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Effect of nanocomposite packaging on postharvest quality and reactive oxygen species metabolism of mushrooms (Flammulina velutipes)
Postharvest Biology and Technology 119 (2016) 49–57
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Effect of nanocomposite packaging on postharvest quality and reactive oxygen species metabolism of mushrooms (Flammulina velutipes) Fang Donglua , Yang Wenjianb , Benard Muinde Kimatua,c , An Xinxina , Hu Qiuhuia , Zhao Liyana,* a b c
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, Jiangsu 210046, PR China Department of Dairy and Food Science and Technology, Egerton University, Nakuru, Kenya
A R T I C L E I N F O
Article history: Received 21 November 2015 Received in revised form 18 April 2016 Accepted 20 April 2016 Available online xxx Keywords: Nanocomposite packaging Flammulina velutipes Preservation ROS NMR
A B S T R A C T
The aim of this study was to analyse the effect of nanocomposite packaging material (Nano-PM) on the physicochemical characteristics and antioxidant capacity of mushrooms (Flammulina velutipes) during 21 days of postharvest storage at 4 C. The results showed that Nano-PM improved retention of nutrients and inhibited the weight loss, respiration and distinct stipe elongation of mushrooms compared with the Normal packing material (Normal-PM). Reactive oxygen species (ROS) levels, including superoxide radical (O2) and hydrogen peroxide (H2O2), increased during storage. Membrane damage, as measured by relative electrolyte leakage and malondi-aldehyde (MDA) contents also increased. However, levels of O2 and H2O2, relative electrolyte leakage and MDA contents were all significantly lower (P < 0.05) in the Nano-PM group than those in the control group. Nano-PM reduced the production of free radicals which cause membrane damage and may therefore be an effective treatment to delay the postharvest growth and caducity in F. velutipes hence increasing its shelf life and at the same time preserving its quality. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Production and consumption of mushrooms have been increasing substantially in the world (Pesce et al., 2015). The golden needle mushroom, Flammulina velutipes, has delicious taste, high nutritional value, and medicinal properties (Yang et al., 2012). However, the shelf-life of mushroom is shorter than that of other supermarket products after harvest because it lacks cuticle to protect it from physical damage, microbial attack, and water loss (Aguirre et al., 2008). The postharvest senescence of fresh products is often associated with increased oxidative damage to proteins, lipids and nucleic acids by ROS and thus their production and removal must be strictly controlled (Mittler, 2002). ROS production can be enhanced in response to various biotic and abiotic stress conditions (Gill and Tuteja, 2010). Postharvest storage can be considered as a kind of abiotic stress for mushrooms as the storage conditions are quite different from the growing conditions, which can lead to electron transport inhibition in mitochondria as well as increased ROS production (Li et al., 2013). Oxidative damage occurs when ROS
* Corresponding author. E-mail address: [email protected] (Z. Liyan). http://dx.doi.org/10.1016/j.postharvbio.2016.04.012 0925-5214/ ã 2016 Elsevier B.V. All rights reserved.
levels exceed the capacity of the defense mechanisms to neutralize them causing “oxidative stress” (Sharma et al., 2012), which then leads to oxidation of lipids and proteins, nucleic acids damage, enzyme inhibition and membrane damage in both cellular and organelle membranes. Therefore, mushrooms need special care, especially during storage so as to retain freshness. Recently, there have been many methods used to extend the shelf life of mushrooms. Cold storage (Dama et al., 2010; Li et al., 2016), UV-C treatment (Murray et al., 2015; Villares et al., 2014), modified atmosphere packaging (MAP) (Oz et al., 2015), bioactive coatings (Liu et al., 2015; Wrona et al., 2015; Echegoyen and Nerín, 2015) and biobased film packaging (Han et al., 2015; Guillaume et al., 2010) have all been applied to extend mushroom shelf-life. Most of them are difficult to operate and achieve commercial application prospect in food industry. If nanotechnology is introduced in the food packaging industry, it can potentially provide solutions to food packaging challenges such as short shelf life (Chaudhry et al., 2008). The incorporation of nanofillers such as silicate, clay, and titanium dioxide (TiO2) to polymers may improve not only its mechanical and barrier properties but also offer other functions and applications in food packaging such as antimicrobial agent, biosensor, and oxygen scavenger (De Azeredo, 2009; Rhim et al., 2013). Panea et al. (2014)
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provided a packaging based on a low density polyethylene (LDPE) blended with a nano-antimicrobial master batch composed of silver (Ag) and zinc oxide (ZnO) and found it had an antimicrobial effect and delayed chicken breast spoilage and lipid oxidation. Knowledge of water mobility and distribution in mushrooms is of major interest in studying the postharvest senescence of this economically important crop (Lagnika et al., 2013). Nuclear magnetic resonance (NMR) spectroscopy is one technique to characterize the state, mobility and distribution of water in fruits and vegetable (Donker et al., 1997; Musse et al., 2010). The objectives of this study were to evaluate the effect of nanocomposite packaging on postharvest senescence of mushrooms and to investigate the influence on free radical metabolism and anti-oxidation system. 2. Materials and methods 2.1. Sample preparation, treatment and storage 2.1.1. Nanocomposite-based packaging materials Nano-powders (30 wt.% of nano-Ag, 35 wt.% of nano-TiO2, 25 wt.% of attapulgite, and 10 wt.% of Nano-SiO2) in the range of 40–60 nm were obtained from a commercial company (Nanjing Haitai Nanotechnology Company, Jiangsu, China). Firstly, a poly ethylene (PE) based nanocomposite master batch containing 15 wt. % of the commercial nano-powder, 46 wt.% of LDPE, 22 wt.% of linear low-density polyethylene (LLDPE), 10 wt.% of dispersing agent, 5 wt.% of lubricant, and 2 wt.% of the cross-linking reagent KH-570 were mixed uniformly with a high-speed mixer for 1 h. After cooling in air, they were extruded to produce a PEnanocomposite master batch using a twin-screw extruder (SHJ20, Nanjing Guangda Chemical Equipment Co. Ltd, Jiangsu, China) with a screw diameter of 22 mm, a screw length/diameter ratio of 42, and a screw speed of 600 rpm. In the second extrusion step, 7.5 kg of the nanocomposite masterbatch, 8 kg of anti-fog masterbatch, and 84.5 kg of PE granules (50 wt.% of LDPE and 50 wt.% of LLDPE) were mixed for 0.5 h, before blowing into a film with a thickness of 40 mm via a plastic extruder (SJ5030/FM1300, Dalian Plastic Machine Factory, Liaoning, China). After cooling, films that measured 40 mm in thickness were used to make bags of 25 25 cm2 with a microcomputer-controlled high-speed bagmaking machine (SDD-A500/1200, Zhaoyuan Packaging Machiner Co., Shandong, China). PE bags of the same thickness and size but without the nanocomposite masterbatch nano-powder were used as the control. 2.1.2. Preparation of mushrooms F. velutipes used in this study was obtained from a commercial farm in Jiangsu, China and transported to the laboratory in half an hour. The mushrooms were selected according to their whiteness, developmental stage, closed veil and shape (stipe length of 10– 15 cm). After storage in darkness at 4 1 C and 90% relative humidity (RH) for 24 h, mushrooms (250 g) were randomly packaged in Nano-PM (24 bags) and Normal-PM (24 bags), which were then sealed with a heat sealer and stored at 4 1 C and 90%– 95% RH for 21 days. Three bags of each packaging were used to analyse the physiological and biochemical indexes every 3 days during the storage. 2.2. Microstructure observation The mushroom segments were pre-fixed in 2.5% (w/w) glutaraldehyde with 67 mmol/L phosphate buffer (pH 7.8) and post-fixed with 1% osmium tetraoxide. The samples were further dehydrated in ethanol and embedded in Quetol-651 resin. Ultrathin sections were stained with 4% (w/w) uranyl acetate and
afterwards by lead citrate (Suzuki et al., 2005). They were examined and photographed using the H-7650 (Hitachi HighTechnologies Corporation, Tokyo, Japan) transmission electron microscope at an accelerating voltage of 80 kV.
2.3. Headspace gas analysis The headspace gas concentrations of O2 and CO2 were analyzed every three days, using a SCY-2A O2 and CO2 analyzer (Xinrui Instrument Co., Shanghai, China). Gas samples were taken from the bags with a 20 mL syringe. 2.4. Physico-chemical analysis 2.4.1. Texture and weight loss The texture of stipes of F. velutipes was measured by a texture analyzer (TA-XT Plus, SMS Co., England). The volodkevich bite jaws (HDP/BS) probe, which simulates the action of an incisor tooth (Azeredo et al., 2006), was used in measuring the texture of stipes of mushrooms for it has been shown that most of the stipes were chewed by fore teeth. Three pieces of stipes of F. velutipes were measured each time at central parts with both ends held. Compression was performed twice (up to 90% sample thickness) at a compression rate of 2 mm/s. Three replications were conducted for each sample. Weight loss was determined by weighing the whole mushrooms before and after the storage period. Weight loss was expressed as the percentage of loss of weight with respect to the initial weight. 2.4.2. Total soluble solids (TSS) and protein content The mushrooms were ground in a mortar and squeezed with a hand press, and the juice was analyzed for the TSS contents. The TSS was measured at 25 C with a refractometer (TZ-62, Optical Instrument Factory Ltd, Guangzhou, China). The protein content was determined using a soluble protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). 2.4.3. Membrane permeability and lipid peroxidation Membrane permeability, expressed by relative electrolyte leakage, was determined by the modified method of Barman et al. (2014). The fresh mushroom discs (3 mm thick, 3 mm diameter, 20 pieces total) were placed in 40 mL distilled water. Conductivity of the suspending solution was measured at once (P0), after 10 min (P1) and thereafter boiling for 10 min (P2) with a DDS-11A electrical conductivity meter (DDS-11A, Kangyi Instrument Co., Shanghai, China). Membrane permeability was expressed according to the following formula: Membrane permeabilityð%Þ ¼ ½ðP1 P0 Þ=ðP2 P0 Þ 100%: The level of lipid peroxidation was expressed as the amount of MDA. Its content was determined with a Maleic Dialdehyde assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s instructions. The unit of MDA was expressed as mol kg1 on a protein basis. 2.4.4. O2 and H2O2 levels O2 content was determined according to the method of Elstner and Heupel (Elstner and Heupel, 1976) with slight modifications. Mushroom sample (4 g) was sliced and homogenized in 36 mL of 50 mmol/L K-phosphate buffer (pH 7.8) for 30s at 4 C. After centrifugation at 10,000g for 20 min at 4 C, the supernatant was used for the determination of free radical levels as follows: 0.5 mL supernatant, 0.5 mL 50 mmol/L K-phosphate buffer, and 1 mmol/L hydroxylamine hydrochloride were mixed
F. Donglu et al. / Postharvest Biology and Technology 119 (2016) 49–57
and incubated at 25 C for 60 min, 1 mL 17 mmol/L p-aminophenyl sulfonic acid (in glacial acetic acid:H2Omol kg1= 3:1) and 7 mmol/ L a-naphthylamine (in glacial acetic acid:H2O = 3:1) were added and the mixture was incubated at 25 C for a further 20 min. Optical density was immediately measured at a wavelength of 530 nm. Na2NO2 was used for standard curve. The assay for H2O2 content was carried out as previously described by Patterson et al. (Patterson et al., 1984). Mushroom sample (2 g) was homogenized in 10 mL 100% (v/v) of acetone at 4 C. After centrifugation for 15 min at 6000 rpm at 4 C, the supernatant was collected. One ml of supernatant was mixed with 0.1 mL 5% (w/v) titanium sulphate and 0.2 mL 25% (v/v) ammonia, then centrifuged for 10 min at 6000 rpm and 4 C. The pellet was dissolved in 3 mL 10% (v/v) H2SO4 and centrifuged for 10 min at 6000 rpm. The absorbance of the supernatant was measured at 410 nm. The H2O2 content was calculated using H2O2 as a standard and expressed as mol kg1 on a fresh weight basis. 2.4.5. Antioxidant enzyme activities For assay enzyme activities, 4 g of mushroom tissue was homogenized with 36 mL of 50 mm Na2HPO4–NaH2PO4 buffer (pH 7.8) containing 0.2 mmol/L ethylene diamine tetraacetic acid (EDTA) and 2% (w/v) insoluble polyvinylpyrrolidone (PVP) under ice cold conditions. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 C and the supernatant was collected and used as the crude enzyme extract for the determination of the following enzyme activities. Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed based on its ability to inhibit photo-reduction of nitroblue tetrazolium (NBT), according to the method reported by (Zhang et al., 2008). One unit of SOD activity was defined as the amount of enzyme that would inhibit 50% of the photo-reduction of NBT at 560 nm. Catalase (CAT; EC 1.11.1.6) activity was assayed according to the method reported by (Aebi, 1984). Each CAT assay solution (3 mL) contained 15 mmol/L phosphate buffer (pH 7.0), 15 mmol/L H2O2, and 0.1 mL enzyme extract. The reaction was initiated by adding the enzyme extract. The change in the absorbance of the reaction solution at 240 nm was read every 40 s. One unit of CAT activity was defined as the amount of enzyme required to decrease the absorbance at 240 nm by 0.1 per minute. Peroxidase (POD, EC1.11.1.7) activity was assayed according to the method reported by (Zhou and Leul, 1999) with some minor modification. The guaiacol-dependent peroxidase activity was measured in the reaction mixture containing 50 mmol/L phosphate buffer (pH 7.0), 1% guaiacol, 0.4% H2O2 and 100 mL enzyme extract. The increase in absorbance reading due to guaiacol oxidation was measured at 470 nm.
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2.6. Statistical analyses Analysis of variance (ANOVA) was performed using the SAS system. To determine statistical differences, comparisons of the means between control and treatment samples were performed using Duncan’s test at a significance level of P < 0.05. 3. Results 3.1. Sensory and microstructure evaluation The sensory quality of mushrooms in different packaging materials was evaluated and the microstructure was observed as well (Figs. 1 and 2F,G). The mushrooms kept in Normal-PM exhibited a certain degree of cap opening and distinct stipe elongation after 21 days of storage at 4 C. There were lots of tiny drops of moisture on the inner surface of Normal-PM. The transmission electron micrographs of the samples showed the microstructural changes of mushroom cells after 21 days of storage. The cells of Nano-PM packed mushroom tissues (Fig. 2F) were more plump and rounded compared with those of Normal-PM packed mushroom tissues (Fig. 2G). Besides, the cell wall of Normal-PM packed mushroom tissues became much thinner and cell membrane breakage was observed at the end of storage. 3.2. Gas analysis Table 1 shows the change in package atmosphere composition over storage time. This variable was clearly influenced by packaging material and storage time, as well as by their interaction. It reflects that mushroom respiration provoked an increase in the concentration of CO2 and the consequent decrease in O2 content, leading to more anaerobic conditions. The concentration of O2 decreased sharply within 72 h and approached a steady state that fluctuated between 2.47–3.93% and 1.63–3.01% in Nano-PM and Normal-PM, respectively. Conversely, the concentration of CO2 in the headspace of all packs increased gradually with increasing storage time. This concentration reached an optimum peak of 11.02% in Normal-PM on the 12th day, which was significantly (P < 0.05) higher than that in the Nano-PM group (7.06%).
2.5. Low field nuclear magnetic resonance analysis Low field nuclear magnetic resonance measurements (LF-NMR) were performed on a 22.4 MHz NMR Analyzer PQ001 (Niumag Co., Ltd., Shanghai, China), according to the method of (Li et al., 2014). Approximately 1.5 g of mushroom samples were placed in a 15 mm NMR diameter glass tube and inserted in the NMR probe. Carr– Purcell–Meiboom–Gill (CPMG) sequences were employed to measure spin–spin relaxation time, T2. The T2 measurements were performed with a t-value of 200 ms (time between 90 and 180 pulse). Data from 8000 echoes were acquired as 32 scan repetitions. The repetition time between two successive scans was 3 s. MultiExp Inv Analysis software (Niumag Co., Ltd., Shanghai, China) was employed for data analysis. Fig. 1. Effects of Nano-PM and Normal-PM on sensory quality of F. velutipes after 21 days of storage.
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Fig. 2. Effects of Nano-PM (&) and Normal-PM (4) on MDA content (A), relative conductity (B), antioxidant enzyme activities (C, D, E) and cell microstructure (F, G) of F. velutipes. Each bar represents the mean SE of the individual samples. *P < 0.05.
F. Donglu et al. / Postharvest Biology and Technology 119 (2016) 49–57
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Table 1 Changes in gas composition (%, V/V) of F. velutipes stored at 4 C for 21 days.* Treatment
Storage time (days) 0
3
6
9
12
15
18
21
N P
O2 21 21
2.47 0.16a 1.63 0.08a
3.50 0.14a 2.47 0.13b
2.63 0.17a 1.70 0.10b
3.90 0.09a 2.63 0.12b
3.91 0.07a 2.80 0.08b
3.40 0.14a 2.43 0.12b
3.93 0.13a 3.01 0.10b
N P
CO2 0 0
8.37 0.18b 10.10 0.22a
7.73 0.16b 9.70 0.28a
8.13 0.17b 9.20 0.25a
7.06 0.21b 11.02 0.32a
7.03 0.11b 8.20 0.26a
7.43 0.15b 8.40 0.02a
7.07 0.19b 7.77 0.05a
Different letters in the same column indicate statistically significant difference at P<0.05 between normal-packing (P) and Nano-packing (N) sample for each time point. * Each value represents the mean of six replicates standard deviation.
3.3. Weight loss, maximum shearing forces, TSS and soluble proteins There's a strong relationship between these experimental indexes and the edible quality of mushrooms. As shown in Table 2, the weight loss and maximum shearing forces increased as the storage period progressed in both treatments. The highest weight loss occurred in the control samples, i.e., 0.62% at the end of the storage period. The maximum shearing forces of mushrooms in Nano-PM were 12.94 N on the 21th day, which was 16.46% lower than the control. The TSS contents of the control mushrooms declined rapidly during the first three days of storage, whereas the mushrooms treated with Nano-PM exhibited a slight decrease during the overall period. The lowest TSS levels were recorded in the two mushroom treatments at the end of the storage period, i.e., 4.83% and 6.02% for Normal-PM and Nano-PM, respectively. Both the Nano-PM and Normal-PM packed mushrooms exhibited slight decreases in their protein contents during the first 18 days, after which the protein decomposed rapidly. The soluble protein contents of the Nano-PM packed mushrooms were 5.40 g kg1 after 21 days of storage at 4 C, which was significantly higher (P < 0.05), than the proteins in Normal-PM packed mushrooms (4.06 g kg1).
(Fig. 2A). Mushrooms packed with Nano-PM maintained a significantly lower MDA content than the control. At the end of storage, the MDA content of Nano-PM packed mushrooms was 2.22 mol kg1, which was 29.3% lower than that in Normal-PM. The relative electrolyte leakage of F. velutipes gradually increased during the whole storage (Fig. 2B). The mushrooms packed in Normal-PM had a higher relative electrolyte leakage after 15 days of storage compared with control samples (P < 0.05). 3.5. O2 and H2O2 contents The O2 content had a marked increase in both control and mushrooms in Nano-PM during the first 9 days of storage. After that, the O2 content in Nano-PM packed mushrooms began to be flat, while in control it continued to rise slowly till the 18th day of storage (Fig. 3A). As shown in Fig. 3B, the H2O2 content took on a fluctuant process during 18 days of storage. The H2O2 content in Nano-PM packed mushrooms were 21.87 and 14.65 mmol kg1 in day 18 and 21 respectively, which were significantly lower than that in control (P < 0.05). The results showed that Nano-PM markedly inhibited the formation of O2 and the accumulation of H2O2 during storage time, compared with the control group. 3.6. Oxygen radical scavenging enzyme activities
3.4. MDA contents and relative electrolyte leakage MDA content increased rapidly during the first 6 days of storage and thereafter continuously increased throughout storage
SOD, CAT and POD are important active free-radical scavenging enzymes (Lee and Lee, 2000). The changes of these enzyme activities were shown in Fig. 2. The mushrooms stored in Nano-PM
Table 2 Weight loss, maximum shearing forces, total soluble solids and soluble proteins of F. velutipes stored at 4 C for 21 days. Treatment
Storage time (days) 0
3
6
9
12
15
18
21
N P
Weight-loss (%) n/a* n/a
0.06 0.01a 0.14 0.05a
0.17 0.09a 0.21 0.02a
0.24 0.02a 0.33 0.08a
0.27 0.02a 0.56 0.11a
0.43 0.10a 0.63 0.11a
0.50 0.09b 0.78 0.12a
0.63 0.05b 0.89 0.13a
N P
Maximum shearing forces (N) 8.43 0.07a 9.70 0.16a 8.43 0.07a 9.80 0.21a
10.29 0.17a 10.39 0.11a
10.88 0.15a 11.27 0.15a
10.29 0.09a 11.56 0.12a
10.88 0.14a 12.45 0.16a
12.35 0.13b 14.21 0.16a
12.94 0.18b 15.49 0.24a
N P
TSS (%) 8.62 0.86 8.62 0.86
8.42 1.07a 6.78 0.50a
7.51 0.24a 6.74 0.56a
7.49 1.05a 6.62 0.22a
7.41 0.91a 6.57 0.77a
6.79 0.63a 6.12 0.02a
6.67 0.99a 5.49 0.48b
6.02 0.49a 4.83 0.15b
N P
Soluble proteins (gkg1) 5.19 0.35 a 5.12 0.49a 5.19 0.35 a 5.08 0.70a
5.62 1.01a 5.38 0.32a
7.25 1.09a 7.05 1.21a
8.84 1.40a 6.82 0.78b
7.09 0.85a 5.32 0.62b
6.07 0.74a 4.41 0.69b
5.40 0.16a 4.06 0.75b
a a
Different letters indicate statistically significant differences at P<0.05 between normal-packing (P) and Nano-packing (N) sample for each time point. * n/a means not applicable. Each value represents the mean of six replicates standard deviation.
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Fig. 3. Effects of Nano-PM (&) and Normal-PM (4) on O2 content (A) and H2O2 content (B) of F. velutipes. Each bar represents the mean SE of the individual samples. *P < 0.05.
Fig. 4. Variations in transverse relaxation time distributions (T2) and amplitude in F. velutipes at the beginning (A) and the end of storage (B) under different treatments (Nano-PM: &, Normal-PM: 4).
maintained remarkably higher SOD activity during storage (Fig. 2C). CAT activity in both treatments increased to a peak on day 6, and then decreased during the rest of the days (Fig. 2D). The significant increase of POD activity was found in Nano-PM packed mushrooms (P < 0.05). The highest enzyme activity was detected in the mushrooms on day 3 (Fig. 2E). After 12 days of storage, there was no significant difference (P > 0.05) in POD activity between Nano-PM treatment and the control.
And the fourth peak in region III was the major part. After 21 days of storage, control and Nano-PM packed samples still display four peaks but with some change. The peaks of the relaxation times in Normal-PM packed mushrooms were smaller and led to a leftward shift compared with Nano-PM packed samples. In region III, it was interesting to find that there was a shoulder between T21 and T22 fractions and they had a tendency to merged into one (Fig. 4B).
3.7. LF-NMR analysis
4. Discussion
The changes in amplitudes of the samples as a function of the relaxation times (T2) in different packing materials are shown in Fig. 4. The relaxation time describes water status in the different compartments of the sample: the mobility, distribution, position of the water and interactions between water and macromolecules (Musse et al., 2010). At the beginning of storage, there were four distinct peaks distributed in three different regions. Region I stands for water in the cell wall; Region II represents cytoplasm water; Region III can be considered as water in the vacuole and extra cellular (Fig. 4A).
Mushrooms are highly perishable and tend to lose quality right after harvest (Wang et al., 2015). In this study, a PE packaging material that contained nano-Ag, nano-TiO2, nano-SiO2, and attapulgite was developed and applied on extending the shelf life of mushrooms. The results showed that treatment with the NanoPM improved the retention of nutritional components of F. velutipes and inhibited the cap development and stripe elongation so that mushrooms in Nano-PM maintained better storage quality than that in control after 21 days of storage (Fig. 1 and Table 2).
F. Donglu et al. / Postharvest Biology and Technology 119 (2016) 49–57
Nano-packaging technology can be considered as one kind of modified atmosphere packaging alternatives, which is an active or passive dynamic process of altering gaseous composition within a package. The nanocomposite packaging with modified atmosphere can be generated inside a package from the natural process of produce respiration and film permeability to achieve the desired gas composition over time (Farber et al., 2003). In our previous study, we tested the physical properties of the nanocomposite material (Fang et al., 2016). From the results of the previous research, we found that the water vapor and oxygen permeability of Nano-PM were 7.22 105 g/m2/s and 4.65 1010 g/m2/s/Pa, respectively, which were 10.5% and 8.0% lower compared with Normal-PM. And its longitudinal strength was enhanced by 26.9%. The patterns of changes for CO2 and O2 concentration in treatment and control mushrooms were similar during storage (Table 1). However, there was a peak of CO2 occurring on day 12th in control samples which was believed to be a result of a sharp rise in post harvest respiration that usually occurs with fruits and vegetables (Eskin and Shahidi, 2012). In Nano-PM packed mushrooms, by contrast, no noticeable peak of respiratory rate was observed after three days of storage. These significant differences in CO2 content during storage between nano-PM and normal-PM could be related to the ethylene scavenging effects of nanoparticle (De Azeredo, 2013). Similar results have been reported by Li et al. (2009), who found that the application of nanocomposite packing could suppress ethylene production during storage time as a result of adding of nano-Ag and nano-TiO2, which could decompose or oxidize ethylene into water and carbon dioxide. The process of fruit ripening and senescence is accompanied by the continuously strengthening of oxidation, which may lead to accumulation of ROS and produce oxidative damage (Rogiers et al., 1998). The accumulation of ROS resulting from an altered balance between ROS production and scavenging capacities will reduce the storage quality and marketability of fruits and vegetables (Hodges et al., 2004). The present study showed that ROS levels (i.e., O2 and H2O2) in control mushrooms continuously increased with fluctuations during storage (Fig. 3). The content of O2 and H2O2 in Nano-PM packed samples were significantly (P < 0.05) lower than those in control at middle storage stage (day 9) and late storage stage (day 18–21). In order to protect cells from ROS-induced injuries, plant tissues possess an antioxidant defence system to scavenge ROS (Duan et al., 2011). SOD could remove O2 by catalyzing this unstable product to O2 and H2O2, and the CAT and POD could dismutate H2O2 into H2O and O2 through different actions, all of which are indispensable for ROS detoxification (Gill and Tuteja, 2010). Jiang et al. (2014) reported that a kind of Nano-
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packing combined with controlled atmosphere induced higher SOD, CAT, and POD activities, thereby limiting oxidative processes in a Chinese herbal medicine called Gynura. In our study, the activities of SOD, CAT and POD in Nano-PM packed mushrooms at the beginning of storage (day 3) were 14.86%, 18.81% and 69.68% respectively higher than that in Normal-PM (Fig. 2). These results indicated that Nano-PM treatment was beneficial in controlling ROS content by activating the antioxidant defence system, thereby alleviating oxidative damage and delaying senescence. Moreover, ROS accumulation in the cell damages membrane structure and function, triggers the membrane lipid peroxidation and induces the membrane breakdown (Juan et al., 2011). Membrane damage in mushrooms increased during storage, assessed by MDA contents and relative electrolyte leakage (Fig. 2A, B) which coincided with the increased ROS levels (Fig. 3). Our present results showed that treatment with NanoPM could reduce the increases in membrane permeability and lipid peroxidation (Fig. 2). Chomkitichai et al. (2014) found that lipoxygenase activity, conjugated diene and MDA contents were positively correlated with ROS levels (r = 0.854–0.939, 0.813– 0.887and 0.894–0.971, respectively), indicating that the extent of membrane lipid peroxidation depended on the levels of ROS. Therefore, I speculated that Nano-PM treatment maintained the membrane integrity via its ability to improve on antioxidant activity. Membrane lipid peroxidation alters membrane properties and results in cell defects such as ion leakage and cellular decompartmentation (Duan et al., 2007; Yang et al., 2009). As a result, the molecular mobility of water and biopolymers in mushrooms, which could be tested by LF-NMR, were also influenced during storage. According to previous studies (Lagnika et al., 2013), the curves can be divided into three distinct regions (I–III) based on the relaxation times (T2). Region I corresponds to the hydration monolayer where water molecules are bound to the product by strong H-bonds. Region II corresponds to the part where water is absorbed as multilayers of molecules of hydrogen bonded to the monolayer. Region III is that of the so-called “free” or solvent water. Water molecules in this region are weakly bound compared to those in regions I and II. This fraction of water is available for mould growth or dissolution of soluble solutes and for enzymatic reaction. Free water is of main interest for storage stability. Hence, region III is of highest relevance. During the 21 days of storage, water changed dynamically. The T22,T23 and T24 relaxation time in control were decreased compared with Nano-PM packed mushrooms at the end of storage (Table 3).
Table 3 Relaxation time distributions (T2) and amplitude before and after 21 days of storage in F. velutipes. Samples Before storage No-treatment
After 21 days of storage Nano-PM
Normal-PM
Peak number
Peak time (T2)
Peak area
Total area
Amplitude
Region
1 2 3 4
1.01 0.08 9.33 0.73 57.22 1.19 305.39 3.82
23.89 1.96 40.63 3.21 155.69 2.68 454.06 10.85
674.27 15.41
13.12 0.98 12.12 1.20 18.30 0.85 101.23 4.23
I II III III
1 2 3 4 1 2 3 4
0.25 0.06 12.33 0.56 65.79 6.24 351.12 3.11 0.25 0.03 7.05 1.26 49.77 5.68 305.39 9.12
98.39 3.57 44.12 2.24 182.35 6.56 572.95 5.87 53.83 2.31 30.01 1.45 190.78 3.38 567.39 2.96
897.81 9.55
15.20 0.54 12.09 0.25 34.87 0.87 127.52 1.22 10.38 1.01 10.75 0.47 25.72 1.81 102.67 2.63
I II III III I I III III
842.01 5.27
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And the T23 and T24 fractions in control samples had a tendency to merge into one (Fig. 4B). In theory, the distribution of proton relaxation times should be equivalent to the distribution of proton fractions in different micro-compartments. However, diffusion of water between neighbouring compartments can average their NMR signals. As a consequence, most water fractions in different micro-compartments cannot be distinguishable in time domain NMR spectrum, only if it exist micro-structural barriers which can hinder the inter-compartment water diffusion (Tang et al., 2000). In previous studies, the membranaceous structures of cell and organelle are considered to act as physical micro-structural barriers for the different water fractions, but they are easily destroyed with the storage duration because of the accumulated ROS damage (Duan et al., 2007; Li et al., 2014). When the physical barriers were broken, the interactions between water and macromolecules which dissolved in free water were enhanced, which would induce the LF-NMR signal change in Fig. 4B. Hence, the LFNMR application in our research made a sidewise approach to prove the effectiveness of Nano-PM on protect the integrity of the biofilm against ROS attack during storage. In conclusion, our present data indicate that the reduction in senescence development by Nano-PM treatment, in mushrooms stored at 4 C, is associated with inhibition of respiratory activity. The maintenance of integrity of biological membrane structure, resulting from the decrease in levels of ROS and the improvement on antioxidant enzymes activity by the treatment is also involved in senescence retardation in mushrooms. Furthermore, these nano-packaging materials have the advantages of simple processing and potential to be applied commercially. Nano-PM could therefore provide an attractive alternative maintain the quality of stored mushrooms. Conflict of interest None. Acknowledgments The authors acknowledge financial support from the Special Fund for Agro-scientific Research in the Public Interest (No. 201303080), the National Natural Science Foundation of China (No. 31401552) and the Natural Science Foundation of Jiangsu Province (No. BK20141009). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Aguirre, L., Frias, J.M., Barry-Ryan, C., Grogan, H., 2008. Assessing the effect of product variability on the management of the quality of mushrooms (Agaricus bisporus). Postharvest Biol. Technol. 49 (2), 247–254. Azeredo, H., Brito, E.S., Moreira, G.E., Farias, V.L., Bruno, L.M., 2006. Effect of drying and storage time on the physico-chemical properties of mango leathers. Int. J. Food Sci. Technol. 41 (6), 635–638. Barman, K., Asrey, R., Pal, R.K., Jha, S.K., Bhatia, K., 2014. Post-harvest nitric oxide treatment reduces chilling injury and enhances the shelf-life of mango (Mangifera indica L.) fruit during low-temperature storage. J. Hortic. Sci. Biotechnol. 89 (3), 253–260. Chaudhry, Q., Scotter, M., Blackburn, J., Ross, B., Boxall, A., Castle, L., Aitken, R., Watkins, R., 2008. Applications and implications of nanotechnologies for the food sector. Food Addit. Contam. 25 (3), 241–258. Chomkitichai, W., Chumyam, A., Rachtanapun, P., Uthaibutra, J., Saengnil, K., 2014. Reduction of reactive oxygen species production and membrane damage during storage of ‘Daw’ longan fruit by chlorine dioxide. Sci. Hortic. 170, 143–149. Dama, C.L., Kumar, S., Mishra, B.K., Shukla, K.B., Mathur, S., Doshi, A., 2010. Antioxidative enzymatic profile of mushrooms stored at low temperature. J. Food Sci. Technol. 47 (6), 650–655. De Azeredo, H.M., 2009. Nanocomposites for food packaging applications. Food Res. Int. 42 (9), 1240–1253. De Azeredo, H.M., 2013. Antimicrobial nanostructures in food packaging. Trends Food Sci. Technol. 30 (1), 56–69.
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Contents lists available at ScienceDirect
Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Effect of nanocomposite packaging on postharvest quality and reactive oxygen species metabolism of mushrooms (Flammulina velutipes) Fang Donglua , Yang Wenjianb , Benard Muinde Kimatua,c , An Xinxina , Hu Qiuhuia , Zhao Liyana,* a b c
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, Jiangsu 210046, PR China Department of Dairy and Food Science and Technology, Egerton University, Nakuru, Kenya
A R T I C L E I N F O
Article history: Received 21 November 2015 Received in revised form 18 April 2016 Accepted 20 April 2016 Available online xxx Keywords: Nanocomposite packaging Flammulina velutipes Preservation ROS NMR
A B S T R A C T
The aim of this study was to analyse the effect of nanocomposite packaging material (Nano-PM) on the physicochemical characteristics and antioxidant capacity of mushrooms (Flammulina velutipes) during 21 days of postharvest storage at 4 C. The results showed that Nano-PM improved retention of nutrients and inhibited the weight loss, respiration and distinct stipe elongation of mushrooms compared with the Normal packing material (Normal-PM). Reactive oxygen species (ROS) levels, including superoxide radical (O2) and hydrogen peroxide (H2O2), increased during storage. Membrane damage, as measured by relative electrolyte leakage and malondi-aldehyde (MDA) contents also increased. However, levels of O2 and H2O2, relative electrolyte leakage and MDA contents were all significantly lower (P < 0.05) in the Nano-PM group than those in the control group. Nano-PM reduced the production of free radicals which cause membrane damage and may therefore be an effective treatment to delay the postharvest growth and caducity in F. velutipes hence increasing its shelf life and at the same time preserving its quality. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Production and consumption of mushrooms have been increasing substantially in the world (Pesce et al., 2015). The golden needle mushroom, Flammulina velutipes, has delicious taste, high nutritional value, and medicinal properties (Yang et al., 2012). However, the shelf-life of mushroom is shorter than that of other supermarket products after harvest because it lacks cuticle to protect it from physical damage, microbial attack, and water loss (Aguirre et al., 2008). The postharvest senescence of fresh products is often associated with increased oxidative damage to proteins, lipids and nucleic acids by ROS and thus their production and removal must be strictly controlled (Mittler, 2002). ROS production can be enhanced in response to various biotic and abiotic stress conditions (Gill and Tuteja, 2010). Postharvest storage can be considered as a kind of abiotic stress for mushrooms as the storage conditions are quite different from the growing conditions, which can lead to electron transport inhibition in mitochondria as well as increased ROS production (Li et al., 2013). Oxidative damage occurs when ROS
* Corresponding author. E-mail address: [email protected] (Z. Liyan). http://dx.doi.org/10.1016/j.postharvbio.2016.04.012 0925-5214/ ã 2016 Elsevier B.V. All rights reserved.
levels exceed the capacity of the defense mechanisms to neutralize them causing “oxidative stress” (Sharma et al., 2012), which then leads to oxidation of lipids and proteins, nucleic acids damage, enzyme inhibition and membrane damage in both cellular and organelle membranes. Therefore, mushrooms need special care, especially during storage so as to retain freshness. Recently, there have been many methods used to extend the shelf life of mushrooms. Cold storage (Dama et al., 2010; Li et al., 2016), UV-C treatment (Murray et al., 2015; Villares et al., 2014), modified atmosphere packaging (MAP) (Oz et al., 2015), bioactive coatings (Liu et al., 2015; Wrona et al., 2015; Echegoyen and Nerín, 2015) and biobased film packaging (Han et al., 2015; Guillaume et al., 2010) have all been applied to extend mushroom shelf-life. Most of them are difficult to operate and achieve commercial application prospect in food industry. If nanotechnology is introduced in the food packaging industry, it can potentially provide solutions to food packaging challenges such as short shelf life (Chaudhry et al., 2008). The incorporation of nanofillers such as silicate, clay, and titanium dioxide (TiO2) to polymers may improve not only its mechanical and barrier properties but also offer other functions and applications in food packaging such as antimicrobial agent, biosensor, and oxygen scavenger (De Azeredo, 2009; Rhim et al., 2013). Panea et al. (2014)
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provided a packaging based on a low density polyethylene (LDPE) blended with a nano-antimicrobial master batch composed of silver (Ag) and zinc oxide (ZnO) and found it had an antimicrobial effect and delayed chicken breast spoilage and lipid oxidation. Knowledge of water mobility and distribution in mushrooms is of major interest in studying the postharvest senescence of this economically important crop (Lagnika et al., 2013). Nuclear magnetic resonance (NMR) spectroscopy is one technique to characterize the state, mobility and distribution of water in fruits and vegetable (Donker et al., 1997; Musse et al., 2010). The objectives of this study were to evaluate the effect of nanocomposite packaging on postharvest senescence of mushrooms and to investigate the influence on free radical metabolism and anti-oxidation system. 2. Materials and methods 2.1. Sample preparation, treatment and storage 2.1.1. Nanocomposite-based packaging materials Nano-powders (30 wt.% of nano-Ag, 35 wt.% of nano-TiO2, 25 wt.% of attapulgite, and 10 wt.% of Nano-SiO2) in the range of 40–60 nm were obtained from a commercial company (Nanjing Haitai Nanotechnology Company, Jiangsu, China). Firstly, a poly ethylene (PE) based nanocomposite master batch containing 15 wt. % of the commercial nano-powder, 46 wt.% of LDPE, 22 wt.% of linear low-density polyethylene (LLDPE), 10 wt.% of dispersing agent, 5 wt.% of lubricant, and 2 wt.% of the cross-linking reagent KH-570 were mixed uniformly with a high-speed mixer for 1 h. After cooling in air, they were extruded to produce a PEnanocomposite master batch using a twin-screw extruder (SHJ20, Nanjing Guangda Chemical Equipment Co. Ltd, Jiangsu, China) with a screw diameter of 22 mm, a screw length/diameter ratio of 42, and a screw speed of 600 rpm. In the second extrusion step, 7.5 kg of the nanocomposite masterbatch, 8 kg of anti-fog masterbatch, and 84.5 kg of PE granules (50 wt.% of LDPE and 50 wt.% of LLDPE) were mixed for 0.5 h, before blowing into a film with a thickness of 40 mm via a plastic extruder (SJ5030/FM1300, Dalian Plastic Machine Factory, Liaoning, China). After cooling, films that measured 40 mm in thickness were used to make bags of 25 25 cm2 with a microcomputer-controlled high-speed bagmaking machine (SDD-A500/1200, Zhaoyuan Packaging Machiner Co., Shandong, China). PE bags of the same thickness and size but without the nanocomposite masterbatch nano-powder were used as the control. 2.1.2. Preparation of mushrooms F. velutipes used in this study was obtained from a commercial farm in Jiangsu, China and transported to the laboratory in half an hour. The mushrooms were selected according to their whiteness, developmental stage, closed veil and shape (stipe length of 10– 15 cm). After storage in darkness at 4 1 C and 90% relative humidity (RH) for 24 h, mushrooms (250 g) were randomly packaged in Nano-PM (24 bags) and Normal-PM (24 bags), which were then sealed with a heat sealer and stored at 4 1 C and 90%– 95% RH for 21 days. Three bags of each packaging were used to analyse the physiological and biochemical indexes every 3 days during the storage. 2.2. Microstructure observation The mushroom segments were pre-fixed in 2.5% (w/w) glutaraldehyde with 67 mmol/L phosphate buffer (pH 7.8) and post-fixed with 1% osmium tetraoxide. The samples were further dehydrated in ethanol and embedded in Quetol-651 resin. Ultrathin sections were stained with 4% (w/w) uranyl acetate and
afterwards by lead citrate (Suzuki et al., 2005). They were examined and photographed using the H-7650 (Hitachi HighTechnologies Corporation, Tokyo, Japan) transmission electron microscope at an accelerating voltage of 80 kV.
2.3. Headspace gas analysis The headspace gas concentrations of O2 and CO2 were analyzed every three days, using a SCY-2A O2 and CO2 analyzer (Xinrui Instrument Co., Shanghai, China). Gas samples were taken from the bags with a 20 mL syringe. 2.4. Physico-chemical analysis 2.4.1. Texture and weight loss The texture of stipes of F. velutipes was measured by a texture analyzer (TA-XT Plus, SMS Co., England). The volodkevich bite jaws (HDP/BS) probe, which simulates the action of an incisor tooth (Azeredo et al., 2006), was used in measuring the texture of stipes of mushrooms for it has been shown that most of the stipes were chewed by fore teeth. Three pieces of stipes of F. velutipes were measured each time at central parts with both ends held. Compression was performed twice (up to 90% sample thickness) at a compression rate of 2 mm/s. Three replications were conducted for each sample. Weight loss was determined by weighing the whole mushrooms before and after the storage period. Weight loss was expressed as the percentage of loss of weight with respect to the initial weight. 2.4.2. Total soluble solids (TSS) and protein content The mushrooms were ground in a mortar and squeezed with a hand press, and the juice was analyzed for the TSS contents. The TSS was measured at 25 C with a refractometer (TZ-62, Optical Instrument Factory Ltd, Guangzhou, China). The protein content was determined using a soluble protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). 2.4.3. Membrane permeability and lipid peroxidation Membrane permeability, expressed by relative electrolyte leakage, was determined by the modified method of Barman et al. (2014). The fresh mushroom discs (3 mm thick, 3 mm diameter, 20 pieces total) were placed in 40 mL distilled water. Conductivity of the suspending solution was measured at once (P0), after 10 min (P1) and thereafter boiling for 10 min (P2) with a DDS-11A electrical conductivity meter (DDS-11A, Kangyi Instrument Co., Shanghai, China). Membrane permeability was expressed according to the following formula: Membrane permeabilityð%Þ ¼ ½ðP1 P0 Þ=ðP2 P0 Þ 100%: The level of lipid peroxidation was expressed as the amount of MDA. Its content was determined with a Maleic Dialdehyde assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s instructions. The unit of MDA was expressed as mol kg1 on a protein basis. 2.4.4. O2 and H2O2 levels O2 content was determined according to the method of Elstner and Heupel (Elstner and Heupel, 1976) with slight modifications. Mushroom sample (4 g) was sliced and homogenized in 36 mL of 50 mmol/L K-phosphate buffer (pH 7.8) for 30s at 4 C. After centrifugation at 10,000g for 20 min at 4 C, the supernatant was used for the determination of free radical levels as follows: 0.5 mL supernatant, 0.5 mL 50 mmol/L K-phosphate buffer, and 1 mmol/L hydroxylamine hydrochloride were mixed
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and incubated at 25 C for 60 min, 1 mL 17 mmol/L p-aminophenyl sulfonic acid (in glacial acetic acid:H2Omol kg1= 3:1) and 7 mmol/ L a-naphthylamine (in glacial acetic acid:H2O = 3:1) were added and the mixture was incubated at 25 C for a further 20 min. Optical density was immediately measured at a wavelength of 530 nm. Na2NO2 was used for standard curve. The assay for H2O2 content was carried out as previously described by Patterson et al. (Patterson et al., 1984). Mushroom sample (2 g) was homogenized in 10 mL 100% (v/v) of acetone at 4 C. After centrifugation for 15 min at 6000 rpm at 4 C, the supernatant was collected. One ml of supernatant was mixed with 0.1 mL 5% (w/v) titanium sulphate and 0.2 mL 25% (v/v) ammonia, then centrifuged for 10 min at 6000 rpm and 4 C. The pellet was dissolved in 3 mL 10% (v/v) H2SO4 and centrifuged for 10 min at 6000 rpm. The absorbance of the supernatant was measured at 410 nm. The H2O2 content was calculated using H2O2 as a standard and expressed as mol kg1 on a fresh weight basis. 2.4.5. Antioxidant enzyme activities For assay enzyme activities, 4 g of mushroom tissue was homogenized with 36 mL of 50 mm Na2HPO4–NaH2PO4 buffer (pH 7.8) containing 0.2 mmol/L ethylene diamine tetraacetic acid (EDTA) and 2% (w/v) insoluble polyvinylpyrrolidone (PVP) under ice cold conditions. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 C and the supernatant was collected and used as the crude enzyme extract for the determination of the following enzyme activities. Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed based on its ability to inhibit photo-reduction of nitroblue tetrazolium (NBT), according to the method reported by (Zhang et al., 2008). One unit of SOD activity was defined as the amount of enzyme that would inhibit 50% of the photo-reduction of NBT at 560 nm. Catalase (CAT; EC 1.11.1.6) activity was assayed according to the method reported by (Aebi, 1984). Each CAT assay solution (3 mL) contained 15 mmol/L phosphate buffer (pH 7.0), 15 mmol/L H2O2, and 0.1 mL enzyme extract. The reaction was initiated by adding the enzyme extract. The change in the absorbance of the reaction solution at 240 nm was read every 40 s. One unit of CAT activity was defined as the amount of enzyme required to decrease the absorbance at 240 nm by 0.1 per minute. Peroxidase (POD, EC1.11.1.7) activity was assayed according to the method reported by (Zhou and Leul, 1999) with some minor modification. The guaiacol-dependent peroxidase activity was measured in the reaction mixture containing 50 mmol/L phosphate buffer (pH 7.0), 1% guaiacol, 0.4% H2O2 and 100 mL enzyme extract. The increase in absorbance reading due to guaiacol oxidation was measured at 470 nm.
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2.6. Statistical analyses Analysis of variance (ANOVA) was performed using the SAS system. To determine statistical differences, comparisons of the means between control and treatment samples were performed using Duncan’s test at a significance level of P < 0.05. 3. Results 3.1. Sensory and microstructure evaluation The sensory quality of mushrooms in different packaging materials was evaluated and the microstructure was observed as well (Figs. 1 and 2F,G). The mushrooms kept in Normal-PM exhibited a certain degree of cap opening and distinct stipe elongation after 21 days of storage at 4 C. There were lots of tiny drops of moisture on the inner surface of Normal-PM. The transmission electron micrographs of the samples showed the microstructural changes of mushroom cells after 21 days of storage. The cells of Nano-PM packed mushroom tissues (Fig. 2F) were more plump and rounded compared with those of Normal-PM packed mushroom tissues (Fig. 2G). Besides, the cell wall of Normal-PM packed mushroom tissues became much thinner and cell membrane breakage was observed at the end of storage. 3.2. Gas analysis Table 1 shows the change in package atmosphere composition over storage time. This variable was clearly influenced by packaging material and storage time, as well as by their interaction. It reflects that mushroom respiration provoked an increase in the concentration of CO2 and the consequent decrease in O2 content, leading to more anaerobic conditions. The concentration of O2 decreased sharply within 72 h and approached a steady state that fluctuated between 2.47–3.93% and 1.63–3.01% in Nano-PM and Normal-PM, respectively. Conversely, the concentration of CO2 in the headspace of all packs increased gradually with increasing storage time. This concentration reached an optimum peak of 11.02% in Normal-PM on the 12th day, which was significantly (P < 0.05) higher than that in the Nano-PM group (7.06%).
2.5. Low field nuclear magnetic resonance analysis Low field nuclear magnetic resonance measurements (LF-NMR) were performed on a 22.4 MHz NMR Analyzer PQ001 (Niumag Co., Ltd., Shanghai, China), according to the method of (Li et al., 2014). Approximately 1.5 g of mushroom samples were placed in a 15 mm NMR diameter glass tube and inserted in the NMR probe. Carr– Purcell–Meiboom–Gill (CPMG) sequences were employed to measure spin–spin relaxation time, T2. The T2 measurements were performed with a t-value of 200 ms (time between 90 and 180 pulse). Data from 8000 echoes were acquired as 32 scan repetitions. The repetition time between two successive scans was 3 s. MultiExp Inv Analysis software (Niumag Co., Ltd., Shanghai, China) was employed for data analysis. Fig. 1. Effects of Nano-PM and Normal-PM on sensory quality of F. velutipes after 21 days of storage.
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Fig. 2. Effects of Nano-PM (&) and Normal-PM (4) on MDA content (A), relative conductity (B), antioxidant enzyme activities (C, D, E) and cell microstructure (F, G) of F. velutipes. Each bar represents the mean SE of the individual samples. *P < 0.05.
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Table 1 Changes in gas composition (%, V/V) of F. velutipes stored at 4 C for 21 days.* Treatment
Storage time (days) 0
3
6
9
12
15
18
21
N P
O2 21 21
2.47 0.16a 1.63 0.08a
3.50 0.14a 2.47 0.13b
2.63 0.17a 1.70 0.10b
3.90 0.09a 2.63 0.12b
3.91 0.07a 2.80 0.08b
3.40 0.14a 2.43 0.12b
3.93 0.13a 3.01 0.10b
N P
CO2 0 0
8.37 0.18b 10.10 0.22a
7.73 0.16b 9.70 0.28a
8.13 0.17b 9.20 0.25a
7.06 0.21b 11.02 0.32a
7.03 0.11b 8.20 0.26a
7.43 0.15b 8.40 0.02a
7.07 0.19b 7.77 0.05a
Different letters in the same column indicate statistically significant difference at P<0.05 between normal-packing (P) and Nano-packing (N) sample for each time point. * Each value represents the mean of six replicates standard deviation.
3.3. Weight loss, maximum shearing forces, TSS and soluble proteins There's a strong relationship between these experimental indexes and the edible quality of mushrooms. As shown in Table 2, the weight loss and maximum shearing forces increased as the storage period progressed in both treatments. The highest weight loss occurred in the control samples, i.e., 0.62% at the end of the storage period. The maximum shearing forces of mushrooms in Nano-PM were 12.94 N on the 21th day, which was 16.46% lower than the control. The TSS contents of the control mushrooms declined rapidly during the first three days of storage, whereas the mushrooms treated with Nano-PM exhibited a slight decrease during the overall period. The lowest TSS levels were recorded in the two mushroom treatments at the end of the storage period, i.e., 4.83% and 6.02% for Normal-PM and Nano-PM, respectively. Both the Nano-PM and Normal-PM packed mushrooms exhibited slight decreases in their protein contents during the first 18 days, after which the protein decomposed rapidly. The soluble protein contents of the Nano-PM packed mushrooms were 5.40 g kg1 after 21 days of storage at 4 C, which was significantly higher (P < 0.05), than the proteins in Normal-PM packed mushrooms (4.06 g kg1).
(Fig. 2A). Mushrooms packed with Nano-PM maintained a significantly lower MDA content than the control. At the end of storage, the MDA content of Nano-PM packed mushrooms was 2.22 mol kg1, which was 29.3% lower than that in Normal-PM. The relative electrolyte leakage of F. velutipes gradually increased during the whole storage (Fig. 2B). The mushrooms packed in Normal-PM had a higher relative electrolyte leakage after 15 days of storage compared with control samples (P < 0.05). 3.5. O2 and H2O2 contents The O2 content had a marked increase in both control and mushrooms in Nano-PM during the first 9 days of storage. After that, the O2 content in Nano-PM packed mushrooms began to be flat, while in control it continued to rise slowly till the 18th day of storage (Fig. 3A). As shown in Fig. 3B, the H2O2 content took on a fluctuant process during 18 days of storage. The H2O2 content in Nano-PM packed mushrooms were 21.87 and 14.65 mmol kg1 in day 18 and 21 respectively, which were significantly lower than that in control (P < 0.05). The results showed that Nano-PM markedly inhibited the formation of O2 and the accumulation of H2O2 during storage time, compared with the control group. 3.6. Oxygen radical scavenging enzyme activities
3.4. MDA contents and relative electrolyte leakage MDA content increased rapidly during the first 6 days of storage and thereafter continuously increased throughout storage
SOD, CAT and POD are important active free-radical scavenging enzymes (Lee and Lee, 2000). The changes of these enzyme activities were shown in Fig. 2. The mushrooms stored in Nano-PM
Table 2 Weight loss, maximum shearing forces, total soluble solids and soluble proteins of F. velutipes stored at 4 C for 21 days. Treatment
Storage time (days) 0
3
6
9
12
15
18
21
N P
Weight-loss (%) n/a* n/a
0.06 0.01a 0.14 0.05a
0.17 0.09a 0.21 0.02a
0.24 0.02a 0.33 0.08a
0.27 0.02a 0.56 0.11a
0.43 0.10a 0.63 0.11a
0.50 0.09b 0.78 0.12a
0.63 0.05b 0.89 0.13a
N P
Maximum shearing forces (N) 8.43 0.07a 9.70 0.16a 8.43 0.07a 9.80 0.21a
10.29 0.17a 10.39 0.11a
10.88 0.15a 11.27 0.15a
10.29 0.09a 11.56 0.12a
10.88 0.14a 12.45 0.16a
12.35 0.13b 14.21 0.16a
12.94 0.18b 15.49 0.24a
N P
TSS (%) 8.62 0.86 8.62 0.86
8.42 1.07a 6.78 0.50a
7.51 0.24a 6.74 0.56a
7.49 1.05a 6.62 0.22a
7.41 0.91a 6.57 0.77a
6.79 0.63a 6.12 0.02a
6.67 0.99a 5.49 0.48b
6.02 0.49a 4.83 0.15b
N P
Soluble proteins (gkg1) 5.19 0.35 a 5.12 0.49a 5.19 0.35 a 5.08 0.70a
5.62 1.01a 5.38 0.32a
7.25 1.09a 7.05 1.21a
8.84 1.40a 6.82 0.78b
7.09 0.85a 5.32 0.62b
6.07 0.74a 4.41 0.69b
5.40 0.16a 4.06 0.75b
a a
Different letters indicate statistically significant differences at P<0.05 between normal-packing (P) and Nano-packing (N) sample for each time point. * n/a means not applicable. Each value represents the mean of six replicates standard deviation.
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F. Donglu et al. / Postharvest Biology and Technology 119 (2016) 49–57
Fig. 3. Effects of Nano-PM (&) and Normal-PM (4) on O2 content (A) and H2O2 content (B) of F. velutipes. Each bar represents the mean SE of the individual samples. *P < 0.05.
Fig. 4. Variations in transverse relaxation time distributions (T2) and amplitude in F. velutipes at the beginning (A) and the end of storage (B) under different treatments (Nano-PM: &, Normal-PM: 4).
maintained remarkably higher SOD activity during storage (Fig. 2C). CAT activity in both treatments increased to a peak on day 6, and then decreased during the rest of the days (Fig. 2D). The significant increase of POD activity was found in Nano-PM packed mushrooms (P < 0.05). The highest enzyme activity was detected in the mushrooms on day 3 (Fig. 2E). After 12 days of storage, there was no significant difference (P > 0.05) in POD activity between Nano-PM treatment and the control.
And the fourth peak in region III was the major part. After 21 days of storage, control and Nano-PM packed samples still display four peaks but with some change. The peaks of the relaxation times in Normal-PM packed mushrooms were smaller and led to a leftward shift compared with Nano-PM packed samples. In region III, it was interesting to find that there was a shoulder between T21 and T22 fractions and they had a tendency to merged into one (Fig. 4B).
3.7. LF-NMR analysis
4. Discussion
The changes in amplitudes of the samples as a function of the relaxation times (T2) in different packing materials are shown in Fig. 4. The relaxation time describes water status in the different compartments of the sample: the mobility, distribution, position of the water and interactions between water and macromolecules (Musse et al., 2010). At the beginning of storage, there were four distinct peaks distributed in three different regions. Region I stands for water in the cell wall; Region II represents cytoplasm water; Region III can be considered as water in the vacuole and extra cellular (Fig. 4A).
Mushrooms are highly perishable and tend to lose quality right after harvest (Wang et al., 2015). In this study, a PE packaging material that contained nano-Ag, nano-TiO2, nano-SiO2, and attapulgite was developed and applied on extending the shelf life of mushrooms. The results showed that treatment with the NanoPM improved the retention of nutritional components of F. velutipes and inhibited the cap development and stripe elongation so that mushrooms in Nano-PM maintained better storage quality than that in control after 21 days of storage (Fig. 1 and Table 2).
F. Donglu et al. / Postharvest Biology and Technology 119 (2016) 49–57
Nano-packaging technology can be considered as one kind of modified atmosphere packaging alternatives, which is an active or passive dynamic process of altering gaseous composition within a package. The nanocomposite packaging with modified atmosphere can be generated inside a package from the natural process of produce respiration and film permeability to achieve the desired gas composition over time (Farber et al., 2003). In our previous study, we tested the physical properties of the nanocomposite material (Fang et al., 2016). From the results of the previous research, we found that the water vapor and oxygen permeability of Nano-PM were 7.22 105 g/m2/s and 4.65 1010 g/m2/s/Pa, respectively, which were 10.5% and 8.0% lower compared with Normal-PM. And its longitudinal strength was enhanced by 26.9%. The patterns of changes for CO2 and O2 concentration in treatment and control mushrooms were similar during storage (Table 1). However, there was a peak of CO2 occurring on day 12th in control samples which was believed to be a result of a sharp rise in post harvest respiration that usually occurs with fruits and vegetables (Eskin and Shahidi, 2012). In Nano-PM packed mushrooms, by contrast, no noticeable peak of respiratory rate was observed after three days of storage. These significant differences in CO2 content during storage between nano-PM and normal-PM could be related to the ethylene scavenging effects of nanoparticle (De Azeredo, 2013). Similar results have been reported by Li et al. (2009), who found that the application of nanocomposite packing could suppress ethylene production during storage time as a result of adding of nano-Ag and nano-TiO2, which could decompose or oxidize ethylene into water and carbon dioxide. The process of fruit ripening and senescence is accompanied by the continuously strengthening of oxidation, which may lead to accumulation of ROS and produce oxidative damage (Rogiers et al., 1998). The accumulation of ROS resulting from an altered balance between ROS production and scavenging capacities will reduce the storage quality and marketability of fruits and vegetables (Hodges et al., 2004). The present study showed that ROS levels (i.e., O2 and H2O2) in control mushrooms continuously increased with fluctuations during storage (Fig. 3). The content of O2 and H2O2 in Nano-PM packed samples were significantly (P < 0.05) lower than those in control at middle storage stage (day 9) and late storage stage (day 18–21). In order to protect cells from ROS-induced injuries, plant tissues possess an antioxidant defence system to scavenge ROS (Duan et al., 2011). SOD could remove O2 by catalyzing this unstable product to O2 and H2O2, and the CAT and POD could dismutate H2O2 into H2O and O2 through different actions, all of which are indispensable for ROS detoxification (Gill and Tuteja, 2010). Jiang et al. (2014) reported that a kind of Nano-
55
packing combined with controlled atmosphere induced higher SOD, CAT, and POD activities, thereby limiting oxidative processes in a Chinese herbal medicine called Gynura. In our study, the activities of SOD, CAT and POD in Nano-PM packed mushrooms at the beginning of storage (day 3) were 14.86%, 18.81% and 69.68% respectively higher than that in Normal-PM (Fig. 2). These results indicated that Nano-PM treatment was beneficial in controlling ROS content by activating the antioxidant defence system, thereby alleviating oxidative damage and delaying senescence. Moreover, ROS accumulation in the cell damages membrane structure and function, triggers the membrane lipid peroxidation and induces the membrane breakdown (Juan et al., 2011). Membrane damage in mushrooms increased during storage, assessed by MDA contents and relative electrolyte leakage (Fig. 2A, B) which coincided with the increased ROS levels (Fig. 3). Our present results showed that treatment with NanoPM could reduce the increases in membrane permeability and lipid peroxidation (Fig. 2). Chomkitichai et al. (2014) found that lipoxygenase activity, conjugated diene and MDA contents were positively correlated with ROS levels (r = 0.854–0.939, 0.813– 0.887and 0.894–0.971, respectively), indicating that the extent of membrane lipid peroxidation depended on the levels of ROS. Therefore, I speculated that Nano-PM treatment maintained the membrane integrity via its ability to improve on antioxidant activity. Membrane lipid peroxidation alters membrane properties and results in cell defects such as ion leakage and cellular decompartmentation (Duan et al., 2007; Yang et al., 2009). As a result, the molecular mobility of water and biopolymers in mushrooms, which could be tested by LF-NMR, were also influenced during storage. According to previous studies (Lagnika et al., 2013), the curves can be divided into three distinct regions (I–III) based on the relaxation times (T2). Region I corresponds to the hydration monolayer where water molecules are bound to the product by strong H-bonds. Region II corresponds to the part where water is absorbed as multilayers of molecules of hydrogen bonded to the monolayer. Region III is that of the so-called “free” or solvent water. Water molecules in this region are weakly bound compared to those in regions I and II. This fraction of water is available for mould growth or dissolution of soluble solutes and for enzymatic reaction. Free water is of main interest for storage stability. Hence, region III is of highest relevance. During the 21 days of storage, water changed dynamically. The T22,T23 and T24 relaxation time in control were decreased compared with Nano-PM packed mushrooms at the end of storage (Table 3).
Table 3 Relaxation time distributions (T2) and amplitude before and after 21 days of storage in F. velutipes. Samples Before storage No-treatment
After 21 days of storage Nano-PM
Normal-PM
Peak number
Peak time (T2)
Peak area
Total area
Amplitude
Region
1 2 3 4
1.01 0.08 9.33 0.73 57.22 1.19 305.39 3.82
23.89 1.96 40.63 3.21 155.69 2.68 454.06 10.85
674.27 15.41
13.12 0.98 12.12 1.20 18.30 0.85 101.23 4.23
I II III III
1 2 3 4 1 2 3 4
0.25 0.06 12.33 0.56 65.79 6.24 351.12 3.11 0.25 0.03 7.05 1.26 49.77 5.68 305.39 9.12
98.39 3.57 44.12 2.24 182.35 6.56 572.95 5.87 53.83 2.31 30.01 1.45 190.78 3.38 567.39 2.96
897.81 9.55
15.20 0.54 12.09 0.25 34.87 0.87 127.52 1.22 10.38 1.01 10.75 0.47 25.72 1.81 102.67 2.63
I II III III I I III III
842.01 5.27
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And the T23 and T24 fractions in control samples had a tendency to merge into one (Fig. 4B). In theory, the distribution of proton relaxation times should be equivalent to the distribution of proton fractions in different micro-compartments. However, diffusion of water between neighbouring compartments can average their NMR signals. As a consequence, most water fractions in different micro-compartments cannot be distinguishable in time domain NMR spectrum, only if it exist micro-structural barriers which can hinder the inter-compartment water diffusion (Tang et al., 2000). In previous studies, the membranaceous structures of cell and organelle are considered to act as physical micro-structural barriers for the different water fractions, but they are easily destroyed with the storage duration because of the accumulated ROS damage (Duan et al., 2007; Li et al., 2014). When the physical barriers were broken, the interactions between water and macromolecules which dissolved in free water were enhanced, which would induce the LF-NMR signal change in Fig. 4B. Hence, the LFNMR application in our research made a sidewise approach to prove the effectiveness of Nano-PM on protect the integrity of the biofilm against ROS attack during storage. In conclusion, our present data indicate that the reduction in senescence development by Nano-PM treatment, in mushrooms stored at 4 C, is associated with inhibition of respiratory activity. The maintenance of integrity of biological membrane structure, resulting from the decrease in levels of ROS and the improvement on antioxidant enzymes activity by the treatment is also involved in senescence retardation in mushrooms. Furthermore, these nano-packaging materials have the advantages of simple processing and potential to be applied commercially. Nano-PM could therefore provide an attractive alternative maintain the quality of stored mushrooms. Conflict of interest None. Acknowledgments The authors acknowledge financial support from the Special Fund for Agro-scientific Research in the Public Interest (No. 201303080), the National Natural Science Foundation of China (No. 31401552) and the Natural Science Foundation of Jiangsu Province (No. BK20141009). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Aguirre, L., Frias, J.M., Barry-Ryan, C., Grogan, H., 2008. Assessing the effect of product variability on the management of the quality of mushrooms (Agaricus bisporus). Postharvest Biol. Technol. 49 (2), 247–254. Azeredo, H., Brito, E.S., Moreira, G.E., Farias, V.L., Bruno, L.M., 2006. Effect of drying and storage time on the physico-chemical properties of mango leathers. Int. J. Food Sci. Technol. 41 (6), 635–638. Barman, K., Asrey, R., Pal, R.K., Jha, S.K., Bhatia, K., 2014. Post-harvest nitric oxide treatment reduces chilling injury and enhances the shelf-life of mango (Mangifera indica L.) fruit during low-temperature storage. J. Hortic. Sci. Biotechnol. 89 (3), 253–260. Chaudhry, Q., Scotter, M., Blackburn, J., Ross, B., Boxall, A., Castle, L., Aitken, R., Watkins, R., 2008. Applications and implications of nanotechnologies for the food sector. Food Addit. Contam. 25 (3), 241–258. Chomkitichai, W., Chumyam, A., Rachtanapun, P., Uthaibutra, J., Saengnil, K., 2014. Reduction of reactive oxygen species production and membrane damage during storage of ‘Daw’ longan fruit by chlorine dioxide. Sci. Hortic. 170, 143–149. Dama, C.L., Kumar, S., Mishra, B.K., Shukla, K.B., Mathur, S., Doshi, A., 2010. Antioxidative enzymatic profile of mushrooms stored at low temperature. J. Food Sci. Technol. 47 (6), 650–655. De Azeredo, H.M., 2009. Nanocomposites for food packaging applications. Food Res. Int. 42 (9), 1240–1253. De Azeredo, H.M., 2013. Antimicrobial nanostructures in food packaging. Trends Food Sci. Technol. 30 (1), 56–69.
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