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Effect of 60Co gamma irradiation on postharvest quality and selected enzyme activities of Volvariella volvacea
Scientia Horticulturae 235 (2018) 382–390
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Effect of 60Co gamma irradiation on postharvest quality and selected enzyme activities of Volvariella volvacea ⁎
Lijuan Hou , Jinsheng Lin, Lin Ma, Shaoxuan Qu, Huiping Li, Ning Jiang
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Institute of Vegetable Crop, Jiangsu Academy of Agricultural Sciences, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing, 210014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fruit body quality Postharvest senescence Volvariella volvacea 60 Co gamma irradiation
Volvariella volvacea fruit bodies were exposed to six different dosage levels (0, 0.2, 0.4, 0.6, 0.8, and 1.0 kGy) of 60 Co gamma ray source, and then stored at 16 °C and 55–60% relative humidity for 7 d. Storage of the 0.8-kGy treatment group resulted in the highest sensory evaluation score, (increase by 51.85% than other treatments). The activity of selected enzymes involved in postharvest deterioration were also studied. The results showed that irradiation treatments have achieved significantly better commercial appearance after 7 d of storage due to slower postharvest mushroom softening, browning, weight loss (10.53%–34.73%) and respiration rate (17.20%48.72%) than control. respectively.Samples irradiated with the 0.8-kGy dose performed better than other treatments. The control showed a significantly higher malonaldehyde (MDA) level than the irradiated samples (5.5%–45.27%). Increased catalase (CAT) activity (P < 0.05) was also observed in the samples receiving doses of 0.8 and 1.0 kGy after storage for 4 and 5 d, respectively. Superoxide (SOD) dismutase activities in the irradiated samples (13.68–40.53%) were significantly higher than those of the control, while the microbial populations decreased in all irradiated samples compared with the control. These findings suggested that irradiating V. volvacea mushrooms with 0.8 kGy of 60Co gamma rays could maintain their quality.
1. Introduction Volvariella volvacea, also known as the straw mushroom or Chinese mushroom, is an edible mushroom that has been cultivated for over 300 years. V. volvacea is one of the most popular cultivated mushrooms in tropical and subtropical regions, and forms fruiting bodies at relatively high temperatures of 28–35 °C (Chen et al., 2003; Bao et al., 2013). V. volvacea is highly perishable and has a short shelf life (1–2 d) due to its high moisture content (∼90%), high respiration rate, and low textural strength (Amuthan et al., 1999). V. volvacea has low cold-resistance, with low temperatures resulting in cryogenic autolysis in both the hyphae and fruiting bodies. Cold storage (4 °C) causes autolysis in V. volvacea mycelia, with the fruiting body becoming pulpy, liquid, and even disintegrating (Chang, 1978). These characteristics reduce the shelf life of V. volvacea considerably compared with those having other mushroom-forming fungi, posing significant challenges for its global distribution and increasing the distribution cost. Accordingly, V. volvacea has the fifth-largest annual worldwide production among mushrooms (Chang, 1999). Therefore, technologies improving postharvest are much needed, such as food irradiation, that can increase shelf life without affecting nutritional value (Farkas and Mohácsi-Farkas, 2011).
Various studies have been carried out to determine the effectiveness of irradiation in improving mushroom quality and shelf life, particularly for Pleurotus eryngii (Akram et al., 2012), Lentinus edodes (Jiang et al., 2010), and Agaricus bisporus (Akram and Kwon, 2010). We recognize that earlier studies have been conducted to successfully enhance the shelf life of V. volvacea (Ye et al., 2000, Xie et al., 2005). However, these existing studies and results cannot be directly applied to our study, since we are using different strains of V. volvacea and different dosage levels. In addition, in existing studies, the effect of 60Co γirradiation on the microbial population and sensory evaluation of V. volvacea has been adequately studied. In this work, we provide comprehensive examination of the effects of different doses of irradiation on the determination indicators of postharvest characteristics of V. volvacea, including the physico-chemical parameters, such as odor, firmness, veil opening, rotting decay, color, respiratory rate, weight loss, browning, and microbiological properties, and biochemical parameters, such as MDA, SOD and CAT, during postharvest storage for 7 d at 16 °C.
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Corresponding authors. E-mail addresses: [email protected], [email protected] (L. Hou), [email protected] (J. Lin), [email protected] (L. Ma), [email protected] (S. Qu), [email protected] (H. Li), [email protected] (N. Jiang). https://doi.org/10.1016/j.scienta.2018.02.074 Received 25 August 2017; Received in revised form 20 February 2018; Accepted 26 February 2018 Available online 20 March 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
2016).
2.1. Mushroom samples
2.5. Respiratory rate
Freshly harvested V. volvacea fruit bodies were purchased from Jiangsu Jiangnan Biotechnology Co. (Danyang, Jiangsu Province, China). The selected mushrooms had good commercial quality in terms of uniformity of shape and color, and did not exhibit mechanical damage or disease. The fruiting bodies were packed in polystyrene foamchip food trays (14 × 10 × 0.45 cm), covered with food-grade polyvinylchloride film, and transported to the irradiation center of Ruidisheng Technology Company (Jiangsu Nanjing, China).
A static-measuring method at a storage temperature of 4 ± 1 °C was adopted (Li et al., 2006). The mushrooms were then sampled randomly, with 50 g of each put into a Petri dish filled with NaOH (10 mL, 0.4 mol/L) and desiccated for 30 min in an airtight desiccator (diameter, 260 mm). The respiratory rate was calculated from the volume of oxalic acid consumed after titration of NaOH with oxalic acid (C2H2O4, 0.2 mol/L). 2.6. Degree of browning
2.2. Gamma irradiation To determine the level of browning of the pellicle, 1g of pellicle from each mushroom, according to different browning indices, was homogenized with 60% methanol (2 mL), insoluble polyvinylpyrrolidone (2 g), and a small amount of silica sand. Then, 60% methanol (8 mL) was added, the sample was centrifuged at 10,000 × g for 20 min, and the supernatant was collected. The supernatant was diluted to 0.02 L using phosphate-buffered saline (pH 6.8, 0.1 mol/L), measured spectrophotometrically at 450 nm (Beckman DU-800), and categorized according to the numerical reading, which indicated the degree of browning (Jiang, 2000).
Plastic boxes containing mushroom trays (110 ± 10 g) were placed on a conveyor and irradiated with 60Co gamma radiation of 0.2, 0.4, 0.6, 0.8, and 1.0 kGy. In the irradiation center (the activity was 1.41 × 1014) of 60Co irradiation device, when irradiated after half time, change the face of the packing box in irradiation, adopting dichromate dosimeters and tracking products absorbed dose. After irradiation, the samples were stored at 16 ± 0.5 °C and 55% relative humidity (RH), and data were recorded for further analysis. After 24 h, triplicate samples were randomly sorted and analyzed based on the following procedure.
2.7. SOD, CAT activities, and MDA content assays 2.3. Sensory evaluation To determine enzymatic activities, mushroom tissues (1.0 g) were homogenized with K3PO4 buffer (9 mL, 50 mM, pH 6.8). The supernatant was collected after centrifugation for 10 min at 10,000×g and 4 °C to assay the SOD and CAT activities. The SOD activities, CAT activities and MDA content were determined using the procedures described by Zhang et al. (2008), Aebi (1984), and Gao et al. (2014), respectively, with a commercially available detection kit (Nanjing Jiancheng Bioengineering Institute, China). Units of SOD and CAT activities were defined as the units of enzyme required to inhibit the rate of nitroblue tetrazolium photoreduction by 50% and decompose 1 μmol of H2O2 at 37 °C, respectively. MDA was extracted using reaction with trichloroacetic acid, and the MDA content was determined spectrophotometrically at 450, 532, and 600 nm, as described by Gao et al. (2014). The SOD and CAT activities of the crude enzyme extract were expressed as U/mg on a protein basis. Protein content was obtained using a total protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute).
Sensory evaluation was conducted by five skilled panelists from the Department Food Science and Technology of Nanjing Agriculture University, China. The samples were ranked for mushroom odor, firmness, surface color, rotting decay, and veil opening according to a 4-point hedonic scale (Table 1). Each evaluation index was divided into four grades, corresponding to four scores, for a total of five evaluation indicators, resulting in a total score out of 20 points (Table 1). The odor index was evaluated by smell, the texture index was evaluated using the thumb and forefinger for straw mushrooms, and degrees of browning, veil opening, and decay were evaluated with the naked eye. This evaluation process was performance three times for each index with continuous observation for 5 d to record the final score. 2.4. Weight loss Weight loss was calculated by periodically weighing the whole mushrooms before and after the storage period, and then dividing the weight change during storage by the initial weight. Data was recorded as the percentage weight loss relative to the initial weight (Fang et al.,
2.8. Microbiological analysis Microbiological analysis of bacterial population, molds, and yeasts was conducted as described by Rodoni et al., 2016. Samples were diluted (1:10) to maintain different concentrations, and the total bacteria was counted by spreading the dilutions on agar plates, which were incubated at 37 °C for 48 h for bacteria counts, and 28 °C for 120 h for mold and yeast counts. Samples were observed daily for analysis, and results are displayed as the number of colony-forming units per gram (CFU g–1).
Table 1 Sensory evaluation indexes and corresponding scores for Volvariella volvacea fruit body. Sensory Evaluation
Scores and Description 1
2
3
4
Odor
No Smell of Fresh
Slight Smell Fresh
Mushroom Flavor
Color
Internal Turn Yellow Extreme Softening Veil Opening
Yellow
A Slight Browning Hard
Typical Mushroom Flavor No Browning
Firmness Veil Opening Rotting Decay
Have Water Stain On The Top Of Mushroom
Change Softening Rupture Of Membranes Have Water Stain On The Bottom Of Mushroom
Crack Shrikage
2.9. Statistical analysis
Very Hard
All determinations for each treatment were conducted at least in triplicate using an entirely randomized design. Data were presented as mean ± standard deviation and subjected to analysis using SPSS IBM 20.0. One-way analysis of variance was used to test the differences between variables for significance. Significantly different means P ≤ 0.05 using Duncan’s multiple comparisons tests. All the results were expressed as means ± standard deviation for 3 replicates.
No Veil Opening No Rotting Decay
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Fig. 1. Sensory characteristics scores of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). The data are mean values ± standard errors (n = 3).
Fig. 2. Sensory evaluation of V. volvacea after gamma irradiation and storage (16 °C and 55% RH).
Fig. 3. Weight loss of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
3. Results
among the other treatments (P < 0.01). The untreated control samples had the lowest sensory evaluation scores, with the treated samples giving scores that were 51.85% (0.8 kGy), 48.11% (1.0 kGy), 18.52% (0.2 kGy), and 3.67% (0.6 kGy) higher, respectively, than those of the control. However, treatment with 0.4 kGy of gamma irradiation gave the same sensory evaluation score as the control. All samples softened as the storage time increases. The firmness of the samples irradiated with 0.8 kGy of gamma radiation changed the least over the 7-d storage period (Fig. 2). Samples irradiated with 0.4 and 0.6 kGy of gamma radiation were softer after 3 d, but showed similar trends to the control during storage. Only the samples irradiated with 0.8 kGy of gamma radiation maintained good quality and firmness over the 7-d storage period. All irradiated samples maintained closed
3.1. Sensory evaluation The sensory quality of mushrooms stored at 16 °C for 7 d was evaluated (Figs. 1 and 2). At the end of each storage period, the untreated control mushrooms suffered from fungus spoilage, discoloration, rotting, browning, and off-odor, and had developed watery exudates. However, samples irradiated with 0.8 kGy of gamma radiation did not exhibit watery exudates, and showed no evidence of fungal spoilage and discoloration at the end of the storage period. Samples irradiated with 0.8 kGy of gamma radiation maintained the best appearance, resulted in the highest sensory evaluation score (Fig. 1) 384
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(p < 0.05). Before 7 d of storage, the samples exposed to 1.0 kGy of gamma radiation exhibited the highest respiratory rates, but the results for all samples were not significant different, while after 7 d of storage, the respiratory rates of the irradiated samples were slightly lower (average of 4.51–20.72%) than that of the control.
veils during storage. 3.2. Weight loss Weight loss during storage of the irradiated and control samples is shown in Fig. 3. The results show that the weight loss of all samples was directly proportional to the storage period. The highest weight loss was observed in the control during the 7-d storage period. The weight loss of the irradiated samples ranged from 10.53–34.74%, which was significantly less (P < 0.05) than that of the control group. Significant weight loss occurred toward the end of the storage period, with the samples treated with 0.8 kGy gamma irradiation showing a 1-d delay in weight loss compared with the control group. The results show that gamma irradiation was reasonably effective at preventing weight loss. This effect was most apparent in the samples irradiated with 0.8 kGy of gamma radiation, which exhibited the smallest weight loss during storage. Weight loss was high in the control, perhaps due to the effect of gamma radiation on the membrane structures, which could have caused water leak into intercellular spaces. The results showed that the weight losses (2.88–4.91%) of the gamma-irradiated samples were still acceptable after 7d of storage. Except for the samples irradiated with 0.6 kGy (weight loss, 5.79%) and 0.4 kGy (weight loss, 6.08%) of gamma radiation, the results were consistent with those of sensory evaluation.
3.4. Degree of browning Browning is a biochemical reaction affecting nutrition and quality during mushroom storage. As shown in Fig. 5, the degree of browning increased initially, then decreased, and finally increased again. The degree of browning increased rapidly at 1d of storage in all samples. Browning of the control, 0.4 kGy, 0.6 kGy, and 1.0 kGy samples reached a second peak after 4d. The percentage of browning in the control group was 122.61% of that after storage for 0d, and was significantly (p < 0.05) higher than those of the gamma-irradiated samples. The percentage of browning in the samples irradiated with 0.8 kGy of gamma radiation was 44.16% after 4d of storage, which was significantly (p < 0.05) lower than that of the control group. The degree of browning of the irradiated samples reached a third peak after storage for 6d. At the end of the storage period, the degree of browning was highest in the control group. 3.5. MDA content
3.3. Respiratory rate The level of lipid peroxidation was determined using the MDA content (Fig. 6). Throughout the 7-d storage period, the MDA content of the gamma-irradiated mushrooms showed a similar trend. From 0–4 d, there was no significant difference among all treatments. After 5d, the control and 0.6 kGy groups had higher MDA contents, which were significantly different (P < 0.05) to that of the 0.2 kGy group. After 6d, there were no significant differences among all treatments, with the control group having the highest MDA content, and the 0.8 kGy and 0.6 kGy groups having the lowest. After 7d, the MDA content of the control group was significantly different from those of the 0.8 kGy and 0.6 kGy groups (p < 0.05). The results showed that the MDA contents of the treated and untreated samples decreased at 2d and 5d, and increased at 4d (Fig. 6). After storage for 2d and 4d, the MDA contents of the treated and untreated samples did not exhibit any significant (p < 0.05) differences. Significant differences (p < 0.01) were observed between the control and samples irradiated with 0.6 kGy of gamma radiation as the MDA levels increased gradually during the first 5d of storage. Samples irradiated with 0.2 kGy of gamma radiation had the lowest MDA content (9.24%), which was significantly (P < 0.01) lower than that of the control. However, at the end of the storage period, the MDA content of the control group was significantly higher than those of the irradiated groups (5.5, 36.57, 45.27, 41.33, and 31.47% for samples irradiated
Changes in the respiratory rate are shown in Fig. 4. The respiratory rate in both control and gamma-irradiated samples decreased significantly from the initial high values. All treatments on day 0 showed respiratory rates of 5.181%. The respiratory rates on day 1 were 5.191% for the control, 4.299% for 0.2 kGy, 3.454% for 0.4 kGy, 2.657% for 0.6 kGy, 3.762% for 0.8 kGy, and 3.126% for 1.0 kGy. The irradiated samples decreased the respiratory rate by 17.02–48.72% compared with the control. The respiratory rate of the control group was significantly higher than those with gamma radiation treatments of 0.6 kGy, 0.8 kGy, and 1.0 kGy (p < 0.05) at 1d, while the respiratory rates of all samples were not significantly different at 2d. After 3d, the respiratory rate of the control was significantly higher than those of the 0.4 kGy, 0.6 kGy, and 0.8 kGy treatments (p < 0.05). After 4d, the control and 0.2 kGy groups showed significantly higher respiratory rates than the 0.8 kGy group (p < 0.05), with the control having the highest respiratory rate, and the 0.8 kGy group having the lowest respiratory rate, while the results of other treatments were not significantly different. After 5d, the control was significantly different to the 0.6 kGy and 0.8 kGy treatments (p < 0.05), but showed no significant differences with the 0.2 kGy, 0.4 kGy, and 1.0 kGy treatments. After 6d, the control samples had a respiratory rate significantly higher than those of all other treatments
Fig. 4. Respiratory rate of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
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Fig. 5. Degree of browning of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
0.6 kGy dosage groups. No significant differences (p < 0.05) were noted in the control, 0.2, 0.8, and 1.0 kGy groups. The control mushrooms had the highest SOD activity, followed by the 0.6, 0.4, 0.2, 1.0, and 0.8 kGy dosage groups, which had activities of 13.68, 22.98, 26.32, 31.58, and 40.53%, respectively, that of the control mushrooms. The decrease in SOD activity after 7 d of storage was significantly (40.53%) lower in the samples treated with 0.8 kGy of gamma radiation than in the control fruit bodies.
with 0.2, 0.4, 0.6, 0.8, and 1.0 kGy of gamma radiation, respectively). The results showed that 0.6 and 0.8 kGy of gamma radiation significantly (p < 0.01) inhibited MDA accumulation during postharvest storage (7d). The MDA contents were positively correlated with the reactive oxygen species (ROS) levels (Chomkitichai et al., 2014), indicating that the extent of membrane lipid peroxidation depended on the level of ROS. Therefore, we speculated that gamma irradiation of 0.6 and 0.8 kGy maintained membrane integrity through its ability to improve antioxidant activity.
3.8. Evolution of microbial populations 3.6. CAT activity The populations of total bacteria (shown in Fig. 9), and yeast and mold (shown Fig. 10) for the straw mushroom were calculated during storage. The populations of total bacteria and mold increased during storage in both the treated and untreated samples. However, gamma irradiation effectively inhibited (p < 0.05) microbial growth compared with the untreated samples. The highest bacteria counts were in the control batches, reaching a mean value of 1.98 × 109 CFU g–1, which was significantly higher (p < 0.01) than the other treatments throughout storage. Treatment with gamma irradiation significantly inhibited the mean bacteria counts during storage, as follows: 0.6 kGy, 0.03 × 109 CFU g–1; 0.4 kGy, 0.0376 × 109 CFU g–1; 0.8 kGy, 0.048 × 109 CFU g–1; 1.0 kGy, 0.436 × 109 CFU g–1; and 0.2 kGy, 0.0396 × 109 CFU g–1. Even at 7d, treatment with 0.8 kGy of gamma radiation significantly inhibited bacterial growth (p < 0.01), decreasing the bacterial counts by 1.976 × 106 CFU g–1, followed by 1.967 × 106 for 0.6 kGy, 1.932 × 106 for 1.0 kGy, 1.44 × 106 for 0.4 kGy, 0.1 × 106 CFU g–1 and for 0.2 kGy. These results showed that increasing irradiation doses had a greater inhibitory effect on the bacteria. After reaching a maximum value, the inhibitory effect on bacterial growth was weakened as the dose increased, but all gamma irradiation treatments significantly reduced the bacterial counts compared with the control. The highest yeast and mold counts were found for control batches,
The catalase (CAT) enzyme activities of the irradiated and control samples over the 7d storage period are shown in Fig. 7. CAT activity increased over from 0–2 d, decreased after 3d, increased again after 5d, decreased again after 6d, and finally increased at 7d. The results showed that 1.0 kGy and 0.8 kGy of 60Co gamma irradiation significantly (p < 0.05) increased the CAT activity of mushrooms at 5 and 4 d, respectively. 3.7. SOD activity SOD activity in both the control and irradiated samples showed a general downward trend throughout the postharvest storage until 4d (Fig. 8). There was no significant difference (p < 0.05) in SOD activity among all treatments. The SOD activity of the mushrooms irradiated with 0.4 kGy of gamma radiation reached 1,070.89 U/g, which was significantly higher (p < 0.05) than those of samples irradiated with 0.6 kGy (533.16 U/g) and 1.0 kGy (587.85 U/g) after 1d. After 2d, the SOD activities of the control and the gamma-irradiated mushrooms showed an increasing trend. The mushrooms treated with 0.4 kGy of gamma radiation exhibited the highest SOD activity, while the mushrooms treated with 0.6 kGy of gamma radiation had the lowest activity. Significant differences (p < 0.05) were observed between the 0.4 and
Fig. 6. MDA content of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
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Fig. 7. CAT activity of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
reaching a mean value of 16.54 × 1011 CFU g–1, which was significantly higher (p < 0.01) than other treatments during storage. The total mold counts in irradiated samples had a similar trend to the bacterial counts, with the mean value of yeast and mold counts after storage increasing as follows: 0.8 kGy, 0.87 × 1011 CFU g–1; 0.6 kGy, 5.03 × 1011 CFU g–1; 0.4 kGy, 7.80 × 1011 CFU g–1; and 1.0 kGy, 8.54 × 1011 CFU g–1. Compared with the control, treatment with 0.2, 0.4, 0.6, and 0.8 kGy of gamma radiation decreased the overall mold and yeast counts by 1.55 × 108, 8.78 × 108, 11.51 × 108, 15.72 × 108, and 8 × 108 CFU g–1, respectively, with 0.8 kGy treatment significantly inhibiting the growth of bacteria and mold. These results showed that V. volvacea exposed to 60Co gamma irradiation resulted in decreased total bacteria, mold, and yeast counts throughout storage compared with that the nonirradiated control.
Compared to the control treatment, an appropriate dose of 60Co gamma irradiation (0.8 kGy) resulted in a higher sensory evaluation score and maintained good quality and firmness over the 7-d storage period. (Figs. 1 and 2). Texture is the main quality measure for consumers when evaluating the freshness of mushrooms (Jiang et al., 2010). Irradiation has been reported to affect different physiological processes that influence water loss and respiratory rate (Benoit et al., 2000; Rivera et al., 2011a,b,c). According to a standard criterion, when a harvested mushroom loses 5–10% of its fresh weight, it begins to wilt and soon becomes unusable (Singh et al., 2010; Fang et al., 2015). We have observed a similar trend in weight loss for irradiated and non-irradiated samples during the 7 days of storage (Fig. 3). Each treatment resulted in different degrees of softening during storage. But, the observed decrease would certainly be higher if samples were not irradiation. The weight loss of the irradiated samples ranged from 10.53–34.74%, which was significantly less (p < 0.05) than that of the control group. Treatment with 0.8 kGy of gamma radiation resulted in the least change over the 7-d storage period (Fig. 3). The respiratory rate is crucial for determining the deterioration rate and the onset of senescence in mushrooms (Lagnika et al., 2013). It was reported that the O2 concentration decreased, and CO2 concentration increased with the storage time (Jiang et al., 2010). Mushrooms have a higher respiratory rate than other vegetables (Kwon et al., 2014). Our result showed that the respiratory rates of the irradiated samples were slightly lower (average of 4.51–20.72%) than that of the control, while after storage for 7d, (Fig. 4) our result similar to those reported previously for Hypsizygus marmoreus (Xing et al., 2007). The reason was that irradiation can reduce the respiratory rate of V.volvacea due to the delayed formation of methionine, thereby inhibiting the formation of endogenous ethylene (Xie et al., 2005)
4. Discussion Mushrooms are highly perishable and tend to lose quality right after harvest (Wang et al., 2015). A series of changes will occur such as browning, weight loss, texture changes, cap opening, stipe elongation, increase respiration rate and microbial attack (Singh et al., 2010). Compared to other mushrooms, the fruiting bodies of V. volvacea suffer from even faster quality deterioration and senescence since that grows at high temperature and humidity. Gamma-irradiation has been shown to be able to extend the postharvest shelf-life not only in fruits and vegetables, but also in fresh mushroom (Beaulieu et al., 2002; Jiang et al., 2010; Akram et al., 2012). The recommended dose is 1–3 kGy for extending the shelf-life of fresh mushroom in different countries (Fernandes et al., 2012). In our experiments, we application six doses of 60 Co γ- radiation to the fruitbodies of V.volvacea, the results found that irradiation treatments delayed the aging process of V. volvacea.
Fig. 8. SOD activity of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
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Fig. 9. Total bacteria counts (CFU g–1) in treated and untreated V. volvacea. Gamma-irradiated samples were stored at 16 °C and 55% RH. Data are mean values (n = 3).
MDA content. Our results showed that gamma radiation treatments reduced the MDA content at 2 d, which was consistent with a previous report regarding the Hypsizygus marmoreus fruit body (Xing et al., 2007). Agaricus bisporus has been reported to be highly efficient at preventing MDA accumulation during early postharvest storage (Duan et al., 2010). A positive correlation has been observed between MDA contents and ROS levels (Chomkitichai et al., 2014), which indicates that the extent of membrane lipid peroxidation also depends on the level of ROS. Therefore, we speculated that gamma radiation of 0.6 and 0.8 kGy maintained the membrane integrity due to its ability to improve antioxidant activity (Fig. 6). Similar results have been reported by Ye et al. (2000). They concluded that irradiation has inhibited the process of membrane peroxidation by maintaining the integrity of the membrane structure to slow down the process of V.volvacea aging. In both studies, the experimental materials are V.volvacea, but we use different strains and different doses. Specifically, they used the strain V84 with 0.8kGy,1.2 kGy and 1.6 kGy, while we used the strain V23, with 0.2 kGy, 0.4 kGy, .6 kGy,0.8 kGy and1.0 kGy. But, both studies obtained the same result: 0.8 kGy can significantly decrease the content of MDA. SOD is the primary enzymatic ROS scavenger that converts superoxide anions into hydrogen peroxide (Roxas et al., 2000), while CAT is an iron-containing enzyme that plays a critical role in H2O2 removal. CAT is believed to reduce postharvest deterioration by maintaining membrane integrity (Hu et al., 2015). Both SOD and CAT protect cells from the destructive effects of reactive oxygen species and constitute key components of the cellular antioxidant defense systems. These combined activities are thought to extend food freshness by protecting the integrity of membranes (Xiong et al., 2009). Our results showed that exposure to 0.8 kGy of gamma radiation caused an increase in CAT
A higher respiratory rate led to rapid nutrient use, which can result in deterioration (Ye et al., 2012). It suggests that the high respiratory rate of the control samples leads to high CO2 levels, which causes the mushroom body to produce toxic free radicals. This, in turn, leads to increase in the activation of protective enzymes, such as SOD and CAT. During prolonged storage, the free radical scavenging capacity gradually decreases, which causes faster decay. Irradiation causes the production of free radicals, which leads to an increase in the SOD activity. As the free radicals are removed, the SOD activity decreases. Browning is among the biochemical reactions affecting nutrition and quality during mushroom storage (Ye et al., 2012). Many researchers reported that browning is a result of two distinct mechanisms of phenol oxidation. One is spontaneous oxidation, the another is activation of tyrosinase, an enzyme belonging to PPO family (Mertinez and Whitaker, 1995; Singh et al., 2010). Our experiments show that the degree of browning of the control group was the highest when compared with the irradiated samples, which reduced the degree of browning at the end of the storage time (Fig. 5). Similar results have shown that use 1.2kGy irradiated to the Pleurotus nebrodensis can naturally lessen the degree of browning (Xiong et al., 2009). Regarding the effects of irradiation on color, all authors reported a delaying in browning and therefore an extension on the mushroom shelf life (Beaulieu et al., 2002; Xiong et al., 2009). Besides PPO, the activity of other enzymes, such as peroxidase (POD) and phenylalamime ammonia-lyase (PAL), is also related to the development of browning pigments (Fernandes et al., 2012). Bacteria, moulds, enzymatic activity and biochemical changes can cause spoilage during storage (Fernandes et al., 2012). MDA is produced by lipid peroxidation (Guillen Sans and Guzman Chozas, 1998). Therefore, the level of lipid peroxidation is usually measured using the
Fig. 10. Total mold and yeast counts (CFU g–1) in treated and untreated V. volvacea. Gamma-irradiated samples were stored at 16 °C and 55% RH. Data are mean values (n = 3).
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work.
(Fig.7) and SOD (Fig.8) activities compared with the control group. After irradiation, the peaks of SOD activity and MDA content appeared in the early stage of storage. We speculated that the generation of free radicals was induced by irradiation. Then, they cause the increase of SOD activity and membrane peroxidation performance. The change of MDA content also follows this similar process (Ye et al., 2000). According to Singh et al. (2010) the large bacterial population in fresh mushrooms is a major factor that significantly diminishes quality by microbial load. In this study, both of the irradiation samples and non-irradiation samples demonstrate consistent increase in the counts of bacterial, molds and yeasts during the storage time (Figs. 9 and 10). However, the treatment with irradiation was more effective in preventing the growth of microbes compared with non-irradiation. Consequently, the microbial degradation that resulted in changes of browning and softening was delayed in the irradiations samples. The mushrooms were decaying and their shelf lives are ended due to microbial spoilage. Most of existing studies reported decrease in microbial population in irradiates samples of different mushroom species, in contrast to increased microbial populations in the control during the storage period (Rivera et al., 2011a,b.c; Akshif et al., 2012). Our results were comparable with non-irradiation results, showing reduction in the populations of bacteria, mold and yeast counts by 98.8% and 95.6%, respectively, which were consistent with the results by Rivera et al. (2011a,b,c) and Dhalsamant et al. (2015). In this study, we have experimented with a wide variety of physicochemical parameters and biochemical parameters, and found that exposing fruit bodies to 0.8kGy was particularly effective. We are currently studying the effects of irradiation treatment on chemical compounds, such as nutrients including proteins, sugars and vitamins, and non- nutrients including phenolics flavonoids and flavor compounds of V.volvacea.
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5. Conclusions Our results have demonstrated that the exposure to appropriate doses of gamma radiation has an clear beneficial effect on maintaining the postharvest texture of harvested V. volvacea fruit bodies. Overall, an irradiation dose of 0.8 kGy was more efficient in reducing the softening rate than doses of 0.2, 0.4, 0.6, and 1.0 kGy during a 7-d sampling period. This indicated that gamma irradiation was reasonably effective in preventing weight loss, lowering the respiratory rate, decreasing the degree of browning, and reducing microbial contamination. 60Co irradiation also minimized the rate of MDA accumulation, with 0.8 kGy of gamma irradiation proving a particularly effective dose, reducing the adverse effects of MDA on the membrane structure of V. volvacea and, therefore, slowing down the aging process. The irradiation-mediated increase in SOD activity allows free radicals to be removed, thereby enabling the repair of mushroom tissues, which results in decreased MDA levels. Gamma irradiation treatments also significantly inhibited the growth of bacteria, mold, and yeast. Based on these results, 0.8 kGy of gamma irradiation could be used to increase the V. volvacea shelf life and maintain mushroom quality. Conflict of interest The authors declare no conflicts of interest. Acknowledgments The authors would like to acknowledge the financial support from the Jiangsu Agriculture Science and Technology Innovation Fund of Jiangsu Province (grant no. CX (13)5012) and the Applied Basic Research Programs of the Science and Technology Commission Foundation of Jiangsu Province (grant no. BK20140742). The authors are grateful to Dr. Hu Qiuhui for providing valuable suggestions for this 389
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1252–1260. Rodoni, L.M., Hasperué, J.H., Ortiz, C.M., Lemoine, M.L., Concellón, A., Vicente, A.R., 2016. Combined use of mild heat treatment and refrigeration to extend the postharvest life of organic pepper sticks, as affected by fruit maturity stage. Postharvest Biol. Technol. 117, 168–176. http://dx.doi.org/10.1016/j.postharvbio.2015.11.016. Rivera, C.S., Venturini, M.E., Oria, R., Blanco, D., 2011b. Selection of a decontamination treatment for fresh Tuber aestivum and Tuber melanosporum truffles packaged in modified atmospheres. Food Control 22, 626–632. http://dx.doi.org/10.1016/j. foodcont.2010.10.015. Rivera, C.S., Blanco, D., Marco, P., Oria, R., VenturiniM, E., 2011c. Effect of electronbeam irradiation on the shelf life, microbial popuations and sensory characteristics pf summer truffles (Tuber melanosporum) packaged under modified atmospheres. Int. J. Food Microbiol. 28, 141–148. http://dx.doi.org/10.1016/j.fm.2010.09.008. Roxas, V.P., Lodhi, S.A., Garrett, D.K., Mahan, J.R., Allen, R.D., 2000. Stress tolerance in transgenic tobacco seedlings that overexpress glutathione s-transferase/glutathione peroxidase. Plant Cell Physiol. 41, 1229–1234. http://dx.doi.org/10.1093/pcp/ pcd051. Singh, P., Langowski, H.C., Wanib, A.A., Saengerlaub, S., 2010. Recent advances in extending the shelf of fresh life of fresh Agaricus mushrooms: a review. J. Sci. Food Agric. 90, 1393–1402. http://dx.doi.org/10.1002/jsfa.3971. Wang, Z.G., Chen, L.J., Yang, H., Wang, A.J., 2015. Effect of exogenous glycine betaine on
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Effect of 60Co gamma irradiation on postharvest quality and selected enzyme activities of Volvariella volvacea ⁎
Lijuan Hou , Jinsheng Lin, Lin Ma, Shaoxuan Qu, Huiping Li, Ning Jiang
T
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Institute of Vegetable Crop, Jiangsu Academy of Agricultural Sciences, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing, 210014, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fruit body quality Postharvest senescence Volvariella volvacea 60 Co gamma irradiation
Volvariella volvacea fruit bodies were exposed to six different dosage levels (0, 0.2, 0.4, 0.6, 0.8, and 1.0 kGy) of 60 Co gamma ray source, and then stored at 16 °C and 55–60% relative humidity for 7 d. Storage of the 0.8-kGy treatment group resulted in the highest sensory evaluation score, (increase by 51.85% than other treatments). The activity of selected enzymes involved in postharvest deterioration were also studied. The results showed that irradiation treatments have achieved significantly better commercial appearance after 7 d of storage due to slower postharvest mushroom softening, browning, weight loss (10.53%–34.73%) and respiration rate (17.20%48.72%) than control. respectively.Samples irradiated with the 0.8-kGy dose performed better than other treatments. The control showed a significantly higher malonaldehyde (MDA) level than the irradiated samples (5.5%–45.27%). Increased catalase (CAT) activity (P < 0.05) was also observed in the samples receiving doses of 0.8 and 1.0 kGy after storage for 4 and 5 d, respectively. Superoxide (SOD) dismutase activities in the irradiated samples (13.68–40.53%) were significantly higher than those of the control, while the microbial populations decreased in all irradiated samples compared with the control. These findings suggested that irradiating V. volvacea mushrooms with 0.8 kGy of 60Co gamma rays could maintain their quality.
1. Introduction Volvariella volvacea, also known as the straw mushroom or Chinese mushroom, is an edible mushroom that has been cultivated for over 300 years. V. volvacea is one of the most popular cultivated mushrooms in tropical and subtropical regions, and forms fruiting bodies at relatively high temperatures of 28–35 °C (Chen et al., 2003; Bao et al., 2013). V. volvacea is highly perishable and has a short shelf life (1–2 d) due to its high moisture content (∼90%), high respiration rate, and low textural strength (Amuthan et al., 1999). V. volvacea has low cold-resistance, with low temperatures resulting in cryogenic autolysis in both the hyphae and fruiting bodies. Cold storage (4 °C) causes autolysis in V. volvacea mycelia, with the fruiting body becoming pulpy, liquid, and even disintegrating (Chang, 1978). These characteristics reduce the shelf life of V. volvacea considerably compared with those having other mushroom-forming fungi, posing significant challenges for its global distribution and increasing the distribution cost. Accordingly, V. volvacea has the fifth-largest annual worldwide production among mushrooms (Chang, 1999). Therefore, technologies improving postharvest are much needed, such as food irradiation, that can increase shelf life without affecting nutritional value (Farkas and Mohácsi-Farkas, 2011).
Various studies have been carried out to determine the effectiveness of irradiation in improving mushroom quality and shelf life, particularly for Pleurotus eryngii (Akram et al., 2012), Lentinus edodes (Jiang et al., 2010), and Agaricus bisporus (Akram and Kwon, 2010). We recognize that earlier studies have been conducted to successfully enhance the shelf life of V. volvacea (Ye et al., 2000, Xie et al., 2005). However, these existing studies and results cannot be directly applied to our study, since we are using different strains of V. volvacea and different dosage levels. In addition, in existing studies, the effect of 60Co γirradiation on the microbial population and sensory evaluation of V. volvacea has been adequately studied. In this work, we provide comprehensive examination of the effects of different doses of irradiation on the determination indicators of postharvest characteristics of V. volvacea, including the physico-chemical parameters, such as odor, firmness, veil opening, rotting decay, color, respiratory rate, weight loss, browning, and microbiological properties, and biochemical parameters, such as MDA, SOD and CAT, during postharvest storage for 7 d at 16 °C.
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Corresponding authors. E-mail addresses: [email protected], [email protected] (L. Hou), [email protected] (J. Lin), [email protected] (L. Ma), [email protected] (S. Qu), [email protected] (H. Li), [email protected] (N. Jiang). https://doi.org/10.1016/j.scienta.2018.02.074 Received 25 August 2017; Received in revised form 20 February 2018; Accepted 26 February 2018 Available online 20 March 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
2016).
2.1. Mushroom samples
2.5. Respiratory rate
Freshly harvested V. volvacea fruit bodies were purchased from Jiangsu Jiangnan Biotechnology Co. (Danyang, Jiangsu Province, China). The selected mushrooms had good commercial quality in terms of uniformity of shape and color, and did not exhibit mechanical damage or disease. The fruiting bodies were packed in polystyrene foamchip food trays (14 × 10 × 0.45 cm), covered with food-grade polyvinylchloride film, and transported to the irradiation center of Ruidisheng Technology Company (Jiangsu Nanjing, China).
A static-measuring method at a storage temperature of 4 ± 1 °C was adopted (Li et al., 2006). The mushrooms were then sampled randomly, with 50 g of each put into a Petri dish filled with NaOH (10 mL, 0.4 mol/L) and desiccated for 30 min in an airtight desiccator (diameter, 260 mm). The respiratory rate was calculated from the volume of oxalic acid consumed after titration of NaOH with oxalic acid (C2H2O4, 0.2 mol/L). 2.6. Degree of browning
2.2. Gamma irradiation To determine the level of browning of the pellicle, 1g of pellicle from each mushroom, according to different browning indices, was homogenized with 60% methanol (2 mL), insoluble polyvinylpyrrolidone (2 g), and a small amount of silica sand. Then, 60% methanol (8 mL) was added, the sample was centrifuged at 10,000 × g for 20 min, and the supernatant was collected. The supernatant was diluted to 0.02 L using phosphate-buffered saline (pH 6.8, 0.1 mol/L), measured spectrophotometrically at 450 nm (Beckman DU-800), and categorized according to the numerical reading, which indicated the degree of browning (Jiang, 2000).
Plastic boxes containing mushroom trays (110 ± 10 g) were placed on a conveyor and irradiated with 60Co gamma radiation of 0.2, 0.4, 0.6, 0.8, and 1.0 kGy. In the irradiation center (the activity was 1.41 × 1014) of 60Co irradiation device, when irradiated after half time, change the face of the packing box in irradiation, adopting dichromate dosimeters and tracking products absorbed dose. After irradiation, the samples were stored at 16 ± 0.5 °C and 55% relative humidity (RH), and data were recorded for further analysis. After 24 h, triplicate samples were randomly sorted and analyzed based on the following procedure.
2.7. SOD, CAT activities, and MDA content assays 2.3. Sensory evaluation To determine enzymatic activities, mushroom tissues (1.0 g) were homogenized with K3PO4 buffer (9 mL, 50 mM, pH 6.8). The supernatant was collected after centrifugation for 10 min at 10,000×g and 4 °C to assay the SOD and CAT activities. The SOD activities, CAT activities and MDA content were determined using the procedures described by Zhang et al. (2008), Aebi (1984), and Gao et al. (2014), respectively, with a commercially available detection kit (Nanjing Jiancheng Bioengineering Institute, China). Units of SOD and CAT activities were defined as the units of enzyme required to inhibit the rate of nitroblue tetrazolium photoreduction by 50% and decompose 1 μmol of H2O2 at 37 °C, respectively. MDA was extracted using reaction with trichloroacetic acid, and the MDA content was determined spectrophotometrically at 450, 532, and 600 nm, as described by Gao et al. (2014). The SOD and CAT activities of the crude enzyme extract were expressed as U/mg on a protein basis. Protein content was obtained using a total protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute).
Sensory evaluation was conducted by five skilled panelists from the Department Food Science and Technology of Nanjing Agriculture University, China. The samples were ranked for mushroom odor, firmness, surface color, rotting decay, and veil opening according to a 4-point hedonic scale (Table 1). Each evaluation index was divided into four grades, corresponding to four scores, for a total of five evaluation indicators, resulting in a total score out of 20 points (Table 1). The odor index was evaluated by smell, the texture index was evaluated using the thumb and forefinger for straw mushrooms, and degrees of browning, veil opening, and decay were evaluated with the naked eye. This evaluation process was performance three times for each index with continuous observation for 5 d to record the final score. 2.4. Weight loss Weight loss was calculated by periodically weighing the whole mushrooms before and after the storage period, and then dividing the weight change during storage by the initial weight. Data was recorded as the percentage weight loss relative to the initial weight (Fang et al.,
2.8. Microbiological analysis Microbiological analysis of bacterial population, molds, and yeasts was conducted as described by Rodoni et al., 2016. Samples were diluted (1:10) to maintain different concentrations, and the total bacteria was counted by spreading the dilutions on agar plates, which were incubated at 37 °C for 48 h for bacteria counts, and 28 °C for 120 h for mold and yeast counts. Samples were observed daily for analysis, and results are displayed as the number of colony-forming units per gram (CFU g–1).
Table 1 Sensory evaluation indexes and corresponding scores for Volvariella volvacea fruit body. Sensory Evaluation
Scores and Description 1
2
3
4
Odor
No Smell of Fresh
Slight Smell Fresh
Mushroom Flavor
Color
Internal Turn Yellow Extreme Softening Veil Opening
Yellow
A Slight Browning Hard
Typical Mushroom Flavor No Browning
Firmness Veil Opening Rotting Decay
Have Water Stain On The Top Of Mushroom
Change Softening Rupture Of Membranes Have Water Stain On The Bottom Of Mushroom
Crack Shrikage
2.9. Statistical analysis
Very Hard
All determinations for each treatment were conducted at least in triplicate using an entirely randomized design. Data were presented as mean ± standard deviation and subjected to analysis using SPSS IBM 20.0. One-way analysis of variance was used to test the differences between variables for significance. Significantly different means P ≤ 0.05 using Duncan’s multiple comparisons tests. All the results were expressed as means ± standard deviation for 3 replicates.
No Veil Opening No Rotting Decay
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Fig. 1. Sensory characteristics scores of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). The data are mean values ± standard errors (n = 3).
Fig. 2. Sensory evaluation of V. volvacea after gamma irradiation and storage (16 °C and 55% RH).
Fig. 3. Weight loss of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
3. Results
among the other treatments (P < 0.01). The untreated control samples had the lowest sensory evaluation scores, with the treated samples giving scores that were 51.85% (0.8 kGy), 48.11% (1.0 kGy), 18.52% (0.2 kGy), and 3.67% (0.6 kGy) higher, respectively, than those of the control. However, treatment with 0.4 kGy of gamma irradiation gave the same sensory evaluation score as the control. All samples softened as the storage time increases. The firmness of the samples irradiated with 0.8 kGy of gamma radiation changed the least over the 7-d storage period (Fig. 2). Samples irradiated with 0.4 and 0.6 kGy of gamma radiation were softer after 3 d, but showed similar trends to the control during storage. Only the samples irradiated with 0.8 kGy of gamma radiation maintained good quality and firmness over the 7-d storage period. All irradiated samples maintained closed
3.1. Sensory evaluation The sensory quality of mushrooms stored at 16 °C for 7 d was evaluated (Figs. 1 and 2). At the end of each storage period, the untreated control mushrooms suffered from fungus spoilage, discoloration, rotting, browning, and off-odor, and had developed watery exudates. However, samples irradiated with 0.8 kGy of gamma radiation did not exhibit watery exudates, and showed no evidence of fungal spoilage and discoloration at the end of the storage period. Samples irradiated with 0.8 kGy of gamma radiation maintained the best appearance, resulted in the highest sensory evaluation score (Fig. 1) 384
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(p < 0.05). Before 7 d of storage, the samples exposed to 1.0 kGy of gamma radiation exhibited the highest respiratory rates, but the results for all samples were not significant different, while after 7 d of storage, the respiratory rates of the irradiated samples were slightly lower (average of 4.51–20.72%) than that of the control.
veils during storage. 3.2. Weight loss Weight loss during storage of the irradiated and control samples is shown in Fig. 3. The results show that the weight loss of all samples was directly proportional to the storage period. The highest weight loss was observed in the control during the 7-d storage period. The weight loss of the irradiated samples ranged from 10.53–34.74%, which was significantly less (P < 0.05) than that of the control group. Significant weight loss occurred toward the end of the storage period, with the samples treated with 0.8 kGy gamma irradiation showing a 1-d delay in weight loss compared with the control group. The results show that gamma irradiation was reasonably effective at preventing weight loss. This effect was most apparent in the samples irradiated with 0.8 kGy of gamma radiation, which exhibited the smallest weight loss during storage. Weight loss was high in the control, perhaps due to the effect of gamma radiation on the membrane structures, which could have caused water leak into intercellular spaces. The results showed that the weight losses (2.88–4.91%) of the gamma-irradiated samples were still acceptable after 7d of storage. Except for the samples irradiated with 0.6 kGy (weight loss, 5.79%) and 0.4 kGy (weight loss, 6.08%) of gamma radiation, the results were consistent with those of sensory evaluation.
3.4. Degree of browning Browning is a biochemical reaction affecting nutrition and quality during mushroom storage. As shown in Fig. 5, the degree of browning increased initially, then decreased, and finally increased again. The degree of browning increased rapidly at 1d of storage in all samples. Browning of the control, 0.4 kGy, 0.6 kGy, and 1.0 kGy samples reached a second peak after 4d. The percentage of browning in the control group was 122.61% of that after storage for 0d, and was significantly (p < 0.05) higher than those of the gamma-irradiated samples. The percentage of browning in the samples irradiated with 0.8 kGy of gamma radiation was 44.16% after 4d of storage, which was significantly (p < 0.05) lower than that of the control group. The degree of browning of the irradiated samples reached a third peak after storage for 6d. At the end of the storage period, the degree of browning was highest in the control group. 3.5. MDA content
3.3. Respiratory rate The level of lipid peroxidation was determined using the MDA content (Fig. 6). Throughout the 7-d storage period, the MDA content of the gamma-irradiated mushrooms showed a similar trend. From 0–4 d, there was no significant difference among all treatments. After 5d, the control and 0.6 kGy groups had higher MDA contents, which were significantly different (P < 0.05) to that of the 0.2 kGy group. After 6d, there were no significant differences among all treatments, with the control group having the highest MDA content, and the 0.8 kGy and 0.6 kGy groups having the lowest. After 7d, the MDA content of the control group was significantly different from those of the 0.8 kGy and 0.6 kGy groups (p < 0.05). The results showed that the MDA contents of the treated and untreated samples decreased at 2d and 5d, and increased at 4d (Fig. 6). After storage for 2d and 4d, the MDA contents of the treated and untreated samples did not exhibit any significant (p < 0.05) differences. Significant differences (p < 0.01) were observed between the control and samples irradiated with 0.6 kGy of gamma radiation as the MDA levels increased gradually during the first 5d of storage. Samples irradiated with 0.2 kGy of gamma radiation had the lowest MDA content (9.24%), which was significantly (P < 0.01) lower than that of the control. However, at the end of the storage period, the MDA content of the control group was significantly higher than those of the irradiated groups (5.5, 36.57, 45.27, 41.33, and 31.47% for samples irradiated
Changes in the respiratory rate are shown in Fig. 4. The respiratory rate in both control and gamma-irradiated samples decreased significantly from the initial high values. All treatments on day 0 showed respiratory rates of 5.181%. The respiratory rates on day 1 were 5.191% for the control, 4.299% for 0.2 kGy, 3.454% for 0.4 kGy, 2.657% for 0.6 kGy, 3.762% for 0.8 kGy, and 3.126% for 1.0 kGy. The irradiated samples decreased the respiratory rate by 17.02–48.72% compared with the control. The respiratory rate of the control group was significantly higher than those with gamma radiation treatments of 0.6 kGy, 0.8 kGy, and 1.0 kGy (p < 0.05) at 1d, while the respiratory rates of all samples were not significantly different at 2d. After 3d, the respiratory rate of the control was significantly higher than those of the 0.4 kGy, 0.6 kGy, and 0.8 kGy treatments (p < 0.05). After 4d, the control and 0.2 kGy groups showed significantly higher respiratory rates than the 0.8 kGy group (p < 0.05), with the control having the highest respiratory rate, and the 0.8 kGy group having the lowest respiratory rate, while the results of other treatments were not significantly different. After 5d, the control was significantly different to the 0.6 kGy and 0.8 kGy treatments (p < 0.05), but showed no significant differences with the 0.2 kGy, 0.4 kGy, and 1.0 kGy treatments. After 6d, the control samples had a respiratory rate significantly higher than those of all other treatments
Fig. 4. Respiratory rate of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
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Fig. 5. Degree of browning of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
0.6 kGy dosage groups. No significant differences (p < 0.05) were noted in the control, 0.2, 0.8, and 1.0 kGy groups. The control mushrooms had the highest SOD activity, followed by the 0.6, 0.4, 0.2, 1.0, and 0.8 kGy dosage groups, which had activities of 13.68, 22.98, 26.32, 31.58, and 40.53%, respectively, that of the control mushrooms. The decrease in SOD activity after 7 d of storage was significantly (40.53%) lower in the samples treated with 0.8 kGy of gamma radiation than in the control fruit bodies.
with 0.2, 0.4, 0.6, 0.8, and 1.0 kGy of gamma radiation, respectively). The results showed that 0.6 and 0.8 kGy of gamma radiation significantly (p < 0.01) inhibited MDA accumulation during postharvest storage (7d). The MDA contents were positively correlated with the reactive oxygen species (ROS) levels (Chomkitichai et al., 2014), indicating that the extent of membrane lipid peroxidation depended on the level of ROS. Therefore, we speculated that gamma irradiation of 0.6 and 0.8 kGy maintained membrane integrity through its ability to improve antioxidant activity.
3.8. Evolution of microbial populations 3.6. CAT activity The populations of total bacteria (shown in Fig. 9), and yeast and mold (shown Fig. 10) for the straw mushroom were calculated during storage. The populations of total bacteria and mold increased during storage in both the treated and untreated samples. However, gamma irradiation effectively inhibited (p < 0.05) microbial growth compared with the untreated samples. The highest bacteria counts were in the control batches, reaching a mean value of 1.98 × 109 CFU g–1, which was significantly higher (p < 0.01) than the other treatments throughout storage. Treatment with gamma irradiation significantly inhibited the mean bacteria counts during storage, as follows: 0.6 kGy, 0.03 × 109 CFU g–1; 0.4 kGy, 0.0376 × 109 CFU g–1; 0.8 kGy, 0.048 × 109 CFU g–1; 1.0 kGy, 0.436 × 109 CFU g–1; and 0.2 kGy, 0.0396 × 109 CFU g–1. Even at 7d, treatment with 0.8 kGy of gamma radiation significantly inhibited bacterial growth (p < 0.01), decreasing the bacterial counts by 1.976 × 106 CFU g–1, followed by 1.967 × 106 for 0.6 kGy, 1.932 × 106 for 1.0 kGy, 1.44 × 106 for 0.4 kGy, 0.1 × 106 CFU g–1 and for 0.2 kGy. These results showed that increasing irradiation doses had a greater inhibitory effect on the bacteria. After reaching a maximum value, the inhibitory effect on bacterial growth was weakened as the dose increased, but all gamma irradiation treatments significantly reduced the bacterial counts compared with the control. The highest yeast and mold counts were found for control batches,
The catalase (CAT) enzyme activities of the irradiated and control samples over the 7d storage period are shown in Fig. 7. CAT activity increased over from 0–2 d, decreased after 3d, increased again after 5d, decreased again after 6d, and finally increased at 7d. The results showed that 1.0 kGy and 0.8 kGy of 60Co gamma irradiation significantly (p < 0.05) increased the CAT activity of mushrooms at 5 and 4 d, respectively. 3.7. SOD activity SOD activity in both the control and irradiated samples showed a general downward trend throughout the postharvest storage until 4d (Fig. 8). There was no significant difference (p < 0.05) in SOD activity among all treatments. The SOD activity of the mushrooms irradiated with 0.4 kGy of gamma radiation reached 1,070.89 U/g, which was significantly higher (p < 0.05) than those of samples irradiated with 0.6 kGy (533.16 U/g) and 1.0 kGy (587.85 U/g) after 1d. After 2d, the SOD activities of the control and the gamma-irradiated mushrooms showed an increasing trend. The mushrooms treated with 0.4 kGy of gamma radiation exhibited the highest SOD activity, while the mushrooms treated with 0.6 kGy of gamma radiation had the lowest activity. Significant differences (p < 0.05) were observed between the 0.4 and
Fig. 6. MDA content of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
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Fig. 7. CAT activity of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
reaching a mean value of 16.54 × 1011 CFU g–1, which was significantly higher (p < 0.01) than other treatments during storage. The total mold counts in irradiated samples had a similar trend to the bacterial counts, with the mean value of yeast and mold counts after storage increasing as follows: 0.8 kGy, 0.87 × 1011 CFU g–1; 0.6 kGy, 5.03 × 1011 CFU g–1; 0.4 kGy, 7.80 × 1011 CFU g–1; and 1.0 kGy, 8.54 × 1011 CFU g–1. Compared with the control, treatment with 0.2, 0.4, 0.6, and 0.8 kGy of gamma radiation decreased the overall mold and yeast counts by 1.55 × 108, 8.78 × 108, 11.51 × 108, 15.72 × 108, and 8 × 108 CFU g–1, respectively, with 0.8 kGy treatment significantly inhibiting the growth of bacteria and mold. These results showed that V. volvacea exposed to 60Co gamma irradiation resulted in decreased total bacteria, mold, and yeast counts throughout storage compared with that the nonirradiated control.
Compared to the control treatment, an appropriate dose of 60Co gamma irradiation (0.8 kGy) resulted in a higher sensory evaluation score and maintained good quality and firmness over the 7-d storage period. (Figs. 1 and 2). Texture is the main quality measure for consumers when evaluating the freshness of mushrooms (Jiang et al., 2010). Irradiation has been reported to affect different physiological processes that influence water loss and respiratory rate (Benoit et al., 2000; Rivera et al., 2011a,b,c). According to a standard criterion, when a harvested mushroom loses 5–10% of its fresh weight, it begins to wilt and soon becomes unusable (Singh et al., 2010; Fang et al., 2015). We have observed a similar trend in weight loss for irradiated and non-irradiated samples during the 7 days of storage (Fig. 3). Each treatment resulted in different degrees of softening during storage. But, the observed decrease would certainly be higher if samples were not irradiation. The weight loss of the irradiated samples ranged from 10.53–34.74%, which was significantly less (p < 0.05) than that of the control group. Treatment with 0.8 kGy of gamma radiation resulted in the least change over the 7-d storage period (Fig. 3). The respiratory rate is crucial for determining the deterioration rate and the onset of senescence in mushrooms (Lagnika et al., 2013). It was reported that the O2 concentration decreased, and CO2 concentration increased with the storage time (Jiang et al., 2010). Mushrooms have a higher respiratory rate than other vegetables (Kwon et al., 2014). Our result showed that the respiratory rates of the irradiated samples were slightly lower (average of 4.51–20.72%) than that of the control, while after storage for 7d, (Fig. 4) our result similar to those reported previously for Hypsizygus marmoreus (Xing et al., 2007). The reason was that irradiation can reduce the respiratory rate of V.volvacea due to the delayed formation of methionine, thereby inhibiting the formation of endogenous ethylene (Xie et al., 2005)
4. Discussion Mushrooms are highly perishable and tend to lose quality right after harvest (Wang et al., 2015). A series of changes will occur such as browning, weight loss, texture changes, cap opening, stipe elongation, increase respiration rate and microbial attack (Singh et al., 2010). Compared to other mushrooms, the fruiting bodies of V. volvacea suffer from even faster quality deterioration and senescence since that grows at high temperature and humidity. Gamma-irradiation has been shown to be able to extend the postharvest shelf-life not only in fruits and vegetables, but also in fresh mushroom (Beaulieu et al., 2002; Jiang et al., 2010; Akram et al., 2012). The recommended dose is 1–3 kGy for extending the shelf-life of fresh mushroom in different countries (Fernandes et al., 2012). In our experiments, we application six doses of 60 Co γ- radiation to the fruitbodies of V.volvacea, the results found that irradiation treatments delayed the aging process of V. volvacea.
Fig. 8. SOD activity of V. volvacea after gamma irradiation and storage (16 °C and 55% RH). Data are mean values ± standard errors (n = 3).
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Fig. 9. Total bacteria counts (CFU g–1) in treated and untreated V. volvacea. Gamma-irradiated samples were stored at 16 °C and 55% RH. Data are mean values (n = 3).
MDA content. Our results showed that gamma radiation treatments reduced the MDA content at 2 d, which was consistent with a previous report regarding the Hypsizygus marmoreus fruit body (Xing et al., 2007). Agaricus bisporus has been reported to be highly efficient at preventing MDA accumulation during early postharvest storage (Duan et al., 2010). A positive correlation has been observed between MDA contents and ROS levels (Chomkitichai et al., 2014), which indicates that the extent of membrane lipid peroxidation also depends on the level of ROS. Therefore, we speculated that gamma radiation of 0.6 and 0.8 kGy maintained the membrane integrity due to its ability to improve antioxidant activity (Fig. 6). Similar results have been reported by Ye et al. (2000). They concluded that irradiation has inhibited the process of membrane peroxidation by maintaining the integrity of the membrane structure to slow down the process of V.volvacea aging. In both studies, the experimental materials are V.volvacea, but we use different strains and different doses. Specifically, they used the strain V84 with 0.8kGy,1.2 kGy and 1.6 kGy, while we used the strain V23, with 0.2 kGy, 0.4 kGy, .6 kGy,0.8 kGy and1.0 kGy. But, both studies obtained the same result: 0.8 kGy can significantly decrease the content of MDA. SOD is the primary enzymatic ROS scavenger that converts superoxide anions into hydrogen peroxide (Roxas et al., 2000), while CAT is an iron-containing enzyme that plays a critical role in H2O2 removal. CAT is believed to reduce postharvest deterioration by maintaining membrane integrity (Hu et al., 2015). Both SOD and CAT protect cells from the destructive effects of reactive oxygen species and constitute key components of the cellular antioxidant defense systems. These combined activities are thought to extend food freshness by protecting the integrity of membranes (Xiong et al., 2009). Our results showed that exposure to 0.8 kGy of gamma radiation caused an increase in CAT
A higher respiratory rate led to rapid nutrient use, which can result in deterioration (Ye et al., 2012). It suggests that the high respiratory rate of the control samples leads to high CO2 levels, which causes the mushroom body to produce toxic free radicals. This, in turn, leads to increase in the activation of protective enzymes, such as SOD and CAT. During prolonged storage, the free radical scavenging capacity gradually decreases, which causes faster decay. Irradiation causes the production of free radicals, which leads to an increase in the SOD activity. As the free radicals are removed, the SOD activity decreases. Browning is among the biochemical reactions affecting nutrition and quality during mushroom storage (Ye et al., 2012). Many researchers reported that browning is a result of two distinct mechanisms of phenol oxidation. One is spontaneous oxidation, the another is activation of tyrosinase, an enzyme belonging to PPO family (Mertinez and Whitaker, 1995; Singh et al., 2010). Our experiments show that the degree of browning of the control group was the highest when compared with the irradiated samples, which reduced the degree of browning at the end of the storage time (Fig. 5). Similar results have shown that use 1.2kGy irradiated to the Pleurotus nebrodensis can naturally lessen the degree of browning (Xiong et al., 2009). Regarding the effects of irradiation on color, all authors reported a delaying in browning and therefore an extension on the mushroom shelf life (Beaulieu et al., 2002; Xiong et al., 2009). Besides PPO, the activity of other enzymes, such as peroxidase (POD) and phenylalamime ammonia-lyase (PAL), is also related to the development of browning pigments (Fernandes et al., 2012). Bacteria, moulds, enzymatic activity and biochemical changes can cause spoilage during storage (Fernandes et al., 2012). MDA is produced by lipid peroxidation (Guillen Sans and Guzman Chozas, 1998). Therefore, the level of lipid peroxidation is usually measured using the
Fig. 10. Total mold and yeast counts (CFU g–1) in treated and untreated V. volvacea. Gamma-irradiated samples were stored at 16 °C and 55% RH. Data are mean values (n = 3).
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work.
(Fig.7) and SOD (Fig.8) activities compared with the control group. After irradiation, the peaks of SOD activity and MDA content appeared in the early stage of storage. We speculated that the generation of free radicals was induced by irradiation. Then, they cause the increase of SOD activity and membrane peroxidation performance. The change of MDA content also follows this similar process (Ye et al., 2000). According to Singh et al. (2010) the large bacterial population in fresh mushrooms is a major factor that significantly diminishes quality by microbial load. In this study, both of the irradiation samples and non-irradiation samples demonstrate consistent increase in the counts of bacterial, molds and yeasts during the storage time (Figs. 9 and 10). However, the treatment with irradiation was more effective in preventing the growth of microbes compared with non-irradiation. Consequently, the microbial degradation that resulted in changes of browning and softening was delayed in the irradiations samples. The mushrooms were decaying and their shelf lives are ended due to microbial spoilage. Most of existing studies reported decrease in microbial population in irradiates samples of different mushroom species, in contrast to increased microbial populations in the control during the storage period (Rivera et al., 2011a,b.c; Akshif et al., 2012). Our results were comparable with non-irradiation results, showing reduction in the populations of bacteria, mold and yeast counts by 98.8% and 95.6%, respectively, which were consistent with the results by Rivera et al. (2011a,b,c) and Dhalsamant et al. (2015). In this study, we have experimented with a wide variety of physicochemical parameters and biochemical parameters, and found that exposing fruit bodies to 0.8kGy was particularly effective. We are currently studying the effects of irradiation treatment on chemical compounds, such as nutrients including proteins, sugars and vitamins, and non- nutrients including phenolics flavonoids and flavor compounds of V.volvacea.
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5. Conclusions Our results have demonstrated that the exposure to appropriate doses of gamma radiation has an clear beneficial effect on maintaining the postharvest texture of harvested V. volvacea fruit bodies. Overall, an irradiation dose of 0.8 kGy was more efficient in reducing the softening rate than doses of 0.2, 0.4, 0.6, and 1.0 kGy during a 7-d sampling period. This indicated that gamma irradiation was reasonably effective in preventing weight loss, lowering the respiratory rate, decreasing the degree of browning, and reducing microbial contamination. 60Co irradiation also minimized the rate of MDA accumulation, with 0.8 kGy of gamma irradiation proving a particularly effective dose, reducing the adverse effects of MDA on the membrane structure of V. volvacea and, therefore, slowing down the aging process. The irradiation-mediated increase in SOD activity allows free radicals to be removed, thereby enabling the repair of mushroom tissues, which results in decreased MDA levels. Gamma irradiation treatments also significantly inhibited the growth of bacteria, mold, and yeast. Based on these results, 0.8 kGy of gamma irradiation could be used to increase the V. volvacea shelf life and maintain mushroom quality. Conflict of interest The authors declare no conflicts of interest. Acknowledgments The authors would like to acknowledge the financial support from the Jiangsu Agriculture Science and Technology Innovation Fund of Jiangsu Province (grant no. CX (13)5012) and the Applied Basic Research Programs of the Science and Technology Commission Foundation of Jiangsu Province (grant no. BK20140742). The authors are grateful to Dr. Hu Qiuhui for providing valuable suggestions for this 389
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