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A novel chitosan alleviates pulp breakdown of harvested longan fruit by suppressing disassembly of cell wall polysaccharides
Carbohydrate Polymers 217 (2019) 126–134
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A novel chitosan alleviates pulp breakdown of harvested longan fruit by suppressing disassembly of cell wall polysaccharides Yifen Lina, Yuzhao Lina, Yixiong Lina, Mengshi Linb, Yihui Chena, Hui Wanga, Hetong Lina, a b
T
⁎
Institute of Postharvest Technology of Agricultural Products, College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China Food Science Program, Division of Food System & Bioengineering, University of Missouri, Columbia, MO 65211-5160, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Longan fruit Pulp breakdown Cell wall polysaccharides Cell wall-disassembling enzymes Chitosan
Longan pulp is an excellent source of polysaccharides and other nutrients that have many health benefits. However, longans is susceptible to pulp breakdown after harvest and loses its nutrition values. To solve this problem, this study aimed to study the effects of a novel chitosan, Kadozan, on pulp breakdown index, contents of pectin, cellulose and hemicelluloses, and activities of enzymes in longan pulp relating to disassembly of polysaccharides (XET, PE, PG, β-Gal, and cellulase). The data illustrated that, compared to the control longans, chitosan-treated longans contained higher amounts of CWM, CSP, ISP, cellulose and hemicelluloses, but exhibited lower pulp breakdown index, lower activities of cell wall-disassembling enzymes, and contained lower WSP amount. These results suggested that Kadozan with a dilution of 1:500 (VKadozan: VKadozan + Water) could significantly decrease activities of disassembling-enzymes and depolymerization of polysaccharides in cell wall, and subsequently alleviate pulp breakdown and prolong storage-life of postharvest longans.
1. Introduction Longan (Dimocarpus longan Lour.) is a unique tropical and subtropical fruit with high nutritional values and many health benefits (Lin, Lin, Chen et al., 2016; Lin, Lin, Lin et al., 2016; Lin, Chen et al., 2018; Lin, Lin, Lin, Chen et al., 2018; Zhang, Zhao, Lai, Chen, & Yang, 2018). Longan is widely cultivated in China, Thailand, Vietnam, India, and other countries (Chen et al., 2015; Lin et al., 2013, 2014). Among them, China accounts for the largest cultivation area and production of longan (Lin et al., 2013). Longan fruit is an excellent source of nutrients including polysaccharides (Holcroft, Lin, & Ketsa, 2005; Yi et al., 2012). However, in China, the harvest season of longan fruit is normally in high temperature and high humidity, causing the quality of longan fruit to deteriorate quickly after harvest (Lin et al., 2014; Lin, Lin, Lin et al., 2016; Lin, Lin, Lin, Ritenour et al., 2017; Lin, Lin, Lin, Shi et al., 2017). For example, the fruit pulp can slowly exude juice and deteriorate, which is commonly known as pulp autolysis or pulp breakdown, which greatly affect its quality and values (Wang et al., 2013; Zhong et al., 2008). Pulp breakdown of longan fruit is caused by the changes of cellular structures and texture (Holcroft et al., 2005), which is due to the degradation of cell wall polysaccharides such as pectin, cellulose, and hemicelluloses (Chen, Hung, Chen, & Lin, 2017; Chen, Sun et al.,
2017). During storage, the activities of xyloglucan endotransglycosylase (XET), pectinesterase (PE), polygalacturonase (PG), β-galactosidase (βGal), and cellulase, and other cell wall hydrolytic enzymes can catalyze the disassembly of cell wall polysaccharides, leading to the destruction of cell wall structure, which are the main causes of texture changes and loss of storability of harvested crops (Chen, Hung et al., 2017; Chen, Sun et al., 2017; Lin, Lin, Lin, Lin et al., 2018). Thus, it is of great theoretical and practical significance to investigate the mechanism of cell wall metabolism in association with pulp breakdown of postharvest longan fruit, and to find effective measures to control pulp breakdown for enhancing the quality and extending the storage period of postharvest longan fruit. Nowadays, novel and environmentally-friendly postharvest technologies are becoming more popular for the preservation of harvested crops because of increasing health consciousness of customers (Chen et al., 2015). In particular, the use of chitosan on disease control and preservation of harvested fruits and vegetables has captured extensive attention (Jiang & Li, 2001; Ma, Yang, Yan, Kennedy, & Meng, 2013; Meng, Yang, Kennedy, & Tian, 2010; Priyadarshi, Sauraj Kumar, & Negi, 2018). Chitosan is obtained by the deacetylation of chitin that is ubiquitous in nature (Romanazzi, Feliziani, Banos, & Sivakumar, 2017; Verlee, Mincke, & Stevens, 2017). Kadozan, a novel chitosan, is a
Abbreviations: PE, pectinesterase; PG, polygalacturonase; β-Gal, β-galactosidase; XET, endotransglycosylase; CWM, cell wall materials; WSP, water-soluble pectin; ISP, ionic-soluble pectin; CSP, covalent-soluble pectin; 1-MCP, 1-methylcyclopropene ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (H. Lin). https://doi.org/10.1016/j.carbpol.2019.04.053 Received 18 February 2019; Received in revised form 13 April 2019; Accepted 13 April 2019 Available online 17 April 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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testing the pulp breakdown evaluation and analysis of related indices of cell wall metabolism.
biodegradable and non-toxic compound that can be conveniently diluted to required concentrations with water without organic solvents and pH adjustment. It has been used to control diseases in harvested litchis caused by Peronophthora litchi (Jiang, Lin, Lin et al., 2018), and to enhance the storability and quality attributes of postharvest litchis (Jiang, Lin, Shi et al., 2018). Nevertheless, there is no report about the effects of Kadozan on the pulp breakdown and cell wall metabolism of postharvest longan fruit. In this study, “Fuyan” longan fruit were selected to investigate the effects of postharvest treatment by Kadozan on the development of pulp breakdown, the changes in content of cell wall polysaccharides, and activities of related degradation enzyme. The results would provide scientific basis for controlling the development of pulp breakdown, prolonging the storage period of postharvest longan fruit, as well as providing guidance for the industry to apply this technology in postharvest longan production.
2.2. Evaluation of pulp breakdown index Fifty fruits were used to evaluate the pulp breakdown index based on the method of Chen et al. (2015). Pulp breakdown was assessed by measuring the extent of the total breakdown area in pulp of 50 individual fruits using the following visual appearance scales: 0, no breakdown; 1, breakdown area < 1/4; 2, 1/4 ≤ breakdown area < 1/ 2; 3, 1/2 ≤ breakdown area < 3/4; 4, breakdown area ≥3/4. The pulp breakdown index was calculated as ∑(breakdown scale × proportion of corresponding fruit within each class). 2.3. Extraction and assays of cell wall materials The amount of cell wall materials (CWM) was measured according to the modified methods of Chen, Hung et al. (2017), Zhang et al. (2012) and Zhang, Zhao et al. (2018). Briefly, 50 g of frozen fruit pulp from 50 longan fruit were homogenized in liquid nitrogen and boiled under a reflux condenser with 200 mL of 80% (v/v) ethanol for 30 min. After centrifugation of the homogenate at 4 000 × g at 4 °C for 20 min, the residues were washed thrice in 200 mL of 80% (v/v) ethanol to clear reducing sugar, and thrice in 200 mL of acetone, and then dissolved in 50 mL of 90% (v/v) dimethyl sulfoxide at 4 °C and then held for 15 h. The filtered residues were washed with distilled water several times and dried at 40 °C for 3 d to constant weight. The estimated CWM yield was expressed as mg g−1.
2. Materials and methods 2.1. Materials and treatments The “Fuyan” longan fruit with commercial maturity were picked from a longan orchard at Nan’an City, Fujian, China, and shipped to our laboratory at Fuzhou, China. The harvested longan fruit were carefully selected to make sure that they were uniform in maturity, size, color and shape, and without any diseases and blemishes. Kadozan, a novel chitosan, was obtained from Shanghai Branch of Lytone Enterprise, Inc., China. It contains 2% chitosan with deacetylated degree of > 95% and molecular mass ranging from 20 to 30 kDa (Jiang, Lin, Shi et al., 2018). Our preliminary experiment evaluated the influence of various concentrations of Kadozan with dilutions of 1:1000, 1:750, 1:500, and 1:250 (VKadozan: VKadozan + Water) for 5 min on pulp breakdown index of longan fruit, in which VKadozan: VKadozan + Water mean the ratio of the volume of Kadozan to the total volume of Kadozan and distilled water. The samples were stockpiled at 85% RH (relative humidity) and 25 °C for 6 days. The results showed that, on storage day 6, pulp breakdown index in different doses-treated longan fruit was in the order as follows: 0.90 (VKadozan: VKadozan + Water = 1:500) < 1.43 (VKadozan: VKadozan + Water = 1:750) < 2.57 (VKadozan: VKadozan + Water = 1:250) < 3.17 (VKadozan: VKadozan + Water = 1:1000) < 3.27 (control), from which it could be found that, compared to the optimum concentration at 1:500 (VKadozan: VKadozan + Water) dilution, the poorer quality was observed in longan fruit treated with chitosan at too high concentration or too low concentration. This result was agreed with previous reports that the poorer quality and storability of harvested fruits like loquats (Ghasemnezhad, Nezhad, & Gerailoo, 2011), citruses (Chien, Sheu, & Lin, 2007) and litchis (Jiang, Lin, Shi et al., 2018) were displayed in chitosan postharvest treatment with relative higher concentration or relative lower concentration than the optimum concentration. Too high concentration of chitosan induced-poorer quality and storability of harvested fruit might result from the injury of fruit caused by the treatment of chitosan at too high concentration (Jiang, Lin, Shi et al., 2018). Consequently, the concentration of the 1:500 (VKadozan: VKadozan + Water) dilution, that is 0.004% chitosan, and immersion for 5 min was chosen for further studies. An amount of longan fruits (N = 150) were used to measure fruit attributes on day 0. Besides, more longan fruits (N = 6 000) were equally distributed into 2 groups. Then one group (3 000 longan fruits) was dipped in Kadozan with the dilution of 1:500 (VKadozan: VKadozan + Water) for 5 min, the other group (3 000 longan fruits) was used as the control group and immersed in distilled-water for 5 min. The treatedfruit were air-dried for about 1 h to remove the moisture on the surface of longan fruit, then packaged in polyethylene bags (50 fruits per bag), and finally stockpiled at 85% RH and 25 °C for 6 days. During storage, 150 fruits (3 bags) from each treatment were randomly selected for
2.4. Extraction and assays of polysaccharides Cell wall polysaccharides including pectin fractions like water-soluble pectin (WSP), ionic-soluble pectin (ISP), covalent-soluble pectin (CSP), cellulose, and hemicelluloses were separated and measured according to the modified methods of Chen, Hung et al. (2017), Chen, Sun et al. (2017), Lin, Lin, Lin, Lin et al. (2018), Wang et al. (2012), Zhang et al. (2012) and Zhang, Lin et al. (2018). 2.4.1. Extraction of cell wall polysaccharides CWM (300 mg) was homogenized and soaked in 20 mL of ultrapurified water for 6 h. The supernatants, designated as WSP, were collected after centrifugation at 10 000 × g for 10 min. The water-insoluble residues were homogenized and immersed in 20 mL of 50 mM sodium acetate buffer (pH 6.8) containing 1 M cyclohexane-trans-l,2diamine tetra-acetate (CDTA) for 6 h to obtain ISP. The CDTA-insoluble residues were extracted with 20 mL of 50 mM sodium carbonate (Na2CO3) containing 20 mM sodium borohydride for 6 h to obtain CSP. The Na2CO3-insoluble residues were then extracted with 50 mM NaOH containing 100 mM sodium borohydride for 6 h. The supernatants were designated as hemicelluloses followed by centrifugation of the homogenate at 10, 000×g for 10 min. The residues were washed thrice with 0.1 M KOH solution and 8 mM sodium sulfite to clear uronic acid and lignin, then immersed in 50 mL of 8 M KOH for 6 h. The residues were washed with 8 M KOH solution after centrifugation. The KOH-insoluble was washed in 99.99% alcohol for three times and dried under reducing pressure to constant weight, which was the yield of cellulose. 2.4.2. Measurement of pectin 0.5 mL extracted supernatant solution (WSP, ISP or CSP) was added to 0.5 mL distilled water with 5 mL concentrated sulfuric acid, and then incubated at 100 °C for 20 min, followed by cooling on ice and combination with 0.2 mL of 7 mM carbazole dissolved in 95% ethanol. The reaction mixture was placed in a dark room for 30 min, and then the absorbance at 530 nm was determined. A calibration curve was obtained using galacturonic acid as a standard. The results were expressed as mg g−1. 127
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was defined as the amount of enzyme that produced 1 μmol glucose per hour.
2.4.3. Measurement of cellulose and hemicelluloses The extracted cellulose solution or hemicelluloses residue was respectively mixed with 30 mL 2 M sulfuric acid for 5 h incubation at 100 °C, followed by cooling on ice and combination with 2 M sulfuric acid to constant volume (100 mL). Afterwards, 2 mL was drawn from 100 mL solution and added to 0.5 mL anthranone with 5 mL concentrated sulfuric acid. After incubation at 100 °C for 5 min and cooling, the absorbance at 620 nm was determined. A calibration curve was obtained using glucose 6-phosphate as a standard. The results were expressed as mg g−1.
2.5.5. β - Galactosidase activity determination The resulting supernatant served as the enzyme source, in which 1 mL was added to 5 mL of 3 mM p-nitrophenyl-β-D-galactopyranoside in 50 mM sodium acetate (pH 4.7) and incubated at 37 °C for 30 min. The reaction was terminated by adding 2 mL of 10 mM Na2CO3 solution, and the release of p-nitrophenol from the artificial substrate (pnitrophenyl-β-D-galactopyranoside) was monitored at 400 nm. Authentic p-nitrophenol was used as the standard. One unit of β-galactosidase activity was defined as the amount of enzyme that produced 1 μmol p-nitrophenol per hour.
2.5. Assays of activities of cell wall-degrading enzymes Xyloglucan endotransglycosylase (XET), pectinesterase (PE), polygalacturonase (PG), β-galactosidase (β-Gal) and cellulase were extracted and the activities were measured based on the methods of Chen, Hung et al. (2017), Chen, Sun et al. (2017); Lin, Lin, Lin, Lin et al. (2018), Zhou, Baumann, Brumer, and Teeri (2006). Five grams of pulp from 10 fruits were ground in 40 mM sodium acetate buffer (pH 5.2) containing 2% (v/v) mercaptoethanol, 100 mM NaCl and 5% (w/v) polyvinyl pyrrolidone. The homogenate was centrifuged for 20 min at 15 000 × g and 4 °C. The supernatants were collected for measuring activities of cell wall-degrading enzymes.
2.5.6. Protein content determination The protein content was measured according to the method of Braford (1976). The activities of XET, PE, PG, β-Gal and cellulase were expressed as U mg−1 protein. 2.6. Statistical analysis All the treatments were performed three times and the data were acquired. The values of the figures were displayed as the mean ± standard error. SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) was used for analyzing the data.
2.5.1. XET activity determination 0.5 mL crude enzyme extract was added to 1.3 mL of 0.1 M sodium acetate buffer (pH 5.0) with 0.2 mL of 0.1% (w/v) xylan. After incubation at 37 °C for 1 h, the reaction was combined with 0.1 M sodium acetate buffer (pH 5.0) to constant volume (10 mL). Then, 2 mL from 10 mL mixture was added to 1.5 mL 3, 5 - dinitrosalicylic acid solution for 5 min boiling water bath. After cooling on ice, the reaction mixture was blended with distilled water to 25 mL for the absorbance determination at 540 nm. A calibration curve was obtained using xylose as standard. Each micromole xylose released per hour was defined as one unit of XET.
3. Results and discussion 3.1. Chitosan-induced alteration in the pulp breakdown index of harvested longan fruit Pulp breakdown is one of phenomena of senescence of longan fruit during storage, which is the result of physiological disorder (Holcroft et al., 2005; Su, Duan, & Jiang, 2006). Many methods such as ATP (Chen et al., 2015), hydrochloric acid (Apai, 2010), and heat treatment (Zhao, Lin, Wang, Lin, & Chen, 2014) have been conducted to inhibit this problem and prolong its storage period. Fig. 1 illustrated that the pulp breakdown index of harvested longan fruit increased continuously from 0 to 6 d during storage. For the control group, the pulp breakdown index of harvested longan fruit increased slightly from 0 to 1 d, but increased rapidly from 1 to 4 d and increased slowly at the end of storage (4–6 d), which reached to 3.79,
2.5.2. PE activity determination The reaction mixture for PE measurement containing 3 mL of the crude enzyme, 5 mL of 0.2 M sodium oxalate and 10 mL of 1% (w/v) pectin was incubated at 40 °C. During the reaction, the pH of the reaction mixture was maintained at 7.4 using 0.01 M NaOH solution. The amount of 0.01 M NaOH added was recorded in 30 min. One unit of PE activity was defined as the amount of enzyme that consumed 1 mmol NaOH solution per hour. 2.5.3. PG activity determination PG activity was determined by measuring the release of reducing sugar using 2-cyanoacetamide. A volume of 1 mL of crude enzyme was added to 2 mL of 0.1% (w/v) polygalacturonic acid and 5 mL of 20 mM sodium acetate buffer (pH 4.0). After incubation at 37 °C for 30 min, the reaction was terminated by adding 2 mL of 10 mM sodium tetraborate. Then, 0.1 mL of 1% (w/v) 2-cyanoacetamide were added to the mixture that was immersed in a boiling water bath for 5 min. After cooling on ice, the absorbance at 276 nm was determined. A calibration curve was obtained using D-galacturonic acid as a standard. One unit of PG activity was expressed as the amount of enzyme that produced 1 μmol galacturonic acid per hour. 2.5.4. Cellulase activity determination A volume of 1 mL of the supernatant was added to 1 mL of 0.25% (w/v) carboxymethyl cellulose and 5 mL of 20 mM sodium acetate buffer (pH 4.0). After incubation at 37 °C for 2 h, the reaction was terminated by 2 mL of 10 mM sodium tetraborate solution and 0.1 mL of 1% (w/v) 2-cyanoacetamide, followed by incubation at 100 °C for 5 min and cooled at 20 °C for 20 min. The final reaction was monitored at 276 nm, with β-D-glucose as standard. One unit of cellulose activity
Fig. 1. Effect of chitosan on the pulp breakdown index of harvested longan fruit. The mark ** represents the significant difference (p < 0.01) between chitosan-treated longan fruit and control longan fruit on each storage day. 128
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chitosan-treated longan fruit (y = −1.5645 x + 13.912, r = −0.971, p < 0.01). These data illustrated that chitosan treatment could effectively inhibit the degradation of CWM and delay pulp breakdown of longan fruit. This result agreed with a previous study conducted by Zhao et al. (2014) in which higher content of CWM maintained in hot water-treated longan fruit contributed to alleviated occurrence of pulp breakdown.
indicating that the longan pulp was almost full decayed. For the chitosan-treated longan fruit, the pulp breakdown index remained unchanged from 0 to 1 d but increased rapidly from 1 to 5 d, followed by a slow rise. Further comparison showed that, in contrast with the control group, chitosan-treated longan fruit displayed a remarkably (p < 0.01) lower pulp breakdown index from storage day 2 to day 6, indicating that chitosan treatment could delay pulp breakdown of harvested longan fruit. This result is in agreement with previous work that chitosan enhanced the firmness, flavor and storability of fresh-cut melons (Poverenov et al., 2018) or Chinese cherry (Xin, Chen, Lai, & Yang, 2017). This result also agreed with Jiang and Li (2001), in which chitosan treatment delayed the color change of longan pericarp and maintained higher contents of total soluble solids, titratable acidity and ascorbic acid in longan pulp, and thus extend the postharvest life of longan fruit.
3.3. Chitosan-induced alterations in cell wall polysaccharides in pulp of harvested longan fruit and its relationship with chitosan-alleviated longan pulp breakdown Pectin, cellulose, and hemicellulose are the main cell wall polysaccharides. Thus, they play major roles in maintaining the texture of harvested fruits (Lin, Lin, Lin, Lin et al., 2018; Wang et al., 2018), which was further investigated in this study.
3.2. Chitosan-induced alteration of pulp CWM in harvested longan fruit and its relationship with chitosan-alleviated longan pulp breakdown
3.3.1. Pectin fractions Pectin mainly exists in intercellular layer and appears in primary cell wall. It plays an important role in maintaining the adhesion and ordered arrangement of cells, and has the greatest effect on the texture and softening of fruit (Broxterman & Schols, 2018). WSP, ISP, and CSP are different forms of pectin involving in different stages of ripening and senescence of fruit. During the process of ripening and senescence, insoluble pectin fractions including CSP and ISP will be converted to WSP (Wang et al., 2018). In this work, the CSP amount in pulp of the control group reduced sharply at the early stage of storage (0– 2 d) and then decreased slowly (Fig. 3A), the ISP content increased slightly at 0– 2 d and then decreased sharply (Fig. 3B), while the WSP content increased slightly at 0– 3 d, followed by a sharp increase (Fig. 3C). The increased WSP is mainly due to the depolymerization of matrix polysaccharides and the convert of CSP to WSP with the severing of ionic bonds. Further analysis showed that the decreased CSP content (Fig. 3A) was negatively correlated with the increased content of ISP (Fig. 3B) and WSP (Fig. 3C) in the first two days, with the correlation coefficient r values of −0.881 and −0.898, respectively. However, the decreased content of CSP (Fig. 3A) and ISP (Fig. 3B) was negatively correlated with the increased WSP content (Fig. 3C) after the first two days, with the r values of −0.973 and −0.953, respectively. These results indicated that CSP was transformed to ISP and WSP at the early stage, while CSP and ISP were continuously transformed to WSP in the middle and late stage. Furthermore, according to the correlation statistics, the increased pulp breakdown index (y) (Fig. 1) was significantly negatively correlated with the content of CSP (x1, Fig. 3A) and ISP (x2, Fig. 3B) in pulp (y = −4.386 x1 + 9.732, r1 = −0.932, p1 < 0.01; y = −23.417 x2 + 6.418, r2 = −0.897, p2 < 0.01, respectively, Table 1), and was significantly positively correlated with the WSP content (x3) (Fig. 3C) in pulp of control fruit (y = 25.253 x3 - 2.388, r3 = 0.978, p3 < 0.01, Table 1). The correlation statistics revealed that the occurrence of pulp breakdown in longan fruit was related to the degradation of pectin such as CSP and ISP. In addition, as displayed in Table 2, for chitosan treated-longan fruit, the pulp breakdown index was also significantly positively correlated with the WSP content, with the r value of 0.944, but negatively correlated with the content of CSP and ISP in pulp, with the r values of −0.900 and −0.816, respectively. Moreover, the content of CSP (Fig. 3A) and ISP (Fig. 3B) in pulp of chitosan treated-longan fruit was higher than that of the control group, but the WSP content (Fig. 3C) increased at a slower pace than that of the control group, which effectively maintained the pectin content in pulp, alleviated the degradation and damage of pulp pectin, controlled the increasing trend of WSP generated from the degradation of ISP and CSP, and slowed down the occurrence of pulp breakdown in postharvest longan fruit. These results were consistent with previous work that an increase in the WSP content as well as a decrease in the content of CSP and ISP caused by
The softening of fruit led to the dissolution of CWM or the depolymerization of its structures (Chen, Hung et al., 2017; Chen, Sun et al., 2017; Lin, Lin, Lin, Lin et al., 2018). The CWM is mainly composed of ˜90% polysaccharides and ˜10% proteins, enzymes, and fatty acids. Cell walls are involved in a series of physiological activities, such as cell support, nutrient supply, material transport, reduction of transpiration and defense functions (Broxterman & Schols, 2018). Therefore, it is of great importance to delay the degradation of CWM and to maintain the integrity of cell wall structures. Fig. 2 displayed that the content of CWM in pulp of control longan fruit decreased rapidly with the storage time progressed. Furthermore, according to the correlation analysis, the content of CWM (x) was significantly negatively correlated with the pulp breakdown index (y) as shown in Fig. 1 (y = −0.473 x + 6.494, r = −0.953, p < 0.01). Therefore, it could be inferred that the continuous decrease in the CWM content in pulp was a major factor promoting the occurrence of pulp breakdown in longan fruit. In addition, further analysis indicated that the CWM content in pulp of chitosan-treated longan fruit was always maintained at a relatively higher level than that of control longan fruit within 0–6 days during storage, demonstrating a significant (p < 0.05) difference between them during the middle and late stage of storage (2– 6 days). Moreover, the decreased CWM (x) was also significantly negatively correlated with the increased pulp breakdown index (y) in
Fig. 2. Effects of chitosan on cell wall materials (CWM) content in pulp of harvested longan fruit. The mark * represents the significant difference (p < 0.05) between chitosan-treated longan fruit and control longan fruit on each storage day. 129
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Fig. 3. Effects of chitosan on contents of covalent-soluble pectin (CSP) (A), ionic-soluble pectin (ISP) (B), and water-soluble pectin (WSP) (C) in pulp of harvested longan fruit. The mark * and ** represents the significant difference (p < 0.05 and p < 0.01, respectively) between chitosan-treated longan fruit and control longan fruit on each storage day.
microfibers. Many studies have shown that, when the pectin is degraded during the process of fruit softening, hemicellulose is constantly modified at the same time, which is manifested as the changes in the content, molecular weight, and substitution and modification of side chain, indicating that hemicellulose also involves in the development of fruit softening (Wang et al., 2013). This work showed that the amounts of cellulose and hemicellulose in pulp of longan fruit decreased during storage (Fig. 4). In addition, according to the correlation analysis, pulp breakdown index (y, Fig. 1) was significantly negatively correlated with the content of cellulose (x1, Fig. 4A) and hemicellulose (x2, Fig. 4B) in pulp of control fruit (y = −2.147 x1 + 9.372, r1 = −0.952, p1 < 0.01; y = −6.832 x2 + 8.652, r2 = −0.983, p2 < 0.01, respectively, Table 1). As displayed in Table 2, the correlations between breakdown index and cellulose or hemicellulose in chitosan-treated longan fruit was the same as that in control fruit, with the r values of −0.973 and −0.990, respectively. It could be inferred that the decreases in the content of cellulose and hemicellulose were also a major promoting factor for the
high-humidity hot air impingement blanching triggered pectin depolymerization and changed the texture profiles of grape fruit (Wang et al., 2018). It was also in agreement with that the decreased WSP content caused by chitosan treatment contributed to the improved quality of citrus fruit (Zhao, Deng, Zhou, Yao, & Zeng, 2018). 3.3.2. Cellulose and hemicellulose Cellulose and hemicellulose are another two types of polysaccharides located in the primary and secondary cell walls. Cellulose, accounting for 20%–30% of cell wall material, is a long linear chain of macromolecular polysaccharide connected by the unit of β-1,4-D-glycosidic bonds. Cellulose generally exists in the form of microfibril, which consists of long chain fibers integrated by a large numbers of hydrogen bonds, making them highly stable and not easy to be degraded (Caffall & Mohnen, 2009). Hemicellulose is mainly located in primary wall and covalently bonded to pectin. A cellulose matrix network has the important function to keep the shape and rigidity of cell wall, which is formed by adhering hemicellulose to cellulose Table 1 Correlation analysis of breakdown index in pulp of control longan fruit.
Breakdown index CWM CSP ISP WSP Cellulose Hemicellulose XET PE PG β-Gal Cellulase
Breakdown index
CWM
1 −0.953 −0.932 −0.897 0.978 −0.952 −0.983 – 0.954 0.927 0.944 0.951
1 – – – – – – – – – –
CSP
ISP
WSP
Cellulose
Hemicellulose
XET
PE
PG
β-Gal
Cellulase
1 – – – – – −0.887 −0.844 −0.887 –
1 – – – – − – – –
1 – – – 0.949 0.943 0.939 –
1 – – – – – −0.975
1 – – – – −0.933
1 – – – –
1 – – –
1 – –
1 –
1
Note: “–” means the correlation analysis is not done. 130
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Table 2 Correlation analysis of breakdown index in pulp of chitosan-treated longan fruit.
Breakdown index CWM CSP ISP WSP Cellulose Hemicellulose XET PE PG β-Gal Cellulase
Breakdown index
CWM
1 −0.971 −0.900 −0.816 0.944 −0.973 −0.990 – 0.962 0.941 0.992 0.932
1 – – – – – – – – – –
CSP
ISP
WSP
Cellulose
Hemicellulose
XET
PE
PG
β-Gal
Cellulase
1 – – – – – −0.862 −0.846 −0.894 –
1 – – – – – – – –
1 – – – 0.963 0.954 0.977 –
1 – – – – – −0.952
1 – – – – −0.962
1 – – – –
1 – – –
1 – –
1 –
1
Note: “–” means the correlation analysis is not done.
pectin, to produce pectic acid (Lin, Lin, Lin, Lin et al., 2018). PG can hydrolyze pectic acid, the degradation product of pectinic acid catalyzed by PE, to galacturonic acid (Zhao et al., 2014). β-Gal catalyzes the breakage of galactoside residue from the non-reducing end of β-1,4galactoside in pectin polysaccharide chain. It can hydrolyze galactoside, thus breaking the cross-linking structure provided by galactoside between cell wall components (Chen, Hung et al., 2017). Cellulase, sometimes also called as glucanase, is a complex family of enzymes including exo-1,4-β-D-glucannase (C1), endo-1,4-β-D-glucanase (Cx), and β-1,4-glucosidase. Under the combination of these enzymes, cellulose is hydrolyzed to amorphous cellulose, cello-oligosaccharide, and glucose. The activity of cellulase is low or undetectable in un-ripened fruit but increases significantly to make the cell wall expansion and loosening during the period of ripening and softening (Lin, Lin, Lin, Lin et al., 2018). In this work, it was found that the activities of XET, PE, PG, β-gal, and cellulase in pulp of the control longan fruit increased with prolonging storage time (Fig. 5). The present results also illustrated that XET activity in pulp of the control longan fruit remained at a high level from storage day 0 to day 2, changed slightly during day 2 and day 3, and then increased slightly (Fig. 5A), while other enzymes (Fig. 5B–E) kept at low activities during the first two days and increased afterwards. These results indicated that the overall structure of pectin-cellulosehemicellulose (PCH) in the cell wall was depolymerized by XET, making it possible for PE, PG, and β-gal to act on pectin. However, opposite results were shown in the pulp of chitosan-treated longan fruit, indicating that chitosan could lower the activity of XET to slow down the depolymerization of overall structure in cell wall, and alleviate pulp breakdown in longan fruit. Furthermore, according to the correlation analysis, the increased activities of PE (y1, Fig. 5B), PG (y2, Fig. 5C) and β-Gal (y3, Fig. 5D) were negatively related to the decreased content of CSP (x1, Fig. 3A) (y1 = −7.2585 x1 + 20.069, r1 = −0.887, p1 < 0.05; y2 = −3.8154
occurrence of pulp breakdown in postharvest longan fruit. However, compared with the control group, chitosan treatment could maintain a significant (P < 0.05) higher level of cellulose from 0 to 6 days and hemicellulose (Fig. 4) from 2 to 6 days, which further maintained cell wall structure in pulp, slowed the process of pulp breakdown, and improved the stability of longan fruit. It was in agreement with a previous study that the improved storability of citrus fruit was due to the increased cellulose content caused by the use of chitosan (Zhao et al., 2018).
3.4. Chitosan-induced alterations in activities of cell wall polysaccharidesdisassembling enzymes in pulp of harvested longan fruit and its relation to chitosan’s suppressing the disassembly of cell wall polysaccharides and alleviating longan pulp breakdown The main reason of fruit softening is the separation and the total structural change of cell wall resulting from the degradation of polysaccharide in cell wall. Moreover, the polysaccharides and structure of cell wall are gradually disassembled and collapsed under the action of cell wall hydrolase like XET, PE, PG, cellulase, and β-Gal (Chen, Hung et al., 2017; Chen, Sun et al., 2017; Lin, Lin, Lin, Lin et al., 2018; Zhou et al., 2006). XET is a newly discovered cell wall hydrolase associated with fruit softening (Zhou et al., 2006). It not only has the function of glycosylation transfer in xyloglucan in which the broken end was transferred to another non-reducing end of xyloglucan polymer, but also has the function of depolymerization and hydrolysis. However, the major role of XET is to depolymerize xyloglucan chain connecting cellulose microfibrils, cause the irreversible rupture of xyloglucan chain, and then give rise to the expansion and softness of cell wall during the process of ripeness and senescence of fruit (Zhong et al., 2008). The main function of PE is to remove the methoxyl group or hydroxyethyl group from pectinic acid, a methyl esterified derivative of galacturonic acid in
Fig. 4. Effects of chitosan on contents of cellulose (A) and hemicellulose (B) in pulp of harvested longan fruit. The mark * represents the significant difference (p < 0.05) between chitosan-treated longan fruit and control longan fruit on each storage day. 131
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Fig. 5. Effect of chitosan on activities of xyloglucan endotransglycosylase (A), pectinesterase (B), polygalacturonase (C), β-galactosidase (D) and cellulose (E) in pulp of harvested longan fruit. The mark * represents the significant difference (p < 0.05) between chitosan-treated longan fruit and control longan fruit on each storage day.
x1 + 10.489, r2 = −0.844, p2 < 0.05; y3 = −71.453 x1 + 214.79, r3 = −0.887, p3 < 0.05; respectively), but positively related to the increased WSP content (x2, Fig. 3C) (y1 = 43.389 x2 −0.3016, r1 = 0.949, p1 < 0.01; y2 = 23.825 x2 − 0.3919, r2 = 0.943, p2 < 0.01; y3 = 422.73 x2 + 15.005, r3 = 0.939, p3 < 0.01, respectively, Table 1) and the increased pulp breakdown index (x3, Fig. 1) (y1 = 1.6645 x3 + 3.9501, r1 = 0.954, p1 < 0.01; y2 = 0.8939 x3 + 1.9804, r2 = 0.927, p2 < 0.01; y3 = 16.21 x3 + 56.442, r3 = 0.944, p3 < 0.01, respectively, Table 1). The correlation results in chitosan-treated longan fruit are similar to that in control longan fruit,
but with different correlation coefficients as displayed in Table 2. These results demonstrated that the increased activities of PE, PG and β-Gal (Fig. 5B–D) enhanced the depolymerization of CSP to WSP (Fig. 3A–C), and thus accelerated the occurrence of pulp breakdown in harvested longan fruit. However, weaker activities of PE, PG, and β-Gal (Fig. 5B–D), lower pulp breakdown index (Fig. 1), as well as higher levels of CSP and ISP (Fig. 3A and B) were exhibited in the pulp of chitosan-treated longan fruit, indicating that chitosan could lower the activities of PE, PG and β-Gal, slow down the degradation of CSP and ISP, and alleviate pulp breakdown of longan fruit. It was in accordance 132
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Fig. 6. The probable mechanism of chitosan’s alleviating pulp breakdown of harvested longan fruit via acting on the disassembly of cell wall polysaccharides.
longan pulp. Moreover, the reduced enzyme activities of cell wall degrading-enzymes (XET, PE, PG, β-Gal and cellulase) were observed in pulp of chitosan-treated longan fruit. These factors acted together to stabilize the cell wall structure of longan pulp and reduce the occurrence of pulp breakdown in longan fruit. Therefore, chitosan treatment delayed the disassembly of cell wall polysaccharides and alleviated pulp breakdown of harvested longan fruit. These results indicate that Kadozan, a novel chitosan formulation, can provide an alternative approach for controlling the pulp breakdown occurrence, keeping nutritional values, and prolonging the storage period of postharvest longan fruit.
with previous reports that application of chitosan resulted in lower levels of cell wall degrading-enzymes like PE, PG, and β-Gal, as well as better cell structural integrity that might improve the storability of dragon fruit (Zahid, Maqbool, Ali, Siddiqui, & Bhatti, 2019) or citrus fruit (Zhao et al., 2018). Additionally, the increased activity of cellulase (y, Fig. 5E) was negatively related to the decreased content of cellulose (x1, Fig. 4A) and hemicelluloses (x2, Fig. 4B) (y = −30.051 x1 + 47.788, r1 = −0.975; p1 < 0.01; y = −9.3053 x2 + 50.492, r2 = −0.933, p2 < 0.01, respectively), but positively related to the increased pulp breakdown index (x3, Fig. 1) (y = 4.2085 x3 + 10.106, r1 = 0.951, p3 < 0.01). As displayed in Table 2, the correlation between cellulase and cellulose or hemicelluloses in chitosan-treated longan fruit is similar to that in control fruit, but with r values of −0.952 and −0.962, respectively. These data indicate that cellulose catalyzed the degradation of cellulose and hemicelluloses and gave rise to the occurrence of pulp breakdown of longan fruit. However, lower cellulase activity (Fig. 5E) and pulp breakdown index (Fig. 1) as well as higher levels of cellulose (Fig. 4A) and hemicelluloses (Fig. 4B) were observed in the pulp of chitosantreated longan fruit, indicating that chitosan could lower cellulase activity, slow down the degradation of cellulose and hemicelluloses, and alleviate pulp breakdown of longan fruit. These results were accorded with previous work that the reduced cellulase activity, caused by hot water or acidic electrolyzed oxidizing water or 1-methylcyclopropene (1-MCP), led to the alleviated occurrence of pulp breakdown of longan fruit (Zhao et al., 2014) or softness of blueberries (Chen, Hung et al., 2017), Huanghua pears (Chen, Sun et al., 2017, and Younai plum fruit (Lin, Lin, Lin, Lin et al., 2018). The above results revealed that postharvest application of chitosan could reduce the enzyme activities relating to the cell wall degradation, that is, inhibit the activities of XET, PE, PG, β-Gal, and cellulose. The chitosan treatment could delay the degradation of cell wall polysaccharides and stabilize the structure of cell wall in longan pulp, alleviate longan pulp breakdown, and thus maintain the quality of longan fruit. Additionally, the approximate cost of Kadozan treatment with the concentration of the 1:500 (VKadozan: VKadozan + Water) dilution, that is 0.004% chitosan, on longans is RMB 0.11 China Yuan (0.016 United States dollar) per kilogram longan fruit. From the above findings, the probable mechanism of chitosan’s alleviating pulp breakdown of harvested longan fruit via acting on the disassembly of cell wall polysaccharides was demonstrated in Fig. 6.
Acknowledgments This work was supported by the National Key Research and Development Program of China (2016YFD0400102), the National Natural Science Foundation of China (Grant Nos. 31701653, 31671914 and 30200192), the Science Fund for Distinguished Young Scholars in Universities at Fujian Province of China (Grant No. Kla18067A), and the Science Fund for Distinguished Young Scholars at Fujian Agriculture and Forestry University of China (Grant No. xjq201619). References Apai, W. (2010). Effects of fruit dipping in hydrochloric acid then rinsing in water on fruit decay and browning of longan fruit. Crop Protection, 29, 1184–1189. Braford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Broxterman, S. E., & Schols, H. A. (2018). Interactions between pectin and cellulose in primary plant cell walls. Carbohydrate Polymers, 192, 263–272. Caffall, K. H., & Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydrate Research, 344, 1879–1900. Chen, M. Y., Lin, H. T., Zhang, S., Lin, Y. F., Chen, Y. H., & Lin, Y. X. (2015). Effects of adenosine triphosphate (ATP) treatment on postharvest physiology, quality and storage behavior of longan fruit. Food and Bioprocess Technology, 8, 971–982. Chen, Y. H., Hung, Y. C., Chen, M. Y., & Lin, H. T. (2017). Effects of acidic electrolyzed oxidizing water on retarding cell wall degradation and delaying softening of blueberries during postharvest storage. LWT - Food Science and Technology, 84, 650–657. Chen, Y. H., Sun, J. Z., Lin, H. T., Hung, Y. C., Zhang, S., Lin, Y. F., et al. (2017). Paperbased 1-MCP treatment suppresses cell wall metabolism and delays softening of Huanghua pears during storage. Journal of the Science of Food and Agriculture, 97, 2547–2552. Chien, P. J., Sheu, F., & Lin, H. R. (2007). Coating citrus (Murcott tangor) fruit with low molecular weight chitosan increases postharvest quality and shelf life. Food Chemistry, 100, 1160–1164. Ghasemnezhad, M., Nezhad, M. A., & Gerailoo, S. (2011). Changes in postharvest quality of loquat (Eriobotrya japonica) fruits influenced by chitosan. Horticulture, Environment and Biotechnology, 52, 40–45. Holcroft, D. M., Lin, H. T., & Ketsa, S. (2005). Harvesting and storage. In C. M. Menzel, & G. K. Waite (Eds.). Litchi and longan: Botany, production and uses (pp. 273–295). Wallingford, UK: CAB International. Jiang, Y. M., & Li, Y. B. (2001). Effect of chitosan coating on postharvest life and quality of longan fruit. Food Chemistry, 73(139-), 143. Jiang, X. J., Lin, H. T., Lin, M. S., Chen, Y. H., Wang, H., Lin, Y. X., et al. (2018). A novel
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A novel chitosan alleviates pulp breakdown of harvested longan fruit by suppressing disassembly of cell wall polysaccharides Yifen Lina, Yuzhao Lina, Yixiong Lina, Mengshi Linb, Yihui Chena, Hui Wanga, Hetong Lina, a b
T
⁎
Institute of Postharvest Technology of Agricultural Products, College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China Food Science Program, Division of Food System & Bioengineering, University of Missouri, Columbia, MO 65211-5160, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Longan fruit Pulp breakdown Cell wall polysaccharides Cell wall-disassembling enzymes Chitosan
Longan pulp is an excellent source of polysaccharides and other nutrients that have many health benefits. However, longans is susceptible to pulp breakdown after harvest and loses its nutrition values. To solve this problem, this study aimed to study the effects of a novel chitosan, Kadozan, on pulp breakdown index, contents of pectin, cellulose and hemicelluloses, and activities of enzymes in longan pulp relating to disassembly of polysaccharides (XET, PE, PG, β-Gal, and cellulase). The data illustrated that, compared to the control longans, chitosan-treated longans contained higher amounts of CWM, CSP, ISP, cellulose and hemicelluloses, but exhibited lower pulp breakdown index, lower activities of cell wall-disassembling enzymes, and contained lower WSP amount. These results suggested that Kadozan with a dilution of 1:500 (VKadozan: VKadozan + Water) could significantly decrease activities of disassembling-enzymes and depolymerization of polysaccharides in cell wall, and subsequently alleviate pulp breakdown and prolong storage-life of postharvest longans.
1. Introduction Longan (Dimocarpus longan Lour.) is a unique tropical and subtropical fruit with high nutritional values and many health benefits (Lin, Lin, Chen et al., 2016; Lin, Lin, Lin et al., 2016; Lin, Chen et al., 2018; Lin, Lin, Lin, Chen et al., 2018; Zhang, Zhao, Lai, Chen, & Yang, 2018). Longan is widely cultivated in China, Thailand, Vietnam, India, and other countries (Chen et al., 2015; Lin et al., 2013, 2014). Among them, China accounts for the largest cultivation area and production of longan (Lin et al., 2013). Longan fruit is an excellent source of nutrients including polysaccharides (Holcroft, Lin, & Ketsa, 2005; Yi et al., 2012). However, in China, the harvest season of longan fruit is normally in high temperature and high humidity, causing the quality of longan fruit to deteriorate quickly after harvest (Lin et al., 2014; Lin, Lin, Lin et al., 2016; Lin, Lin, Lin, Ritenour et al., 2017; Lin, Lin, Lin, Shi et al., 2017). For example, the fruit pulp can slowly exude juice and deteriorate, which is commonly known as pulp autolysis or pulp breakdown, which greatly affect its quality and values (Wang et al., 2013; Zhong et al., 2008). Pulp breakdown of longan fruit is caused by the changes of cellular structures and texture (Holcroft et al., 2005), which is due to the degradation of cell wall polysaccharides such as pectin, cellulose, and hemicelluloses (Chen, Hung, Chen, & Lin, 2017; Chen, Sun et al.,
2017). During storage, the activities of xyloglucan endotransglycosylase (XET), pectinesterase (PE), polygalacturonase (PG), β-galactosidase (βGal), and cellulase, and other cell wall hydrolytic enzymes can catalyze the disassembly of cell wall polysaccharides, leading to the destruction of cell wall structure, which are the main causes of texture changes and loss of storability of harvested crops (Chen, Hung et al., 2017; Chen, Sun et al., 2017; Lin, Lin, Lin, Lin et al., 2018). Thus, it is of great theoretical and practical significance to investigate the mechanism of cell wall metabolism in association with pulp breakdown of postharvest longan fruit, and to find effective measures to control pulp breakdown for enhancing the quality and extending the storage period of postharvest longan fruit. Nowadays, novel and environmentally-friendly postharvest technologies are becoming more popular for the preservation of harvested crops because of increasing health consciousness of customers (Chen et al., 2015). In particular, the use of chitosan on disease control and preservation of harvested fruits and vegetables has captured extensive attention (Jiang & Li, 2001; Ma, Yang, Yan, Kennedy, & Meng, 2013; Meng, Yang, Kennedy, & Tian, 2010; Priyadarshi, Sauraj Kumar, & Negi, 2018). Chitosan is obtained by the deacetylation of chitin that is ubiquitous in nature (Romanazzi, Feliziani, Banos, & Sivakumar, 2017; Verlee, Mincke, & Stevens, 2017). Kadozan, a novel chitosan, is a
Abbreviations: PE, pectinesterase; PG, polygalacturonase; β-Gal, β-galactosidase; XET, endotransglycosylase; CWM, cell wall materials; WSP, water-soluble pectin; ISP, ionic-soluble pectin; CSP, covalent-soluble pectin; 1-MCP, 1-methylcyclopropene ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (H. Lin). https://doi.org/10.1016/j.carbpol.2019.04.053 Received 18 February 2019; Received in revised form 13 April 2019; Accepted 13 April 2019 Available online 17 April 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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testing the pulp breakdown evaluation and analysis of related indices of cell wall metabolism.
biodegradable and non-toxic compound that can be conveniently diluted to required concentrations with water without organic solvents and pH adjustment. It has been used to control diseases in harvested litchis caused by Peronophthora litchi (Jiang, Lin, Lin et al., 2018), and to enhance the storability and quality attributes of postharvest litchis (Jiang, Lin, Shi et al., 2018). Nevertheless, there is no report about the effects of Kadozan on the pulp breakdown and cell wall metabolism of postharvest longan fruit. In this study, “Fuyan” longan fruit were selected to investigate the effects of postharvest treatment by Kadozan on the development of pulp breakdown, the changes in content of cell wall polysaccharides, and activities of related degradation enzyme. The results would provide scientific basis for controlling the development of pulp breakdown, prolonging the storage period of postharvest longan fruit, as well as providing guidance for the industry to apply this technology in postharvest longan production.
2.2. Evaluation of pulp breakdown index Fifty fruits were used to evaluate the pulp breakdown index based on the method of Chen et al. (2015). Pulp breakdown was assessed by measuring the extent of the total breakdown area in pulp of 50 individual fruits using the following visual appearance scales: 0, no breakdown; 1, breakdown area < 1/4; 2, 1/4 ≤ breakdown area < 1/ 2; 3, 1/2 ≤ breakdown area < 3/4; 4, breakdown area ≥3/4. The pulp breakdown index was calculated as ∑(breakdown scale × proportion of corresponding fruit within each class). 2.3. Extraction and assays of cell wall materials The amount of cell wall materials (CWM) was measured according to the modified methods of Chen, Hung et al. (2017), Zhang et al. (2012) and Zhang, Zhao et al. (2018). Briefly, 50 g of frozen fruit pulp from 50 longan fruit were homogenized in liquid nitrogen and boiled under a reflux condenser with 200 mL of 80% (v/v) ethanol for 30 min. After centrifugation of the homogenate at 4 000 × g at 4 °C for 20 min, the residues were washed thrice in 200 mL of 80% (v/v) ethanol to clear reducing sugar, and thrice in 200 mL of acetone, and then dissolved in 50 mL of 90% (v/v) dimethyl sulfoxide at 4 °C and then held for 15 h. The filtered residues were washed with distilled water several times and dried at 40 °C for 3 d to constant weight. The estimated CWM yield was expressed as mg g−1.
2. Materials and methods 2.1. Materials and treatments The “Fuyan” longan fruit with commercial maturity were picked from a longan orchard at Nan’an City, Fujian, China, and shipped to our laboratory at Fuzhou, China. The harvested longan fruit were carefully selected to make sure that they were uniform in maturity, size, color and shape, and without any diseases and blemishes. Kadozan, a novel chitosan, was obtained from Shanghai Branch of Lytone Enterprise, Inc., China. It contains 2% chitosan with deacetylated degree of > 95% and molecular mass ranging from 20 to 30 kDa (Jiang, Lin, Shi et al., 2018). Our preliminary experiment evaluated the influence of various concentrations of Kadozan with dilutions of 1:1000, 1:750, 1:500, and 1:250 (VKadozan: VKadozan + Water) for 5 min on pulp breakdown index of longan fruit, in which VKadozan: VKadozan + Water mean the ratio of the volume of Kadozan to the total volume of Kadozan and distilled water. The samples were stockpiled at 85% RH (relative humidity) and 25 °C for 6 days. The results showed that, on storage day 6, pulp breakdown index in different doses-treated longan fruit was in the order as follows: 0.90 (VKadozan: VKadozan + Water = 1:500) < 1.43 (VKadozan: VKadozan + Water = 1:750) < 2.57 (VKadozan: VKadozan + Water = 1:250) < 3.17 (VKadozan: VKadozan + Water = 1:1000) < 3.27 (control), from which it could be found that, compared to the optimum concentration at 1:500 (VKadozan: VKadozan + Water) dilution, the poorer quality was observed in longan fruit treated with chitosan at too high concentration or too low concentration. This result was agreed with previous reports that the poorer quality and storability of harvested fruits like loquats (Ghasemnezhad, Nezhad, & Gerailoo, 2011), citruses (Chien, Sheu, & Lin, 2007) and litchis (Jiang, Lin, Shi et al., 2018) were displayed in chitosan postharvest treatment with relative higher concentration or relative lower concentration than the optimum concentration. Too high concentration of chitosan induced-poorer quality and storability of harvested fruit might result from the injury of fruit caused by the treatment of chitosan at too high concentration (Jiang, Lin, Shi et al., 2018). Consequently, the concentration of the 1:500 (VKadozan: VKadozan + Water) dilution, that is 0.004% chitosan, and immersion for 5 min was chosen for further studies. An amount of longan fruits (N = 150) were used to measure fruit attributes on day 0. Besides, more longan fruits (N = 6 000) were equally distributed into 2 groups. Then one group (3 000 longan fruits) was dipped in Kadozan with the dilution of 1:500 (VKadozan: VKadozan + Water) for 5 min, the other group (3 000 longan fruits) was used as the control group and immersed in distilled-water for 5 min. The treatedfruit were air-dried for about 1 h to remove the moisture on the surface of longan fruit, then packaged in polyethylene bags (50 fruits per bag), and finally stockpiled at 85% RH and 25 °C for 6 days. During storage, 150 fruits (3 bags) from each treatment were randomly selected for
2.4. Extraction and assays of polysaccharides Cell wall polysaccharides including pectin fractions like water-soluble pectin (WSP), ionic-soluble pectin (ISP), covalent-soluble pectin (CSP), cellulose, and hemicelluloses were separated and measured according to the modified methods of Chen, Hung et al. (2017), Chen, Sun et al. (2017), Lin, Lin, Lin, Lin et al. (2018), Wang et al. (2012), Zhang et al. (2012) and Zhang, Lin et al. (2018). 2.4.1. Extraction of cell wall polysaccharides CWM (300 mg) was homogenized and soaked in 20 mL of ultrapurified water for 6 h. The supernatants, designated as WSP, were collected after centrifugation at 10 000 × g for 10 min. The water-insoluble residues were homogenized and immersed in 20 mL of 50 mM sodium acetate buffer (pH 6.8) containing 1 M cyclohexane-trans-l,2diamine tetra-acetate (CDTA) for 6 h to obtain ISP. The CDTA-insoluble residues were extracted with 20 mL of 50 mM sodium carbonate (Na2CO3) containing 20 mM sodium borohydride for 6 h to obtain CSP. The Na2CO3-insoluble residues were then extracted with 50 mM NaOH containing 100 mM sodium borohydride for 6 h. The supernatants were designated as hemicelluloses followed by centrifugation of the homogenate at 10, 000×g for 10 min. The residues were washed thrice with 0.1 M KOH solution and 8 mM sodium sulfite to clear uronic acid and lignin, then immersed in 50 mL of 8 M KOH for 6 h. The residues were washed with 8 M KOH solution after centrifugation. The KOH-insoluble was washed in 99.99% alcohol for three times and dried under reducing pressure to constant weight, which was the yield of cellulose. 2.4.2. Measurement of pectin 0.5 mL extracted supernatant solution (WSP, ISP or CSP) was added to 0.5 mL distilled water with 5 mL concentrated sulfuric acid, and then incubated at 100 °C for 20 min, followed by cooling on ice and combination with 0.2 mL of 7 mM carbazole dissolved in 95% ethanol. The reaction mixture was placed in a dark room for 30 min, and then the absorbance at 530 nm was determined. A calibration curve was obtained using galacturonic acid as a standard. The results were expressed as mg g−1. 127
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was defined as the amount of enzyme that produced 1 μmol glucose per hour.
2.4.3. Measurement of cellulose and hemicelluloses The extracted cellulose solution or hemicelluloses residue was respectively mixed with 30 mL 2 M sulfuric acid for 5 h incubation at 100 °C, followed by cooling on ice and combination with 2 M sulfuric acid to constant volume (100 mL). Afterwards, 2 mL was drawn from 100 mL solution and added to 0.5 mL anthranone with 5 mL concentrated sulfuric acid. After incubation at 100 °C for 5 min and cooling, the absorbance at 620 nm was determined. A calibration curve was obtained using glucose 6-phosphate as a standard. The results were expressed as mg g−1.
2.5.5. β - Galactosidase activity determination The resulting supernatant served as the enzyme source, in which 1 mL was added to 5 mL of 3 mM p-nitrophenyl-β-D-galactopyranoside in 50 mM sodium acetate (pH 4.7) and incubated at 37 °C for 30 min. The reaction was terminated by adding 2 mL of 10 mM Na2CO3 solution, and the release of p-nitrophenol from the artificial substrate (pnitrophenyl-β-D-galactopyranoside) was monitored at 400 nm. Authentic p-nitrophenol was used as the standard. One unit of β-galactosidase activity was defined as the amount of enzyme that produced 1 μmol p-nitrophenol per hour.
2.5. Assays of activities of cell wall-degrading enzymes Xyloglucan endotransglycosylase (XET), pectinesterase (PE), polygalacturonase (PG), β-galactosidase (β-Gal) and cellulase were extracted and the activities were measured based on the methods of Chen, Hung et al. (2017), Chen, Sun et al. (2017); Lin, Lin, Lin, Lin et al. (2018), Zhou, Baumann, Brumer, and Teeri (2006). Five grams of pulp from 10 fruits were ground in 40 mM sodium acetate buffer (pH 5.2) containing 2% (v/v) mercaptoethanol, 100 mM NaCl and 5% (w/v) polyvinyl pyrrolidone. The homogenate was centrifuged for 20 min at 15 000 × g and 4 °C. The supernatants were collected for measuring activities of cell wall-degrading enzymes.
2.5.6. Protein content determination The protein content was measured according to the method of Braford (1976). The activities of XET, PE, PG, β-Gal and cellulase were expressed as U mg−1 protein. 2.6. Statistical analysis All the treatments were performed three times and the data were acquired. The values of the figures were displayed as the mean ± standard error. SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) was used for analyzing the data.
2.5.1. XET activity determination 0.5 mL crude enzyme extract was added to 1.3 mL of 0.1 M sodium acetate buffer (pH 5.0) with 0.2 mL of 0.1% (w/v) xylan. After incubation at 37 °C for 1 h, the reaction was combined with 0.1 M sodium acetate buffer (pH 5.0) to constant volume (10 mL). Then, 2 mL from 10 mL mixture was added to 1.5 mL 3, 5 - dinitrosalicylic acid solution for 5 min boiling water bath. After cooling on ice, the reaction mixture was blended with distilled water to 25 mL for the absorbance determination at 540 nm. A calibration curve was obtained using xylose as standard. Each micromole xylose released per hour was defined as one unit of XET.
3. Results and discussion 3.1. Chitosan-induced alteration in the pulp breakdown index of harvested longan fruit Pulp breakdown is one of phenomena of senescence of longan fruit during storage, which is the result of physiological disorder (Holcroft et al., 2005; Su, Duan, & Jiang, 2006). Many methods such as ATP (Chen et al., 2015), hydrochloric acid (Apai, 2010), and heat treatment (Zhao, Lin, Wang, Lin, & Chen, 2014) have been conducted to inhibit this problem and prolong its storage period. Fig. 1 illustrated that the pulp breakdown index of harvested longan fruit increased continuously from 0 to 6 d during storage. For the control group, the pulp breakdown index of harvested longan fruit increased slightly from 0 to 1 d, but increased rapidly from 1 to 4 d and increased slowly at the end of storage (4–6 d), which reached to 3.79,
2.5.2. PE activity determination The reaction mixture for PE measurement containing 3 mL of the crude enzyme, 5 mL of 0.2 M sodium oxalate and 10 mL of 1% (w/v) pectin was incubated at 40 °C. During the reaction, the pH of the reaction mixture was maintained at 7.4 using 0.01 M NaOH solution. The amount of 0.01 M NaOH added was recorded in 30 min. One unit of PE activity was defined as the amount of enzyme that consumed 1 mmol NaOH solution per hour. 2.5.3. PG activity determination PG activity was determined by measuring the release of reducing sugar using 2-cyanoacetamide. A volume of 1 mL of crude enzyme was added to 2 mL of 0.1% (w/v) polygalacturonic acid and 5 mL of 20 mM sodium acetate buffer (pH 4.0). After incubation at 37 °C for 30 min, the reaction was terminated by adding 2 mL of 10 mM sodium tetraborate. Then, 0.1 mL of 1% (w/v) 2-cyanoacetamide were added to the mixture that was immersed in a boiling water bath for 5 min. After cooling on ice, the absorbance at 276 nm was determined. A calibration curve was obtained using D-galacturonic acid as a standard. One unit of PG activity was expressed as the amount of enzyme that produced 1 μmol galacturonic acid per hour. 2.5.4. Cellulase activity determination A volume of 1 mL of the supernatant was added to 1 mL of 0.25% (w/v) carboxymethyl cellulose and 5 mL of 20 mM sodium acetate buffer (pH 4.0). After incubation at 37 °C for 2 h, the reaction was terminated by 2 mL of 10 mM sodium tetraborate solution and 0.1 mL of 1% (w/v) 2-cyanoacetamide, followed by incubation at 100 °C for 5 min and cooled at 20 °C for 20 min. The final reaction was monitored at 276 nm, with β-D-glucose as standard. One unit of cellulose activity
Fig. 1. Effect of chitosan on the pulp breakdown index of harvested longan fruit. The mark ** represents the significant difference (p < 0.01) between chitosan-treated longan fruit and control longan fruit on each storage day. 128
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chitosan-treated longan fruit (y = −1.5645 x + 13.912, r = −0.971, p < 0.01). These data illustrated that chitosan treatment could effectively inhibit the degradation of CWM and delay pulp breakdown of longan fruit. This result agreed with a previous study conducted by Zhao et al. (2014) in which higher content of CWM maintained in hot water-treated longan fruit contributed to alleviated occurrence of pulp breakdown.
indicating that the longan pulp was almost full decayed. For the chitosan-treated longan fruit, the pulp breakdown index remained unchanged from 0 to 1 d but increased rapidly from 1 to 5 d, followed by a slow rise. Further comparison showed that, in contrast with the control group, chitosan-treated longan fruit displayed a remarkably (p < 0.01) lower pulp breakdown index from storage day 2 to day 6, indicating that chitosan treatment could delay pulp breakdown of harvested longan fruit. This result is in agreement with previous work that chitosan enhanced the firmness, flavor and storability of fresh-cut melons (Poverenov et al., 2018) or Chinese cherry (Xin, Chen, Lai, & Yang, 2017). This result also agreed with Jiang and Li (2001), in which chitosan treatment delayed the color change of longan pericarp and maintained higher contents of total soluble solids, titratable acidity and ascorbic acid in longan pulp, and thus extend the postharvest life of longan fruit.
3.3. Chitosan-induced alterations in cell wall polysaccharides in pulp of harvested longan fruit and its relationship with chitosan-alleviated longan pulp breakdown Pectin, cellulose, and hemicellulose are the main cell wall polysaccharides. Thus, they play major roles in maintaining the texture of harvested fruits (Lin, Lin, Lin, Lin et al., 2018; Wang et al., 2018), which was further investigated in this study.
3.2. Chitosan-induced alteration of pulp CWM in harvested longan fruit and its relationship with chitosan-alleviated longan pulp breakdown
3.3.1. Pectin fractions Pectin mainly exists in intercellular layer and appears in primary cell wall. It plays an important role in maintaining the adhesion and ordered arrangement of cells, and has the greatest effect on the texture and softening of fruit (Broxterman & Schols, 2018). WSP, ISP, and CSP are different forms of pectin involving in different stages of ripening and senescence of fruit. During the process of ripening and senescence, insoluble pectin fractions including CSP and ISP will be converted to WSP (Wang et al., 2018). In this work, the CSP amount in pulp of the control group reduced sharply at the early stage of storage (0– 2 d) and then decreased slowly (Fig. 3A), the ISP content increased slightly at 0– 2 d and then decreased sharply (Fig. 3B), while the WSP content increased slightly at 0– 3 d, followed by a sharp increase (Fig. 3C). The increased WSP is mainly due to the depolymerization of matrix polysaccharides and the convert of CSP to WSP with the severing of ionic bonds. Further analysis showed that the decreased CSP content (Fig. 3A) was negatively correlated with the increased content of ISP (Fig. 3B) and WSP (Fig. 3C) in the first two days, with the correlation coefficient r values of −0.881 and −0.898, respectively. However, the decreased content of CSP (Fig. 3A) and ISP (Fig. 3B) was negatively correlated with the increased WSP content (Fig. 3C) after the first two days, with the r values of −0.973 and −0.953, respectively. These results indicated that CSP was transformed to ISP and WSP at the early stage, while CSP and ISP were continuously transformed to WSP in the middle and late stage. Furthermore, according to the correlation statistics, the increased pulp breakdown index (y) (Fig. 1) was significantly negatively correlated with the content of CSP (x1, Fig. 3A) and ISP (x2, Fig. 3B) in pulp (y = −4.386 x1 + 9.732, r1 = −0.932, p1 < 0.01; y = −23.417 x2 + 6.418, r2 = −0.897, p2 < 0.01, respectively, Table 1), and was significantly positively correlated with the WSP content (x3) (Fig. 3C) in pulp of control fruit (y = 25.253 x3 - 2.388, r3 = 0.978, p3 < 0.01, Table 1). The correlation statistics revealed that the occurrence of pulp breakdown in longan fruit was related to the degradation of pectin such as CSP and ISP. In addition, as displayed in Table 2, for chitosan treated-longan fruit, the pulp breakdown index was also significantly positively correlated with the WSP content, with the r value of 0.944, but negatively correlated with the content of CSP and ISP in pulp, with the r values of −0.900 and −0.816, respectively. Moreover, the content of CSP (Fig. 3A) and ISP (Fig. 3B) in pulp of chitosan treated-longan fruit was higher than that of the control group, but the WSP content (Fig. 3C) increased at a slower pace than that of the control group, which effectively maintained the pectin content in pulp, alleviated the degradation and damage of pulp pectin, controlled the increasing trend of WSP generated from the degradation of ISP and CSP, and slowed down the occurrence of pulp breakdown in postharvest longan fruit. These results were consistent with previous work that an increase in the WSP content as well as a decrease in the content of CSP and ISP caused by
The softening of fruit led to the dissolution of CWM or the depolymerization of its structures (Chen, Hung et al., 2017; Chen, Sun et al., 2017; Lin, Lin, Lin, Lin et al., 2018). The CWM is mainly composed of ˜90% polysaccharides and ˜10% proteins, enzymes, and fatty acids. Cell walls are involved in a series of physiological activities, such as cell support, nutrient supply, material transport, reduction of transpiration and defense functions (Broxterman & Schols, 2018). Therefore, it is of great importance to delay the degradation of CWM and to maintain the integrity of cell wall structures. Fig. 2 displayed that the content of CWM in pulp of control longan fruit decreased rapidly with the storage time progressed. Furthermore, according to the correlation analysis, the content of CWM (x) was significantly negatively correlated with the pulp breakdown index (y) as shown in Fig. 1 (y = −0.473 x + 6.494, r = −0.953, p < 0.01). Therefore, it could be inferred that the continuous decrease in the CWM content in pulp was a major factor promoting the occurrence of pulp breakdown in longan fruit. In addition, further analysis indicated that the CWM content in pulp of chitosan-treated longan fruit was always maintained at a relatively higher level than that of control longan fruit within 0–6 days during storage, demonstrating a significant (p < 0.05) difference between them during the middle and late stage of storage (2– 6 days). Moreover, the decreased CWM (x) was also significantly negatively correlated with the increased pulp breakdown index (y) in
Fig. 2. Effects of chitosan on cell wall materials (CWM) content in pulp of harvested longan fruit. The mark * represents the significant difference (p < 0.05) between chitosan-treated longan fruit and control longan fruit on each storage day. 129
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Fig. 3. Effects of chitosan on contents of covalent-soluble pectin (CSP) (A), ionic-soluble pectin (ISP) (B), and water-soluble pectin (WSP) (C) in pulp of harvested longan fruit. The mark * and ** represents the significant difference (p < 0.05 and p < 0.01, respectively) between chitosan-treated longan fruit and control longan fruit on each storage day.
microfibers. Many studies have shown that, when the pectin is degraded during the process of fruit softening, hemicellulose is constantly modified at the same time, which is manifested as the changes in the content, molecular weight, and substitution and modification of side chain, indicating that hemicellulose also involves in the development of fruit softening (Wang et al., 2013). This work showed that the amounts of cellulose and hemicellulose in pulp of longan fruit decreased during storage (Fig. 4). In addition, according to the correlation analysis, pulp breakdown index (y, Fig. 1) was significantly negatively correlated with the content of cellulose (x1, Fig. 4A) and hemicellulose (x2, Fig. 4B) in pulp of control fruit (y = −2.147 x1 + 9.372, r1 = −0.952, p1 < 0.01; y = −6.832 x2 + 8.652, r2 = −0.983, p2 < 0.01, respectively, Table 1). As displayed in Table 2, the correlations between breakdown index and cellulose or hemicellulose in chitosan-treated longan fruit was the same as that in control fruit, with the r values of −0.973 and −0.990, respectively. It could be inferred that the decreases in the content of cellulose and hemicellulose were also a major promoting factor for the
high-humidity hot air impingement blanching triggered pectin depolymerization and changed the texture profiles of grape fruit (Wang et al., 2018). It was also in agreement with that the decreased WSP content caused by chitosan treatment contributed to the improved quality of citrus fruit (Zhao, Deng, Zhou, Yao, & Zeng, 2018). 3.3.2. Cellulose and hemicellulose Cellulose and hemicellulose are another two types of polysaccharides located in the primary and secondary cell walls. Cellulose, accounting for 20%–30% of cell wall material, is a long linear chain of macromolecular polysaccharide connected by the unit of β-1,4-D-glycosidic bonds. Cellulose generally exists in the form of microfibril, which consists of long chain fibers integrated by a large numbers of hydrogen bonds, making them highly stable and not easy to be degraded (Caffall & Mohnen, 2009). Hemicellulose is mainly located in primary wall and covalently bonded to pectin. A cellulose matrix network has the important function to keep the shape and rigidity of cell wall, which is formed by adhering hemicellulose to cellulose Table 1 Correlation analysis of breakdown index in pulp of control longan fruit.
Breakdown index CWM CSP ISP WSP Cellulose Hemicellulose XET PE PG β-Gal Cellulase
Breakdown index
CWM
1 −0.953 −0.932 −0.897 0.978 −0.952 −0.983 – 0.954 0.927 0.944 0.951
1 – – – – – – – – – –
CSP
ISP
WSP
Cellulose
Hemicellulose
XET
PE
PG
β-Gal
Cellulase
1 – – – – – −0.887 −0.844 −0.887 –
1 – – – – − – – –
1 – – – 0.949 0.943 0.939 –
1 – – – – – −0.975
1 – – – – −0.933
1 – – – –
1 – – –
1 – –
1 –
1
Note: “–” means the correlation analysis is not done. 130
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Table 2 Correlation analysis of breakdown index in pulp of chitosan-treated longan fruit.
Breakdown index CWM CSP ISP WSP Cellulose Hemicellulose XET PE PG β-Gal Cellulase
Breakdown index
CWM
1 −0.971 −0.900 −0.816 0.944 −0.973 −0.990 – 0.962 0.941 0.992 0.932
1 – – – – – – – – – –
CSP
ISP
WSP
Cellulose
Hemicellulose
XET
PE
PG
β-Gal
Cellulase
1 – – – – – −0.862 −0.846 −0.894 –
1 – – – – – – – –
1 – – – 0.963 0.954 0.977 –
1 – – – – – −0.952
1 – – – – −0.962
1 – – – –
1 – – –
1 – –
1 –
1
Note: “–” means the correlation analysis is not done.
pectin, to produce pectic acid (Lin, Lin, Lin, Lin et al., 2018). PG can hydrolyze pectic acid, the degradation product of pectinic acid catalyzed by PE, to galacturonic acid (Zhao et al., 2014). β-Gal catalyzes the breakage of galactoside residue from the non-reducing end of β-1,4galactoside in pectin polysaccharide chain. It can hydrolyze galactoside, thus breaking the cross-linking structure provided by galactoside between cell wall components (Chen, Hung et al., 2017). Cellulase, sometimes also called as glucanase, is a complex family of enzymes including exo-1,4-β-D-glucannase (C1), endo-1,4-β-D-glucanase (Cx), and β-1,4-glucosidase. Under the combination of these enzymes, cellulose is hydrolyzed to amorphous cellulose, cello-oligosaccharide, and glucose. The activity of cellulase is low or undetectable in un-ripened fruit but increases significantly to make the cell wall expansion and loosening during the period of ripening and softening (Lin, Lin, Lin, Lin et al., 2018). In this work, it was found that the activities of XET, PE, PG, β-gal, and cellulase in pulp of the control longan fruit increased with prolonging storage time (Fig. 5). The present results also illustrated that XET activity in pulp of the control longan fruit remained at a high level from storage day 0 to day 2, changed slightly during day 2 and day 3, and then increased slightly (Fig. 5A), while other enzymes (Fig. 5B–E) kept at low activities during the first two days and increased afterwards. These results indicated that the overall structure of pectin-cellulosehemicellulose (PCH) in the cell wall was depolymerized by XET, making it possible for PE, PG, and β-gal to act on pectin. However, opposite results were shown in the pulp of chitosan-treated longan fruit, indicating that chitosan could lower the activity of XET to slow down the depolymerization of overall structure in cell wall, and alleviate pulp breakdown in longan fruit. Furthermore, according to the correlation analysis, the increased activities of PE (y1, Fig. 5B), PG (y2, Fig. 5C) and β-Gal (y3, Fig. 5D) were negatively related to the decreased content of CSP (x1, Fig. 3A) (y1 = −7.2585 x1 + 20.069, r1 = −0.887, p1 < 0.05; y2 = −3.8154
occurrence of pulp breakdown in postharvest longan fruit. However, compared with the control group, chitosan treatment could maintain a significant (P < 0.05) higher level of cellulose from 0 to 6 days and hemicellulose (Fig. 4) from 2 to 6 days, which further maintained cell wall structure in pulp, slowed the process of pulp breakdown, and improved the stability of longan fruit. It was in agreement with a previous study that the improved storability of citrus fruit was due to the increased cellulose content caused by the use of chitosan (Zhao et al., 2018).
3.4. Chitosan-induced alterations in activities of cell wall polysaccharidesdisassembling enzymes in pulp of harvested longan fruit and its relation to chitosan’s suppressing the disassembly of cell wall polysaccharides and alleviating longan pulp breakdown The main reason of fruit softening is the separation and the total structural change of cell wall resulting from the degradation of polysaccharide in cell wall. Moreover, the polysaccharides and structure of cell wall are gradually disassembled and collapsed under the action of cell wall hydrolase like XET, PE, PG, cellulase, and β-Gal (Chen, Hung et al., 2017; Chen, Sun et al., 2017; Lin, Lin, Lin, Lin et al., 2018; Zhou et al., 2006). XET is a newly discovered cell wall hydrolase associated with fruit softening (Zhou et al., 2006). It not only has the function of glycosylation transfer in xyloglucan in which the broken end was transferred to another non-reducing end of xyloglucan polymer, but also has the function of depolymerization and hydrolysis. However, the major role of XET is to depolymerize xyloglucan chain connecting cellulose microfibrils, cause the irreversible rupture of xyloglucan chain, and then give rise to the expansion and softness of cell wall during the process of ripeness and senescence of fruit (Zhong et al., 2008). The main function of PE is to remove the methoxyl group or hydroxyethyl group from pectinic acid, a methyl esterified derivative of galacturonic acid in
Fig. 4. Effects of chitosan on contents of cellulose (A) and hemicellulose (B) in pulp of harvested longan fruit. The mark * represents the significant difference (p < 0.05) between chitosan-treated longan fruit and control longan fruit on each storage day. 131
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Fig. 5. Effect of chitosan on activities of xyloglucan endotransglycosylase (A), pectinesterase (B), polygalacturonase (C), β-galactosidase (D) and cellulose (E) in pulp of harvested longan fruit. The mark * represents the significant difference (p < 0.05) between chitosan-treated longan fruit and control longan fruit on each storage day.
x1 + 10.489, r2 = −0.844, p2 < 0.05; y3 = −71.453 x1 + 214.79, r3 = −0.887, p3 < 0.05; respectively), but positively related to the increased WSP content (x2, Fig. 3C) (y1 = 43.389 x2 −0.3016, r1 = 0.949, p1 < 0.01; y2 = 23.825 x2 − 0.3919, r2 = 0.943, p2 < 0.01; y3 = 422.73 x2 + 15.005, r3 = 0.939, p3 < 0.01, respectively, Table 1) and the increased pulp breakdown index (x3, Fig. 1) (y1 = 1.6645 x3 + 3.9501, r1 = 0.954, p1 < 0.01; y2 = 0.8939 x3 + 1.9804, r2 = 0.927, p2 < 0.01; y3 = 16.21 x3 + 56.442, r3 = 0.944, p3 < 0.01, respectively, Table 1). The correlation results in chitosan-treated longan fruit are similar to that in control longan fruit,
but with different correlation coefficients as displayed in Table 2. These results demonstrated that the increased activities of PE, PG and β-Gal (Fig. 5B–D) enhanced the depolymerization of CSP to WSP (Fig. 3A–C), and thus accelerated the occurrence of pulp breakdown in harvested longan fruit. However, weaker activities of PE, PG, and β-Gal (Fig. 5B–D), lower pulp breakdown index (Fig. 1), as well as higher levels of CSP and ISP (Fig. 3A and B) were exhibited in the pulp of chitosan-treated longan fruit, indicating that chitosan could lower the activities of PE, PG and β-Gal, slow down the degradation of CSP and ISP, and alleviate pulp breakdown of longan fruit. It was in accordance 132
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Fig. 6. The probable mechanism of chitosan’s alleviating pulp breakdown of harvested longan fruit via acting on the disassembly of cell wall polysaccharides.
longan pulp. Moreover, the reduced enzyme activities of cell wall degrading-enzymes (XET, PE, PG, β-Gal and cellulase) were observed in pulp of chitosan-treated longan fruit. These factors acted together to stabilize the cell wall structure of longan pulp and reduce the occurrence of pulp breakdown in longan fruit. Therefore, chitosan treatment delayed the disassembly of cell wall polysaccharides and alleviated pulp breakdown of harvested longan fruit. These results indicate that Kadozan, a novel chitosan formulation, can provide an alternative approach for controlling the pulp breakdown occurrence, keeping nutritional values, and prolonging the storage period of postharvest longan fruit.
with previous reports that application of chitosan resulted in lower levels of cell wall degrading-enzymes like PE, PG, and β-Gal, as well as better cell structural integrity that might improve the storability of dragon fruit (Zahid, Maqbool, Ali, Siddiqui, & Bhatti, 2019) or citrus fruit (Zhao et al., 2018). Additionally, the increased activity of cellulase (y, Fig. 5E) was negatively related to the decreased content of cellulose (x1, Fig. 4A) and hemicelluloses (x2, Fig. 4B) (y = −30.051 x1 + 47.788, r1 = −0.975; p1 < 0.01; y = −9.3053 x2 + 50.492, r2 = −0.933, p2 < 0.01, respectively), but positively related to the increased pulp breakdown index (x3, Fig. 1) (y = 4.2085 x3 + 10.106, r1 = 0.951, p3 < 0.01). As displayed in Table 2, the correlation between cellulase and cellulose or hemicelluloses in chitosan-treated longan fruit is similar to that in control fruit, but with r values of −0.952 and −0.962, respectively. These data indicate that cellulose catalyzed the degradation of cellulose and hemicelluloses and gave rise to the occurrence of pulp breakdown of longan fruit. However, lower cellulase activity (Fig. 5E) and pulp breakdown index (Fig. 1) as well as higher levels of cellulose (Fig. 4A) and hemicelluloses (Fig. 4B) were observed in the pulp of chitosantreated longan fruit, indicating that chitosan could lower cellulase activity, slow down the degradation of cellulose and hemicelluloses, and alleviate pulp breakdown of longan fruit. These results were accorded with previous work that the reduced cellulase activity, caused by hot water or acidic electrolyzed oxidizing water or 1-methylcyclopropene (1-MCP), led to the alleviated occurrence of pulp breakdown of longan fruit (Zhao et al., 2014) or softness of blueberries (Chen, Hung et al., 2017), Huanghua pears (Chen, Sun et al., 2017, and Younai plum fruit (Lin, Lin, Lin, Lin et al., 2018). The above results revealed that postharvest application of chitosan could reduce the enzyme activities relating to the cell wall degradation, that is, inhibit the activities of XET, PE, PG, β-Gal, and cellulose. The chitosan treatment could delay the degradation of cell wall polysaccharides and stabilize the structure of cell wall in longan pulp, alleviate longan pulp breakdown, and thus maintain the quality of longan fruit. Additionally, the approximate cost of Kadozan treatment with the concentration of the 1:500 (VKadozan: VKadozan + Water) dilution, that is 0.004% chitosan, on longans is RMB 0.11 China Yuan (0.016 United States dollar) per kilogram longan fruit. From the above findings, the probable mechanism of chitosan’s alleviating pulp breakdown of harvested longan fruit via acting on the disassembly of cell wall polysaccharides was demonstrated in Fig. 6.
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