atmosphere, either antimicrobial or antioxidant (Nerín et al., 2006). Cinnamon essential oil is well known for its antimicrobial activity (López, Sánchez, Batlle & Nerín, 2005; Rodríguez, Nerín & Batlle, 2008). It has also been reported to have antioxidant activity, derived mostly from its phenolic components (El-Baroty, ElBaky, Farag & Saleh, 2010). As for many other free radicals, ·OH can be neutralized if a hydrogen atom is provided. The scavenging activities of phenolic substances may be due to hydrogen donation during hydroxyl substitutions (Mathew & Abraham, 2006). For this reason, antioxidant behavior is associated with phenolic compounds. The aim of this work was to determine the performance of two active papers containing cinnamon essential oil as an active agent to delay postharvest deterioration in mushrooms (Agaris bisporus). Repsol (Spain) provided the active material using a patented technology (WO2007144444-A1). 2. Experimental 2.1. Active paper manufacture
3
Sheets of active paper were provided by Rylesa-Repsol (Madrid, Spain). Active paraffin formulations (based on solid and emulsion paraffin), containing the same amount of cinnamon essential oil, were prepared in a joint project with the University Research Group GUIA (University of Zaragoza, Spain). This methodology is protected by PCT patent WO2007144444-A1. For the solid paraffin, manufacturing involved mixing at about 110 ºC for 5 minutes. For the paraffin emulsions the manufacturing process did not include heating. Repsol (Spain) applied the active coatings to Kraft paper [100 g/m2] at a pilot plant scale. Grammage was controlled by weight. 2.2. Sample preparation Macroperforated trays were used to avoid condensation inside the packaging. There were 20 macroperforations in each tray, eight at the bottom (19mm x 5mm) and 12 in the lid (6 mm x 9 mm). Two different coverings of the macroperforated trays were studied: a) type one, in which only the bottom was covered with active paper and b) type two in which both the bottom and the walls were covered with paper. Paper coated by the same paraffin formula but without cinnamon essential oil were used as blanks. Three trays of each type of material containing 12 mushrooms each were monitored. Samples were maintained at 8ºC during the assays. 2.3. Quality assessment Weight loss in the mushrooms was monitored before treatment and during storage to evaluate the effect of active packaging. Loss of water with time was gravimetrically assessed. Active and control trays containing the mushrooms were weighed at specific time intervals (daily for the first five days of storage and every two days afterwards). Three trays for each condition were tested. Cumulative weight losses were expressed as
4
percentage loss of the initial weight. Browning of the mushrooms was evaluated by taking daily images of each tray, always under the same light. 2.4. Antioxidant capacity Free radical-scavenging activity of active papers was measured as follows: A 60 mg/L solution of DPPH (1,1-diphenyl-2-picrylhydrazyl) in methanol was prepared. A square of 2x2 cm of active paper was introduced in 20 mL of the DPPH solution in 30 mL vials. The vials were kept in the dark to avoid light interaction. Decrease in absorbance at 515 nm was measured in a UV-Vis spectrometer (UV-1700 PharmaSpec, Shimadzu). A sample with just DPPH solution was monitored as a control and a blank of paraffin paper without active components was also measured. For the antioxidant activity of cinnamon essential oil and its main components 5mL of DPPH solution in methanol (100 mg/L) was introduced in 20 mL vials and 100 µl of different concentrations of the components were added. Absorbance at 515 nm was measured at different reaction times. All samples and measurements were made in triplicate. Standard deviations were below 5% in all cases. Percentage of DPPH inhibition was calculated using the following equation: (DPPH_concentrationcontrol – DPPH_concentrationsample)*100/DPPH concentrationcontrol. 2.5. Release of volatile compounds from active papers Analysis of cinnamaldehyde release was carried out by headspace-solid phase microextraction – gas chromatography – mass spectrometry (HS-SPME-GC-MS). A SPME fiber coated with polydimethylsiloxane (PDMS) (Supelco, Bellefonte, PA, USA) with 100µm thickness was used. A 3 x 3 cm section of the paper was placed into a 20mL vial and the vial was sealed. Samples were pre-heated for 5 minutes at 40ºC and then the fiber was exposed to the headspace for 5 minutes at 40 ºC. Desorption was carried out in the gas chromatograph injection port for 2 minutes. An HP 6890N gas
5
chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to a 5975-B MS detector was used. A BPX5 capillary column (30m×0.25mm×0.25µm, SGE) was used with the following temperature program: at 45 ºC for 1 minute, 2 ºCmin−1 up to 85 ºC, then heated to 170 ºC at 5 ºCmin−1 and finally heated to 200 ºC at 15 ºCmin−1 and maintained at 200ºC for 2 minutes. Helium was the carrier gas at a constant flow rate of 1.0mLmin−1 from Carburos Metálicos (Zaragoza, Spain). Injection was carried out at 250 ◦C in splitless mode. Interface temperature was 280 ◦C. The mass spectrometer was operated in electronic impact mode (70 eV) and masses were scanned over an m/z range of 55–400 m/z. Compounds were identified, by matching their mass spectra with the US National Institute of Standards and Technology (Gaithersburg, MD, USA) commercial library. Retention time and fragmentation spectra of pure standards were obtained for confirmation. Verbenone was used as an internal standard because of its similarity to the phenolic compounds of interest, specifically retention time and chromatographic behavior. It was dissolved in acetone to a final concentration of 2460 µg/g and 25 µL were added to the vial in which the active papers were introduced, so that the final concentration in the headspace was such that the area for the verbenone peak was similar to the area of the cinnamaldehyde peak in the middle of the calibration curve. 2.6. Enzyme assay Tyrosinase catalyzes the reaction between two substrates, a phenolic compound and oxygen. The enzyme activity was monitored by dopachrome formation at 475 nm (Jimenez et al., 2001) accompanying the oxidation of the substrate L-DOPA. The reaction media (3 mL) for o-diphenolase activity contained 0.5 mmol/L L-DOPA, the indicated concentration of inhibitor and 0.67% DMSO. The final concentration of mushroom tyrosinase was 6.67 µg/mL. In this method, 2.96 mL substrate solution in sodium phosphate buffer was mixed with 20 µL of different concentrations of inhibitor 6
dissolved in DMSO at 30ºC for 5 minutes. Then, 20 µL of the aqueous solution of mushroom tyrosinase were added and the solution was immediately monitored for 5 minutes (after a lag period of 5 seconds) for the formation of dopachrome by measuring the linear increase in absorbance at 475 nm. The reaction was carried out at a constant temperature of 30ºC in a UV-Vis spectrometer (UV-1700 PharmaSpec, Shimadzu). 3. Results and discussion 3.1. Antioxidant evaluation and shelf life First of all, volatile compounds present in the cinnamon essential oil used for the preparation of the active papers were analyzed by HSPME-GC-MS. Numerous compounds, including antioxidant compounds, were detected, as shown in Figure 1. In addition to cinnamaldehyde (94.65%), D-Limonene (3.55%) and eugenol (1.8%) were detected by this method. D-Limonene is a monoterpene present in citrus fruit and its antioxidant activity has been described by several authors (Malhotra, Suri & Tuli, 2009; Moon & Shibamoto, 2009). Gülçin (2011) and Pezo et al. (2007) studied the antioxidant activity of eugenol, which is a major phenolic component from clove essential oil. Release of cinnamaldehyde from the two active papers, based on emulsion and solid paraffin, was measured by HSPME-GC-MS during the nine days of experiment. For this purpose, the bottoms of three macroperforated trays were covered with each kind of active paper. 2 x 2 cm subsamples were taken from each tray at different time intervals. The results obtained are shown in Figure 2. As can be seen, cinnamaldehyde concentration in the vial headspace was higher for the emulsion than for the solid. This performance is in agreement with the radical scavenging activity and with the quality assessment of the mushrooms, which is shown below. The amount of cinnamaldehyde in the headspace was similar for the nine days of experiment; papers did not lose significant activity during this time. The amount of cinnamaldehyde released from the
7
emulsion ranged between 12 and 18 µg/mL, and around 4 µg/mL was released from the solid paraffin; no peak corresponding to eugenol was detected from either. Both papers have the same amount of cinnamon essential oil in the formulation (6% of the paraffin in weight) so it seems that in the formulation prepared as an aqueous emulsion, cinnamaldehyde was released more readily than from solid paraffin, as might be expected. This behavior may be due to the lower solubility of cinnamon in the aqueous emulsion than in the solid paraffin. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the two active papers (emulsion and paraffin based), expressed as percentage of DPPH inhibition, are given in Figure 3. As can be concluded from the DPPH experiments, both active papers presented antioxidant capacity. At the beginning of the experiment DPPH inhibition was similar for both active papers. With time, active paper based on emulsion paraffin exhibited a higher increase in percentage of DPPH inhibition until the difference stabilized at around 6% greater for the emulsion, which was maintained for more than a day. Paraffin paper without cinnamon essential oil also exhibited antioxidant activity (Figure 3), but to a much lesser extent. This is due to the fact that the lignin phenolic compounds present in the kraft paper have also antioxidant activity (Faustino, Bautista & Duarte, 2010; Perez-Perez & Rodríguez-Malaver, 2005). For both active and non-active papers, paraffin is applied on one side (the one in contact with mushrooms) and the other remains uncovered. There is a direct relationship between DPPH inhibition and the concentrations of cinnamon essential oil and its components, as shown in Figure 4. In Figure 4 DPPH inhibition after four hours, at different concentrations of active component, are shown for essential oil, cinnamaldehyde and limonene, while for eugenol it is shown only after
8
five minutes and at lower concentrations due to its faster reaction with DPPH. The scavenging activity of eugenol increased with increasing concentrations up to 200 µg/g and then leveled off with further increase in concentration. The correlation coefficients of the regression lines at these reaction times were 0.99 for the essential oil, 0.97 for eugenol in the linear region of the curve, 0.95 for cinnamaldehyde and 0.83 for limonene. The EC50 values for these substances were 46.22 µg/mL for eugenol at 5 minutes and 3606 µg/mL for cinnamaldehyde and 3480 µg/mL for limonene at 24 hours. These results suggest most of the radical scavenging activity of cinnamon essential oil comes from eugenol, as already described by Mathew & Abraham (2006). However, as no peak corresponding to eugenol was detected in the headspace, the radical scavenging activity of the papers can be ascribed to cinnamaldehyde. That said, some direct radical scavenging activity may be due to eugenol where the papers came into contact with the mushrooms. From a visual inspection of the trays, browning of the mushrooms was observed from day one of the experiment. This is probably due to the amount of oxygen entering the packaging through the macroperforated trays that the cinnamaldehyde is unable to counteract completely. It is worth noting that this kind of trays is not the most appropriate for mushrooms commercialization but they were selected to avoid condensation and accurately measure weight loss in samples. In addition, due to the number of mushrooms needed for the replicates, only one layer of mushrooms was introduced to each tray, which left considerable free space exposing the mushrooms to a large volume of air. The key point of this experiment is the radical scavenging properties, which facilitate antioxidant protection of the mushrooms, even in the presence of oxygen. This antioxidant concept was first proposed by Nerín et al. (Nerín, 2012; Pezo, Salafranca & Nerín, 2006; Nerín, Tovar & Salafranca, 2008; Montero-
9
Prado, Rodríguez-Lafuente & Nerín, 2011; and Colón & Nerín, 2012) and explains how radical scavenging materials can act as antioxidant in the presence of molecular oxygen. First and last day appearance of mushrooms are shown in Figures 5a and 5b respectively. It was evident that presence of cinnamon in the formulations inhibited both browning and loss in volume (related to weight loss) of mushrooms. Also, if we compared both active packages, mushrooms appeared whiter when emulsion paraffin was used, which is in agreement with the results measuring antioxidant activity and cinnamaldehyde release. 3.2. Enzymatic inhibition Mushrooms browning is mostly associated with the activity of tyrosinase. The kinetic behavior of mushroom tyrosinase during the oxidation of L-DOPA was studied. Under the condition employed in the present investigation, the oxidation reaction followed Michaelis-Menten kinetics. The kinetic parameters for mushroom tyrosinase obtained from a Lineweaver-Burk plot show that Km was 0.44 mmol/L and Vmax 292.7 ∆A/min. Tyrosinase inhibition of cinnamon essential oil was tested, and the results are listed in Table 1. Both cinnamaldehyde and eugenol showed dose-dependent inhibition of LDOPA oxidation; limonene did not inhibit this reaction. However, cinnamaldehyde showed increased inhibition compared with eugenol, as indicated by its lower IC50. It can also be seen from the table that the ratio of inhibition constant (KI) of eugenol against cinnamaldehyde was 3.06, indicating inhibition of mushroom tyrosinase by cinnamaldehyde is about three-fold more potent than that of eugenol alone. From these results it can be concluded that reduction in browning was due to the presence of cinnamaldehyde in the protective atmosphere provided by the cinnamon essential oil papers.
10
The inhibition kinetics of eugenol and cinnamaldehyde were also analyzed by a Lineweaver-Burk plot as shown in Figure 6. The three lines obtained from the uninhibited enzyme and from the two different concentrations of cinnamaldehyde (Figure 6.a) intersected on the horizontal axis. This result indicates that cinnamaldehyde is a non-competitive inhibitor for L-DOPA oxidation by mushroom tyrosinase. On the other hand, the results illustrated in Figure 6.b show that eugenol is a competitive inhibitor because increasing the eugenol concentration resulted in a family of lines with a common intercept on the 1/v axis but with different slopes. 3.3. Weight losses There were also significant differences in weight loss for the two active papers. At the end of the experiment (day nine) weight losses ranged from 59.82% for the emulsion without cinnamaldehyde (type one – bottom only) to 30% for the emulsion with cinnamaldehyde (type two - bottom and sides). From the results (Figure 7) we can conclude that weight loss was significantly less in the presence of the essential oil. Between active papers there was a slight difference and the performance was better (around 2% less weight loss) with emulsion paper. Also, independent of the formulation, for type two samples (bottom and sides) weight loss was less (around 10%) than for type one (bottom only). This may have arisen from decreased oxygen where perforations were covered at the bottom and sides as well as increased exposure to the essential oil due to the larger active paper surface. It should be emphasized that previous studies have shown that cinnamaldehyde also has antimicrobial properties. These properties were not specifically studied, but microbial growth was not observed during the study (nine days), which is much longer than expected for the mushrooms to be microbe-free.
11
4. Conclusions From the results obtained we can conclude that preservation of mushrooms against oxidation can be achieved through an appropriate active packaging. This work described the performance of active paper containing cinnamon essential oil specifically designed for this purpose. The active material acted as a reservoir for cinnamaldehyde subsequently released and shown to be responsible for the antioxidant behavior observed with respect to tyrosinase inhibition and, with eugenol, for radical scavenging activity. Based on these results, we conclude that the patented active paper is able to extend the shelf life of mushrooms. Acknowledgements The authors acknowledge the company Repsol (Spain) for providing the active materials and for financial help. Yolanda Echegoyen acknowledges the Spanish Ministry of Science and Technology for the concession of a Juan de la Cierva Contract. Thanks are also given to Gobierno de Aragón and Fondo Social Europeo for financial help given to GUIA group T-10. References Chang, S. T., and & Quimio, T. H. (1984). Tropical mushroom biological nature and cultivation method. The Chinese University Press, Hong Kong, 493 p. Chouliara, E., Karatapanis, A., Savvaidis, I. N., & Kontominas, M. G. (2007). Combined effect of oregano essential oil and modified atmosphere packaging on shelf life extension of fresh chicken breast meat, stored at 4 ◦C. Food Microbiology, 24, 607– 617. Colón, M., & Nerín, C. (2012). Role of catechins in the antioxidant capacity of an active film containing green tea, green coffee and grapefruit extracts. Journal of Agricultural and Food Chemistry, 60, 9842-9849.
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El-Baroty, G. S., El-Baky, H. H. A., Farag, R. S., & Saleh, M. A. (2010). Characterization of antioxidant and antimicrobial compounds of cinnamon and ginger essential oils. African Journal of Biochemistry Research , 4, 167-174. Escriche Roberto, I., Galotto López, M. J., Gómez Ariza, M. R., & Serra Belenguer, J. A. (2001). Effect of ozone treatment and storage temperature on physicochemical properties of mushrooms (Agaris bisporus). Food science and technology international, 7, 251-258 Farag, R. S., Badei, A. Z. M. A., Hewedi, F. M., El-Baroty, G. S. A. (1989). Antioxidant activity of some spice essential oils on linolenic acid oxidation in aqueous media. Journal of American Oil Chemists’ Society, 66, 792–799. Faustino, H., Gil, N., Baptista, C., Duarte, A. P. (2010). Antioxidant activity of lignin phenolic compounds extracted from kraft and sulphite black liquors. Molecules, 15, 9308-9322. Gómez-Estaca, J., Lacey, A. L. D., López-Caballero, M. E., Gómez-Guillén, M. C., & Montero, P., (2010). Biodegradable gelatin-chitosan films incorporated with essential oils as antimicrobial agents for fish preservation. Food Microbiology, 27, 889–896. Gülçin, I. (2011). Antioxidant activity of eugtenol: a structure-activity relationship study. Journal of Medicinal Food, 14, 975-985. Holley, R. A., & Patel, D., (2005). Improvement in shelf life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiology, 22, 273– 292. Jiang, T., Feng, L., & Zheng, X. (2012) Effect of chitosan coating enriched with thyme oil on postharvest quality and shelf life of shiitake mushroom (Lentinus edodes). Journal of Agricultural and Food Chemistry, 60, 188-196.
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Jimenez, M., Chazarra, S., Escribano, J., Cabanes, J., & Garcia-Carmina, F. (2001) Competitive Inhibition of Mushroom Tyrosinase by 4-Substituted Benzaldehydes. Journal of Agricultural and Food Chemistry, 49, 4060-4063. López, P., Sánchez, C., Batlle, R., & Nerín, C. (2005). Solid- and Vapor-phase antimicrobial activities of six essential oils: susceptibility of selected foodborne bacterial and fungal strains. Journal of Agricultural and Food Chemistry, 2005, 53, 6939-6946. Malhotra, S., Suri, S., & Tuli, R. (2009). Antioxidant activity of citrus cultivars and chemical composition of Citrus karna essential oil. Planta Medica, 75, 62-64. Martínez, M. V., & Whitaker, J. R. (1995). The biochemisty and control of enzymatic browning. Trends in Food Science & Technology, 6, 195–200. Mathew, S., T. Emilia & Abraham, T. E. (2006). Studies on the antioxidant activities of cinnamon (Cinnamomum verum) bark extracts, through various in vitro models. Food Chemistry, 94, 520-528. Montero-Prado, P., Rodríguez-Lafuente, A., & Nerín, C. (2011). Active label-based packaging to extend the shelf life of “Calanda” peach fruit: Changes in fruit quality and enzymatic activity. Postharvest biology and technology, 60, 211-219. Moon, J.-K., & Shibamoto, T. (2009). Antioxidant assays for plant and food components. Journal of Agricultural and Food Chemistry, 57, 1655-1666. Nerín, C. (2012). Antioxidant active packaging and antioxidant edible films. In E.A. Decker, R.J. Elias, & D.J. McClements (Eds.) Oxidation in Foods and Beverages and Antioxidant Applications, vol. 2 – Management in Different Industry Sectors, (pp. 496515). Woodhead Publishing, Cambridge, United Kingdom. Nerín, C., Tovar, L., & Salafranca, J. (2008). Behaviour of a new antioxidant active film versus oxidizable model compounds. Journal of Food Engineering, 84, 313-320.
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Nerín, C., Tovar, L., Djenane, D., Camo, J., Salafranca, J., Beltrán, J. A., & Roncalés, P. Stabilization of beef meat by a new active packaging containing natural antioxidants. Journal of Agricultural and Food Chemistry, 2006, 54, 7840-7846. Nerya, O., Ben-Arie, R., Luzzatto, T., Musa, R., Khativ, S. and & Vaya, J. (2006). Prevention of Agaricus bisporus postharvest browning with tyrosinase inhibitors. Postharvest Biology and Technology, 39, 272-277. Parke, D. V., & Lewis, D. F. V., (1992). Safety aspects of food preservatives. Food Additives and Contaminants, 9, 561–577. Perez-Perez, E., & Rodríguez-Malaver, A. J. (2005). Antioxidant capacity of Kraft black liquor from the pulp and paper industry. Journal of Environmental Biology, 26, 603-608. Pezo, D; Salafranca, J; Nerín, C. (2007). Development of an automatic multiple dynamic hollow fiber liquid-phase microextraction procedure for specific migration analysis of new active food packagings containing essential oils. Journal of Chromatography A, 1174, 85-94. Pezo, D., Salafranca, J., & Nerín, C. (2008). Determination of the antioxidant capacity of active food packagings by in situ gas-phase hydroxyl radical generation and highperformance
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Chromatography A, 1178, 126-133. Qiu, L., Chen, Q.-H., Zhuang, J.-X., Zhong, X., Zhou, J.-J., Guo, Y.-J., and & Chen, Q.X. (2009). Inhibitory effects of α-cyano-4- hydroxycinnamic acid on the activity of mushroom tyrosinase. Food Chemistry, 112, 609-613. Rodríguez, A., Nerín, C., & Battle, R. (2008). New cinnamon-based active paper packaging against Rhizopus stolonifer food spoilage. Journal of Agricultural and Food Chemistry, 2008, 56, 6364–6369.
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Whitaker, J. R., and & Lee, C. Y. (1995). Recent advances in chemistry of enzymatic browning: an overview. In C. Y. Lee, & J. R. Whitaker (Eds.). Enzymatic Browning and Its Prevention. (pp. 2–7). Washington, DC: ACS Symposium Series 600, American Chemical Society.
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Figure captions Figure 1. GC-MS chromatogram of the cinnamon essential oil used in this study. Figure 2. Cinnamaldehyde released to the vapor phase of the tray during the 9 days of experiment for the different active papers. Figure 3. DPPH inhibition for the different active and non-active papers. Figure 4. DPPH inhibition for cinnamon essential oil and its main components. Figure 5. Appearance of the mushrooms the first and last day of experiment. a) Type 1 with emulsion paraffin sample. b) Type 2 with solid paraffin sample. Figure 6. Lineweaver-Burk plots for the catalysis of L-DOPA by mushroom tyrosinase (6.67 µg/mL) at 30ºC, pH 6.8 at different inhibitor concentrations. a) cinnamaldehyde; b) eugenol. Figure 7. Evolution in weight loss (percentage) over storage time for the different samples.
17
Table 1. Inhibition of mushroom tyrosinase by the main components of cinnamon essential oil.
Compound Cinnamaldehyde Eugenol
Type of inhibition
Inhibition constant (mmol/L) 2.78 8.51
Noncompetitive Competitive
18
IC50 (mmol/L) 4.73 12.83
Figure 1. Yolanda Echegoyen
Abundance 3.6e+07 3.4e+07 3.2e+07 3e+07 2.8e+07 2.6e+07
Cinnamaldehyde
2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07
Eugenol
8000000
Limonene
6000000 4000000 2000000
Time-->
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
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28.00
30.00
32.00
34.00
36.00
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40.00
Figure 2. Yolanda Echegoyen
20 Emulsion
Cinnamaldehyde concentration (mg/ml)
18
Solid
16 14 12 10 8 6 4 2 0 0
1
2
3
4
5 Time (days)
6
7
8
9
10
Figure 3. Yolanda Echegoyen
Figure 4. Yolanda Echegoyen
Figure 5a. Yolanda Echegoyen
Figure 5b. Yolanda Echegoyen
Figure 6
Figure 7. Yolanda Echegoyen
80 70
Weight loss (%)
60
Emulsion (type 1)
Emulsion (type 2)
Emulsion+cinnamon (type 1)
Emulsion+cinnamon (type 2)
Solid (type 1)
Solid (type 2)
Solid+cinnamon (type 1)
Solid+cinnamon (type 2)
50 40 30 20 10 0 0
2
4
6 Time (days)
8
10
Highlights - The antioxidant capacity of two different active papers with cinnamon essential oil was studied. - The use of the active paper increased the shelf life of mushrooms. - Papers based on emulsion paraffin were more active against mushroom browning and weight loss. - Scavenging capacity of the DPPH radical was mainly related with eugenol. - Tyrosinase inhibition was mainly due to cinnamaldehyde release.
19
S0308-8146(14)01247-3 http://dx.doi.org/10.1016/j.foodchem.2014.08.032 FOCH 16255
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 April 2013 1 July 2014 10 August 2014
Please cite this article as: Echegoyen, Y., Nerín, C., Performance of an active paper based on cinnamon essential oil in mushrooms quality, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.08.032
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PERFORMANCE OF AN
ACTIVE
PAPER
BASED
ON
CINNAMON
ESSENTIAL OIL IN MUSHROOMS QUALITY. Y. Echegoyen, C. Nerín∗ I3A, Department of Analytical Chemistry, University of Zaragoza, C/ María de Luna 3, 50018, Zaragoza, Spain Abstract The antioxidant capacity of two active papers (based on solid and emulsion paraffin) with cinnamon essential oil was studied. Mushroom samples were introduced in macroperforated PET trays covered with the active papers, and weight loss and browning monitored for nine days. The antioxidant capacity of the different papers was evaluated based on scavenging 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and tyrosinase inhibition kinetics, and the release of aromatic volatile oils was determined by HSPMEGC-MS. Differences in performance were observed: the active papers were more efficient at avoiding weight loss and mushroom browning when compared to the nonactive paraffin-based papers. The efficiency increased when the bottom and walls of the trays were covered rather than the bottom alone. Better results were observed when cinnamon was incorporated as emulsion paraffin instead of a solid. Keywords: mushroom quality, active packaging, cinnamon essential oil, antioxidant capacity. 1. Introduction Half of the world’s fruit and vegetable crops are lost due to postharvest deterioration. Browning disorders in agricultural products are normally induced by dehydration, mechanical injury or wound deterioration (Whitaker & Lee, 1995). Polyphenol oxidase
∗
To whom correspondence should be addressed: [email protected]; Tlf: +34976761873; Fax: +34976762388; I3A, Department of Analytical Chemistry, University of Zaragoza, C/ María de Luna 3, 50018, Zaragoza, Spain.
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(PPO) is the major enzyme involved in browning reactions. PPO catalyzes the oxidation of phenolic compounds to o-quinones, resulting in brown pigmentation (Chang & Quimio 1984). Most research on browning of white button mushroom (Agaricus bisporus) has associated browning with PPO or related enzymes, specifically tyrosinase, an enzyme belonging to the PPO family (Nerya, Ben-Arie, Luzzatto, Musa, Khativ & Vaya, 2006; Qiu et al., 2009). Cool storage conditions slow the rate of deterioration (Escriche-Roberto, Galotto-López, Gómez-Ariza & Serra-Belenguer, 2001), but temperatures that are too low may cause chilling injury. The most important factors determining the rate of enzymatic browning of fruit and vegetables are the concentrations of active PPO and phenolic compounds, pH, temperature and oxygen availability of the tissue (Martínez & Whitaker, 1995). Extending the shelf life of mushrooms is difficult for a number of reasons: it is a delicate fresh product and the handling can seriously damage the tissue, facilitating the appearance of black or brown spots, which decrease their value. Mushrooms have a high respiration rate and produce a large amount of water vapor, which increases the decay of mushrooms in closed packaging systems. Mushrooms are fungi and, therefore, naturally have a short shelf life. The initial problem is the browning, more evident in the gills or lamella than in the cap. As fresh products, mushrooms cannot be treated with antioxidants to protect them against oxidation, active packaging is a smart solution to extend their shelf life. The use of essential oils to extend the shelf life is becoming popular since consumers have become more conscious about potential health problems associated with synthetic preservatives (Gómez-Estaca, Lacey, López-Caballero & Gómez-Guillén, 2010; Holley & Patel, 2005). Synthetic preservatives, added to food items as antimicrobials and/or antioxidants, are generally-recognized-as-safe and, usually, have a beneficial effect on
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food preservation. However, there have been problems concerning the safety of some chemicals, including potential allergens (e.g. benzoic acid and sulphites) and the formation of carcinogenic compounds such as nitrosamines from nitrites, and the possible
rodent
carcinogenicity
of
butylated
hydroxyanisole
and
butylated
hydroxytoluene (Parke & Lewis, 1992). Previous studies with essential oils as food additives have revealed them to be advantageous, as observed by an increase in food shelf life (Chouliara, Karatapanis, Savvaidis & Kontominas, 2007; Jiang, Feng & Zheng, 2012). The amount of essential oils, however, determined the acceptability of products since strong aromas might be imparted to the product. One alternative to the direct incorporation of essential oils in foodstuffs is active packaging where volatile compounds in the essential oil create a protective atmosphere, either antimicrobial or antioxidant (Nerín et al., 2006). Cinnamon essential oil is well known for its antimicrobial activity (López, Sánchez, Batlle & Nerín, 2005; Rodríguez, Nerín & Batlle, 2008). It has also been reported to have antioxidant activity, derived mostly from its phenolic components (El-Baroty, ElBaky, Farag & Saleh, 2010). As for many other free radicals, ·OH can be neutralized if a hydrogen atom is provided. The scavenging activities of phenolic substances may be due to hydrogen donation during hydroxyl substitutions (Mathew & Abraham, 2006). For this reason, antioxidant behavior is associated with phenolic compounds. The aim of this work was to determine the performance of two active papers containing cinnamon essential oil as an active agent to delay postharvest deterioration in mushrooms (Agaris bisporus). Repsol (Spain) provided the active material using a patented technology (WO2007144444-A1). 2. Experimental 2.1. Active paper manufacture
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Sheets of active paper were provided by Rylesa-Repsol (Madrid, Spain). Active paraffin formulations (based on solid and emulsion paraffin), containing the same amount of cinnamon essential oil, were prepared in a joint project with the University Research Group GUIA (University of Zaragoza, Spain). This methodology is protected by PCT patent WO2007144444-A1. For the solid paraffin, manufacturing involved mixing at about 110 ºC for 5 minutes. For the paraffin emulsions the manufacturing process did not include heating. Repsol (Spain) applied the active coatings to Kraft paper [100 g/m2] at a pilot plant scale. Grammage was controlled by weight. 2.2. Sample preparation Macroperforated trays were used to avoid condensation inside the packaging. There were 20 macroperforations in each tray, eight at the bottom (19mm x 5mm) and 12 in the lid (6 mm x 9 mm). Two different coverings of the macroperforated trays were studied: a) type one, in which only the bottom was covered with active paper and b) type two in which both the bottom and the walls were covered with paper. Paper coated by the same paraffin formula but without cinnamon essential oil were used as blanks. Three trays of each type of material containing 12 mushrooms each were monitored. Samples were maintained at 8ºC during the assays. 2.3. Quality assessment Weight loss in the mushrooms was monitored before treatment and during storage to evaluate the effect of active packaging. Loss of water with time was gravimetrically assessed. Active and control trays containing the mushrooms were weighed at specific time intervals (daily for the first five days of storage and every two days afterwards). Three trays for each condition were tested. Cumulative weight losses were expressed as
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percentage loss of the initial weight. Browning of the mushrooms was evaluated by taking daily images of each tray, always under the same light. 2.4. Antioxidant capacity Free radical-scavenging activity of active papers was measured as follows: A 60 mg/L solution of DPPH (1,1-diphenyl-2-picrylhydrazyl) in methanol was prepared. A square of 2x2 cm of active paper was introduced in 20 mL of the DPPH solution in 30 mL vials. The vials were kept in the dark to avoid light interaction. Decrease in absorbance at 515 nm was measured in a UV-Vis spectrometer (UV-1700 PharmaSpec, Shimadzu). A sample with just DPPH solution was monitored as a control and a blank of paraffin paper without active components was also measured. For the antioxidant activity of cinnamon essential oil and its main components 5mL of DPPH solution in methanol (100 mg/L) was introduced in 20 mL vials and 100 µl of different concentrations of the components were added. Absorbance at 515 nm was measured at different reaction times. All samples and measurements were made in triplicate. Standard deviations were below 5% in all cases. Percentage of DPPH inhibition was calculated using the following equation: (DPPH_concentrationcontrol – DPPH_concentrationsample)*100/DPPH concentrationcontrol. 2.5. Release of volatile compounds from active papers Analysis of cinnamaldehyde release was carried out by headspace-solid phase microextraction – gas chromatography – mass spectrometry (HS-SPME-GC-MS). A SPME fiber coated with polydimethylsiloxane (PDMS) (Supelco, Bellefonte, PA, USA) with 100µm thickness was used. A 3 x 3 cm section of the paper was placed into a 20mL vial and the vial was sealed. Samples were pre-heated for 5 minutes at 40ºC and then the fiber was exposed to the headspace for 5 minutes at 40 ºC. Desorption was carried out in the gas chromatograph injection port for 2 minutes. An HP 6890N gas
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chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to a 5975-B MS detector was used. A BPX5 capillary column (30m×0.25mm×0.25µm, SGE) was used with the following temperature program: at 45 ºC for 1 minute, 2 ºCmin−1 up to 85 ºC, then heated to 170 ºC at 5 ºCmin−1 and finally heated to 200 ºC at 15 ºCmin−1 and maintained at 200ºC for 2 minutes. Helium was the carrier gas at a constant flow rate of 1.0mLmin−1 from Carburos Metálicos (Zaragoza, Spain). Injection was carried out at 250 ◦C in splitless mode. Interface temperature was 280 ◦C. The mass spectrometer was operated in electronic impact mode (70 eV) and masses were scanned over an m/z range of 55–400 m/z. Compounds were identified, by matching their mass spectra with the US National Institute of Standards and Technology (Gaithersburg, MD, USA) commercial library. Retention time and fragmentation spectra of pure standards were obtained for confirmation. Verbenone was used as an internal standard because of its similarity to the phenolic compounds of interest, specifically retention time and chromatographic behavior. It was dissolved in acetone to a final concentration of 2460 µg/g and 25 µL were added to the vial in which the active papers were introduced, so that the final concentration in the headspace was such that the area for the verbenone peak was similar to the area of the cinnamaldehyde peak in the middle of the calibration curve. 2.6. Enzyme assay Tyrosinase catalyzes the reaction between two substrates, a phenolic compound and oxygen. The enzyme activity was monitored by dopachrome formation at 475 nm (Jimenez et al., 2001) accompanying the oxidation of the substrate L-DOPA. The reaction media (3 mL) for o-diphenolase activity contained 0.5 mmol/L L-DOPA, the indicated concentration of inhibitor and 0.67% DMSO. The final concentration of mushroom tyrosinase was 6.67 µg/mL. In this method, 2.96 mL substrate solution in sodium phosphate buffer was mixed with 20 µL of different concentrations of inhibitor 6
dissolved in DMSO at 30ºC for 5 minutes. Then, 20 µL of the aqueous solution of mushroom tyrosinase were added and the solution was immediately monitored for 5 minutes (after a lag period of 5 seconds) for the formation of dopachrome by measuring the linear increase in absorbance at 475 nm. The reaction was carried out at a constant temperature of 30ºC in a UV-Vis spectrometer (UV-1700 PharmaSpec, Shimadzu). 3. Results and discussion 3.1. Antioxidant evaluation and shelf life First of all, volatile compounds present in the cinnamon essential oil used for the preparation of the active papers were analyzed by HSPME-GC-MS. Numerous compounds, including antioxidant compounds, were detected, as shown in Figure 1. In addition to cinnamaldehyde (94.65%), D-Limonene (3.55%) and eugenol (1.8%) were detected by this method. D-Limonene is a monoterpene present in citrus fruit and its antioxidant activity has been described by several authors (Malhotra, Suri & Tuli, 2009; Moon & Shibamoto, 2009). Gülçin (2011) and Pezo et al. (2007) studied the antioxidant activity of eugenol, which is a major phenolic component from clove essential oil. Release of cinnamaldehyde from the two active papers, based on emulsion and solid paraffin, was measured by HSPME-GC-MS during the nine days of experiment. For this purpose, the bottoms of three macroperforated trays were covered with each kind of active paper. 2 x 2 cm subsamples were taken from each tray at different time intervals. The results obtained are shown in Figure 2. As can be seen, cinnamaldehyde concentration in the vial headspace was higher for the emulsion than for the solid. This performance is in agreement with the radical scavenging activity and with the quality assessment of the mushrooms, which is shown below. The amount of cinnamaldehyde in the headspace was similar for the nine days of experiment; papers did not lose significant activity during this time. The amount of cinnamaldehyde released from the
7
emulsion ranged between 12 and 18 µg/mL, and around 4 µg/mL was released from the solid paraffin; no peak corresponding to eugenol was detected from either. Both papers have the same amount of cinnamon essential oil in the formulation (6% of the paraffin in weight) so it seems that in the formulation prepared as an aqueous emulsion, cinnamaldehyde was released more readily than from solid paraffin, as might be expected. This behavior may be due to the lower solubility of cinnamon in the aqueous emulsion than in the solid paraffin. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the two active papers (emulsion and paraffin based), expressed as percentage of DPPH inhibition, are given in Figure 3. As can be concluded from the DPPH experiments, both active papers presented antioxidant capacity. At the beginning of the experiment DPPH inhibition was similar for both active papers. With time, active paper based on emulsion paraffin exhibited a higher increase in percentage of DPPH inhibition until the difference stabilized at around 6% greater for the emulsion, which was maintained for more than a day. Paraffin paper without cinnamon essential oil also exhibited antioxidant activity (Figure 3), but to a much lesser extent. This is due to the fact that the lignin phenolic compounds present in the kraft paper have also antioxidant activity (Faustino, Bautista & Duarte, 2010; Perez-Perez & Rodríguez-Malaver, 2005). For both active and non-active papers, paraffin is applied on one side (the one in contact with mushrooms) and the other remains uncovered. There is a direct relationship between DPPH inhibition and the concentrations of cinnamon essential oil and its components, as shown in Figure 4. In Figure 4 DPPH inhibition after four hours, at different concentrations of active component, are shown for essential oil, cinnamaldehyde and limonene, while for eugenol it is shown only after
8
five minutes and at lower concentrations due to its faster reaction with DPPH. The scavenging activity of eugenol increased with increasing concentrations up to 200 µg/g and then leveled off with further increase in concentration. The correlation coefficients of the regression lines at these reaction times were 0.99 for the essential oil, 0.97 for eugenol in the linear region of the curve, 0.95 for cinnamaldehyde and 0.83 for limonene. The EC50 values for these substances were 46.22 µg/mL for eugenol at 5 minutes and 3606 µg/mL for cinnamaldehyde and 3480 µg/mL for limonene at 24 hours. These results suggest most of the radical scavenging activity of cinnamon essential oil comes from eugenol, as already described by Mathew & Abraham (2006). However, as no peak corresponding to eugenol was detected in the headspace, the radical scavenging activity of the papers can be ascribed to cinnamaldehyde. That said, some direct radical scavenging activity may be due to eugenol where the papers came into contact with the mushrooms. From a visual inspection of the trays, browning of the mushrooms was observed from day one of the experiment. This is probably due to the amount of oxygen entering the packaging through the macroperforated trays that the cinnamaldehyde is unable to counteract completely. It is worth noting that this kind of trays is not the most appropriate for mushrooms commercialization but they were selected to avoid condensation and accurately measure weight loss in samples. In addition, due to the number of mushrooms needed for the replicates, only one layer of mushrooms was introduced to each tray, which left considerable free space exposing the mushrooms to a large volume of air. The key point of this experiment is the radical scavenging properties, which facilitate antioxidant protection of the mushrooms, even in the presence of oxygen. This antioxidant concept was first proposed by Nerín et al. (Nerín, 2012; Pezo, Salafranca & Nerín, 2006; Nerín, Tovar & Salafranca, 2008; Montero-
9
Prado, Rodríguez-Lafuente & Nerín, 2011; and Colón & Nerín, 2012) and explains how radical scavenging materials can act as antioxidant in the presence of molecular oxygen. First and last day appearance of mushrooms are shown in Figures 5a and 5b respectively. It was evident that presence of cinnamon in the formulations inhibited both browning and loss in volume (related to weight loss) of mushrooms. Also, if we compared both active packages, mushrooms appeared whiter when emulsion paraffin was used, which is in agreement with the results measuring antioxidant activity and cinnamaldehyde release. 3.2. Enzymatic inhibition Mushrooms browning is mostly associated with the activity of tyrosinase. The kinetic behavior of mushroom tyrosinase during the oxidation of L-DOPA was studied. Under the condition employed in the present investigation, the oxidation reaction followed Michaelis-Menten kinetics. The kinetic parameters for mushroom tyrosinase obtained from a Lineweaver-Burk plot show that Km was 0.44 mmol/L and Vmax 292.7 ∆A/min. Tyrosinase inhibition of cinnamon essential oil was tested, and the results are listed in Table 1. Both cinnamaldehyde and eugenol showed dose-dependent inhibition of LDOPA oxidation; limonene did not inhibit this reaction. However, cinnamaldehyde showed increased inhibition compared with eugenol, as indicated by its lower IC50. It can also be seen from the table that the ratio of inhibition constant (KI) of eugenol against cinnamaldehyde was 3.06, indicating inhibition of mushroom tyrosinase by cinnamaldehyde is about three-fold more potent than that of eugenol alone. From these results it can be concluded that reduction in browning was due to the presence of cinnamaldehyde in the protective atmosphere provided by the cinnamon essential oil papers.
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The inhibition kinetics of eugenol and cinnamaldehyde were also analyzed by a Lineweaver-Burk plot as shown in Figure 6. The three lines obtained from the uninhibited enzyme and from the two different concentrations of cinnamaldehyde (Figure 6.a) intersected on the horizontal axis. This result indicates that cinnamaldehyde is a non-competitive inhibitor for L-DOPA oxidation by mushroom tyrosinase. On the other hand, the results illustrated in Figure 6.b show that eugenol is a competitive inhibitor because increasing the eugenol concentration resulted in a family of lines with a common intercept on the 1/v axis but with different slopes. 3.3. Weight losses There were also significant differences in weight loss for the two active papers. At the end of the experiment (day nine) weight losses ranged from 59.82% for the emulsion without cinnamaldehyde (type one – bottom only) to 30% for the emulsion with cinnamaldehyde (type two - bottom and sides). From the results (Figure 7) we can conclude that weight loss was significantly less in the presence of the essential oil. Between active papers there was a slight difference and the performance was better (around 2% less weight loss) with emulsion paper. Also, independent of the formulation, for type two samples (bottom and sides) weight loss was less (around 10%) than for type one (bottom only). This may have arisen from decreased oxygen where perforations were covered at the bottom and sides as well as increased exposure to the essential oil due to the larger active paper surface. It should be emphasized that previous studies have shown that cinnamaldehyde also has antimicrobial properties. These properties were not specifically studied, but microbial growth was not observed during the study (nine days), which is much longer than expected for the mushrooms to be microbe-free.
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4. Conclusions From the results obtained we can conclude that preservation of mushrooms against oxidation can be achieved through an appropriate active packaging. This work described the performance of active paper containing cinnamon essential oil specifically designed for this purpose. The active material acted as a reservoir for cinnamaldehyde subsequently released and shown to be responsible for the antioxidant behavior observed with respect to tyrosinase inhibition and, with eugenol, for radical scavenging activity. Based on these results, we conclude that the patented active paper is able to extend the shelf life of mushrooms. Acknowledgements The authors acknowledge the company Repsol (Spain) for providing the active materials and for financial help. Yolanda Echegoyen acknowledges the Spanish Ministry of Science and Technology for the concession of a Juan de la Cierva Contract. Thanks are also given to Gobierno de Aragón and Fondo Social Europeo for financial help given to GUIA group T-10. References Chang, S. T., and & Quimio, T. H. (1984). Tropical mushroom biological nature and cultivation method. The Chinese University Press, Hong Kong, 493 p. Chouliara, E., Karatapanis, A., Savvaidis, I. N., & Kontominas, M. G. (2007). Combined effect of oregano essential oil and modified atmosphere packaging on shelf life extension of fresh chicken breast meat, stored at 4 ◦C. Food Microbiology, 24, 607– 617. Colón, M., & Nerín, C. (2012). Role of catechins in the antioxidant capacity of an active film containing green tea, green coffee and grapefruit extracts. Journal of Agricultural and Food Chemistry, 60, 9842-9849.
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El-Baroty, G. S., El-Baky, H. H. A., Farag, R. S., & Saleh, M. A. (2010). Characterization of antioxidant and antimicrobial compounds of cinnamon and ginger essential oils. African Journal of Biochemistry Research , 4, 167-174. Escriche Roberto, I., Galotto López, M. J., Gómez Ariza, M. R., & Serra Belenguer, J. A. (2001). Effect of ozone treatment and storage temperature on physicochemical properties of mushrooms (Agaris bisporus). Food science and technology international, 7, 251-258 Farag, R. S., Badei, A. Z. M. A., Hewedi, F. M., El-Baroty, G. S. A. (1989). Antioxidant activity of some spice essential oils on linolenic acid oxidation in aqueous media. Journal of American Oil Chemists’ Society, 66, 792–799. Faustino, H., Gil, N., Baptista, C., Duarte, A. P. (2010). Antioxidant activity of lignin phenolic compounds extracted from kraft and sulphite black liquors. Molecules, 15, 9308-9322. Gómez-Estaca, J., Lacey, A. L. D., López-Caballero, M. E., Gómez-Guillén, M. C., & Montero, P., (2010). Biodegradable gelatin-chitosan films incorporated with essential oils as antimicrobial agents for fish preservation. Food Microbiology, 27, 889–896. Gülçin, I. (2011). Antioxidant activity of eugtenol: a structure-activity relationship study. Journal of Medicinal Food, 14, 975-985. Holley, R. A., & Patel, D., (2005). Improvement in shelf life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiology, 22, 273– 292. Jiang, T., Feng, L., & Zheng, X. (2012) Effect of chitosan coating enriched with thyme oil on postharvest quality and shelf life of shiitake mushroom (Lentinus edodes). Journal of Agricultural and Food Chemistry, 60, 188-196.
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Jimenez, M., Chazarra, S., Escribano, J., Cabanes, J., & Garcia-Carmina, F. (2001) Competitive Inhibition of Mushroom Tyrosinase by 4-Substituted Benzaldehydes. Journal of Agricultural and Food Chemistry, 49, 4060-4063. López, P., Sánchez, C., Batlle, R., & Nerín, C. (2005). Solid- and Vapor-phase antimicrobial activities of six essential oils: susceptibility of selected foodborne bacterial and fungal strains. Journal of Agricultural and Food Chemistry, 2005, 53, 6939-6946. Malhotra, S., Suri, S., & Tuli, R. (2009). Antioxidant activity of citrus cultivars and chemical composition of Citrus karna essential oil. Planta Medica, 75, 62-64. Martínez, M. V., & Whitaker, J. R. (1995). The biochemisty and control of enzymatic browning. Trends in Food Science & Technology, 6, 195–200. Mathew, S., T. Emilia & Abraham, T. E. (2006). Studies on the antioxidant activities of cinnamon (Cinnamomum verum) bark extracts, through various in vitro models. Food Chemistry, 94, 520-528. Montero-Prado, P., Rodríguez-Lafuente, A., & Nerín, C. (2011). Active label-based packaging to extend the shelf life of “Calanda” peach fruit: Changes in fruit quality and enzymatic activity. Postharvest biology and technology, 60, 211-219. Moon, J.-K., & Shibamoto, T. (2009). Antioxidant assays for plant and food components. Journal of Agricultural and Food Chemistry, 57, 1655-1666. Nerín, C. (2012). Antioxidant active packaging and antioxidant edible films. In E.A. Decker, R.J. Elias, & D.J. McClements (Eds.) Oxidation in Foods and Beverages and Antioxidant Applications, vol. 2 – Management in Different Industry Sectors, (pp. 496515). Woodhead Publishing, Cambridge, United Kingdom. Nerín, C., Tovar, L., & Salafranca, J. (2008). Behaviour of a new antioxidant active film versus oxidizable model compounds. Journal of Food Engineering, 84, 313-320.
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Nerín, C., Tovar, L., Djenane, D., Camo, J., Salafranca, J., Beltrán, J. A., & Roncalés, P. Stabilization of beef meat by a new active packaging containing natural antioxidants. Journal of Agricultural and Food Chemistry, 2006, 54, 7840-7846. Nerya, O., Ben-Arie, R., Luzzatto, T., Musa, R., Khativ, S. and & Vaya, J. (2006). Prevention of Agaricus bisporus postharvest browning with tyrosinase inhibitors. Postharvest Biology and Technology, 39, 272-277. Parke, D. V., & Lewis, D. F. V., (1992). Safety aspects of food preservatives. Food Additives and Contaminants, 9, 561–577. Perez-Perez, E., & Rodríguez-Malaver, A. J. (2005). Antioxidant capacity of Kraft black liquor from the pulp and paper industry. Journal of Environmental Biology, 26, 603-608. Pezo, D; Salafranca, J; Nerín, C. (2007). Development of an automatic multiple dynamic hollow fiber liquid-phase microextraction procedure for specific migration analysis of new active food packagings containing essential oils. Journal of Chromatography A, 1174, 85-94. Pezo, D., Salafranca, J., & Nerín, C. (2008). Determination of the antioxidant capacity of active food packagings by in situ gas-phase hydroxyl radical generation and highperformance
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Chromatography A, 1178, 126-133. Qiu, L., Chen, Q.-H., Zhuang, J.-X., Zhong, X., Zhou, J.-J., Guo, Y.-J., and & Chen, Q.X. (2009). Inhibitory effects of α-cyano-4- hydroxycinnamic acid on the activity of mushroom tyrosinase. Food Chemistry, 112, 609-613. Rodríguez, A., Nerín, C., & Battle, R. (2008). New cinnamon-based active paper packaging against Rhizopus stolonifer food spoilage. Journal of Agricultural and Food Chemistry, 2008, 56, 6364–6369.
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Whitaker, J. R., and & Lee, C. Y. (1995). Recent advances in chemistry of enzymatic browning: an overview. In C. Y. Lee, & J. R. Whitaker (Eds.). Enzymatic Browning and Its Prevention. (pp. 2–7). Washington, DC: ACS Symposium Series 600, American Chemical Society.
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Figure captions Figure 1. GC-MS chromatogram of the cinnamon essential oil used in this study. Figure 2. Cinnamaldehyde released to the vapor phase of the tray during the 9 days of experiment for the different active papers. Figure 3. DPPH inhibition for the different active and non-active papers. Figure 4. DPPH inhibition for cinnamon essential oil and its main components. Figure 5. Appearance of the mushrooms the first and last day of experiment. a) Type 1 with emulsion paraffin sample. b) Type 2 with solid paraffin sample. Figure 6. Lineweaver-Burk plots for the catalysis of L-DOPA by mushroom tyrosinase (6.67 µg/mL) at 30ºC, pH 6.8 at different inhibitor concentrations. a) cinnamaldehyde; b) eugenol. Figure 7. Evolution in weight loss (percentage) over storage time for the different samples.
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Table 1. Inhibition of mushroom tyrosinase by the main components of cinnamon essential oil.
Compound Cinnamaldehyde Eugenol
Type of inhibition
Inhibition constant (mmol/L) 2.78 8.51
Noncompetitive Competitive
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IC50 (mmol/L) 4.73 12.83
Figure 1. Yolanda Echegoyen
Abundance 3.6e+07 3.4e+07 3.2e+07 3e+07 2.8e+07 2.6e+07
Cinnamaldehyde
2.4e+07 2.2e+07 2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07 1e+07
Eugenol
8000000
Limonene
6000000 4000000 2000000
Time-->
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
Figure 2. Yolanda Echegoyen
20 Emulsion
Cinnamaldehyde concentration (mg/ml)
18
Solid
16 14 12 10 8 6 4 2 0 0
1
2
3
4
5 Time (days)
6
7
8
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10
Figure 3. Yolanda Echegoyen
Figure 4. Yolanda Echegoyen
Figure 5a. Yolanda Echegoyen
Figure 5b. Yolanda Echegoyen
Figure 6
Figure 7. Yolanda Echegoyen
80 70
Weight loss (%)
60
Emulsion (type 1)
Emulsion (type 2)
Emulsion+cinnamon (type 1)
Emulsion+cinnamon (type 2)
Solid (type 1)
Solid (type 2)
Solid+cinnamon (type 1)
Solid+cinnamon (type 2)
50 40 30 20 10 0 0
2
4
6 Time (days)
8
10
Highlights - The antioxidant capacity of two different active papers with cinnamon essential oil was studied. - The use of the active paper increased the shelf life of mushrooms. - Papers based on emulsion paraffin were more active against mushroom browning and weight loss. - Scavenging capacity of the DPPH radical was mainly related with eugenol. - Tyrosinase inhibition was mainly due to cinnamaldehyde release.
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