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Synergistic interaction of natamycin with carboxymethyl chitosan for controlling Alternata alternara, a cause of black spot rot in postharvest jujube fruit
Postharvest Biology and Technology 156 (2019) 110919
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Synergistic interaction of natamycin with carboxymethyl chitosan for controlling Alternata alternara, a cause of black spot rot in postharvest jujube fruit
T
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Liang Gonga,b,1, Zhiyong Zhaoc,1, Chunxiao Yina, Vijai Kumar Guptad, Xianhui Zhangc, , Yueming Jianga,b a Key Laboratory of Plant Resource Conservation and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China b Key Laboratory of Post-Harvest Handling of Fruits, Ministry of Agriculture, Guangzhou 510650, China c Xinjiang Academy of Agricultural and Reclamation Science, Shihezi 832000, China d Department of Chemistry and Biotechnology, ERA Chair of Green Chemistry, Tallinn University of Technology, Tallinn 12618, Estonia
A R T I C LE I N FO
A B S T R A C T
Keywords: Jujube fruit Natamycin Carboxymethyl chitosan Alternata alternara ITS sequencing
Black spot rot of jujube, caused by Alternata alternara, is an important disease affecting jujube tree and fruit production. In this study, the mixtures of natamycin (NATA) and carboxymethyl chitosan (CMCS) were investigated for their combined and/or individual bioactivity against A. alternara and the quality maintenance of postharvest jujube fruit. The combined effectiveness between NATA and CMCS was calculated by Wadley’s method, and the results indicated synergistic interactions (SR ≥ 1.5) between NATA and CMCS up to the ratios of 1: 100 and 1: 500. Then, an emulsion was developed by mixing NATA and CMCS and was characterized by determining the particle size and the zeta potential. The results suggested that the emulsion has great physical stability after storage for 60 d at 25 °C. Furthermore, the emulsion was applied to the postharvest treatment of fresh jujube fruit. The results indicated that the treatment significantly decreased the fruit respiration rate and ethylene production, reduced postharvest natural decay, promoted fruit firmness, and delayed loss of titratable acidity and vitamin C during the storage 40 d at 10 °C, maintaining a better quality of the jujube fruit. Finally, high-throughput sequencing was used to determine the sequences of the ITS2 gene in ITS5-1737F variable regions between the treated and control jujube fruit. The results showed that the emulsion reduced the entire fungal counts and altered the absolute abundance of plant pathogens, including Alternaria species, in the jujube fruit. Taken together, these results suggested that the formulation incorporating NATA and CMCS represents an alternative to control postharvest diseases and to improve the shelf life quality of jujube fruit.
1. Introduction Black spot rot caused by Alternata alternara is an important disease of postharvest jujube fruit. A. alternara is a low-temperature tolerant pathogen that can germinate and grow even in −2 °C conditions; it can infect wide-ranging plant hosts and lead to serious damage on the surface of the jujube fruit (Yuan et al., 2019), causing dark brown lesions. In addition, A. alternara can produce mycotoxins including tenuazonic acid, alternariol methylether and alternariol, which can lead to food safety issues and threaten human and animal health (De Berardis et al., 2018; Hu et al., 2017). Hence, the discovery of an effective method for the control of A. alternata is crucial to the sustainable
development of the jujube industry. Instead of using chemical fungicides, scientists would prefer to create environmentally acceptable alternatives that are safe in food. Li et al. (2018) reported that ethyl p-coumarate exerts antifungal activity against A. alternata with a half-inhibition concentration of 176.8 μg/ mL. Antagonistic yeast, mainly including Aureobasidium pullulans, was found to reduce A. alternata infection and tenuazonic acid production in grapes (Prendes et al., 2018). Induced resistance was activated by γaminobutyric acid in tomato, which could reduce the infection of A. alternata (Yang et al., 2017). In addition, food additives were generally recognized as safe substances and hence widely utilized for control of A. alternata in postharvest fruits and vegetables (Rodriguez-Garcia et al.,
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Corresponding authors. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.postharvbio.2019.05.020 Received 22 January 2019; Received in revised form 22 May 2019; Accepted 24 May 2019 Available online 05 July 2019 0925-5214/ © 2019 Published by Elsevier B.V.
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2.2. In vitro experiments
2016; Fagundes et al., 2013). Natamycin (NATA) is a polyene macrolide antibiotic that is produced by fermentation of Streptomyces spp. In China, NATA may be applied to cheese, meat and fruit juice with amounts not exceeding 300 μg/mL. It has been reported that NATA combined with azithromycin had a synergistic effect against Aspergillus flavus and Fusarium solani in clinical trials (Guo et al., 2018). Sradhanjali et al. (2018) also demonstrated the occurrence of synergy between NATA and voriconazole against clinical isolates, including Fusarium, Candida, Aspergillus and Curvularia spp. (Sradhanjali et al., 2018). Chemical fungicides, such as fludioxonil or cyprodinil, and dip treatments of transplants with NATA were highly effective for managing crown rot of strawberry caused by Colletotrichum acutatum (Haack et al., 2018). However, the use of NATA as a food additive is strictly regulated on the basis of the Chinese national standard (GB2760-2014). To improve the efficiency of treatment, we have to optimize the components of the formulation in the finished product. Chitosan is another widely used food additive and a suitable alternative to synthetic fungicides for treating postharvest fruits and vegetables because of its good antimicrobial properties and induced resistance for sustainable crop protection (Romanazzi et al., 2017). It has been reported that chitosan-based edible coatings on postharvest fruits and vegetables can control moisture transfer, inhibit the respiration rate and oxidation processes, and ultimately extend shelf life (Kerch, 2015). According to a database search, the combined use of NATA with chitosan in an edible coating could extend the shelf life of Hami melon, causing decreases in weight loss and decay (Cong et al., 2007). The effects of vacuum packaging with chitosan and NATA on phyllo pastry quality were distinct, with significant reductions in microbial species populations and an extension of shelf life eleven days (Tsiraki et al., 2018). Tsiraki et al. (2017) reported that the application of chitosan and NATA on a dough-based wheat product could result in a doubling of the shelf life and allow the product to retain excellent sensorial characteristics (Tsiraki et al., 2017). However, the interaction of chitosan and NATA against A. alternata in postharvest fresh jujube fruit has never been reported. Chinese winter jujube (Ziziphus jujuba Mill. cv. Dongzao) is one of the most upscale fresh foods in China, and it has high nutritional value with amino acids, triterpene acids, multiplex vitamins and trace elements (Shi et al., 2018). A. alternara can seriously affect the yield and quality of fresh jujube, and its infection may occur both at harvest and postharvest stages. In this paper, possible synergistic interactions of NATA and carboxymethyl chitosan against A. alternara were investigated according to Wadley’s method. A formulation containing a suitable ratio of NATA and carboxymethyl chitosan (CMCS) was developed and utilized to maintain the nutritional quality features of the postharvest jujube fruit, including firmness, total soluble solids, titratable acidity and vitamin C. In addition, metagenomic studies of the fruit material were performed to investigate the changes in fungal populations after the treatment. This study provides a promising formulation for protecting jujube fruit during postharvest storage.
The individual and combinative antifungal activity of NATA and CMCS were assessed by the radial growth test on PDA according to our previous report (Gong et al., 2016). In brief, five concentration series were assigned for each individual and combined substance, and each one was performed with three biological repeats. After incubation for 7 d, the diameter of the mycelial growth was measured with a ruler (mm), and the percentage of growth inhibition (I) was calculated as follows: I (%) = [(C − T)/(C − C0)] × 100. T indicates the mycelial diameter of the treated group; C indicates the mycelial diameter of the control group; C0 indicates the initial diameter of the fungal agar discs (5 mm). The results of the interactions between NATA (A) and CMCS (B) were calculated based on Wadley methods (Amoucha and Cohen, 1988), which is applied for estimating the optimum ratio of the components in mixtures to achieve the highest control effectiveness. The procedures of this method are shown as follows, firstly the EC50 (median effect concentration) practical values of the mixtures and single components were calculated by using the GraphPad Prism 5 software with the above mentioned I value, and then compared with the EC50 theoretical value as follows:
a b ⎞ + EC50 theoreticalvalue = (a+b)/⎜⎛ ⎟ EC EC (A)50 (B)50 ⎠ ⎝ a and b are the ratios of the components in the mixture; Finally, the synergistic ratio (SR) was calculated and evaluated as follows: SR = EC50 theoretical value / EC50 practical value; SR ≥ 1.5 means synergism; SR ≤ 0.5 means antagonism; and 05 < SR < 1.5 means an additive response. 2.3. Emulsion preparation and characterization analysis NATA (5%, w/v) suspension concentrate was prepared according to our patent description (Application No. 201910064513.1); then, the emulsion was obtained by dissolving each substance in deionized water to the final formulation of CMCS (1000 mg/L) and NATA (10 mg/L). The zeta potential and mean particle diameter of the emulsion were determined by zeta potential analyzer (Brookhaven Instruments Ltd., Texas, USA). Emulsion samples were diluted 1:10 using deionized water. Samples were measured at day 1, day 30 and day 60 after the preparation. The data were reported according to the result of three independent replicates. 2.4. In vivo experiments experiments The fresh winter jujube (Z. jujuba Mill. cv. Dongzao) were purchased from Shangdong Jinan, China in October 2017, and those were harvested at commercial maturity stage, with less than 25% red color on the skin. The fruits were further selected with uniformity in size, color, as well as no mechanical damage and fungal infection; after being washed with clean water and air-dried, the fruit were dipped in the above-mentioned final formulation for 5 min. The group that was only treated with water was used as a negative control. After air drying, the treated fruit was stored for 40 d at 10 °C and 85 to 95% relative humidity. The experiment was performed in three replicates, with 100 jujube fruits per replicate. The decay index of the jujube fruits after the postharvest storage was calculated according to our previous report (Gong et al., 2016).
2. Material and methods 2.1. Chemical agents and fungal isolates NATA (wt % > 95%) was purchased from Fruida medicinal Co. (Shangdong, China). CMCS was obtained from Yuanyebio Co., Ltd. (Shanghai, China); the powder contained 80% carboxylated chitosan. All the other chemicals were of analytical grade and were obtained from Guangzhou Chemical Reagent Co., Ltd. (Guangzhou, China). Alternata alternara was derived from a single spore isolated from Xinjiang jujubes, which was further identified according to its TEF1-α sequence (Kahl et al., 2015). The pathogens were cultured on potato dextrose agar (PDA) medium with a regular photoperiod of 12 h light/ 12 h dark at 25 °C.
2.5. Determination of ethylene production and respiration rate of the fruit For each treatment, 500 g of the fruit was sealed in 5 L gas-tight jars at 25 °C. After 2 h, 1 mL gas was removed from the headspace using a syringe and injected into a gas chromatograph (SQ-206, Beijing, China) 2
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equipped with an activated alumina column and a flame ionization detector for ethylene determination and a thermal conductivity detector for CO2 determination. The rates of ethylene production and respiration were calculated based on the report (Hua et al., 2014). Three replications for each treatment were performed.
Table 1 Efficacy of natamycin and carboxymethyl chitosan for controlling mycelial growth of Alternata alternara on potato dextrose agar. Compounds and ratios
Regression equation of virulence (y=)
R2
natamycin (A) carboxymethyl chitosan (B) A : B = 1 : 50 A : B = 1 : 100 A : B = 1 : 300 A : B = 1 : 500
0.1351X+86.55 42.15X+40.77
0.9643 0.8783
1.000X+64.17 0.01191X+83.30 0.1673X+55 1.000X+53.67
0.9221 0.9916 0.8073 0.81
EC50 theoretical value
2.6. Fruit quality determination Firmness, soluble solids content, titratable acidity and vitamin C content of the fruit were determined during the storage process. Flesh firmness was determined on the position near the equator of the fruit using a handheld fruit firmness tester (GY4, China), which was equipped with a cylindrical plunger 7.9 mm in diameter. Soluble solids content (SSC) and titratable acidity (TA) were determined by a digital refractometer PAL-BX/ACID1 (ATAGO, Japan) with the measurement accuracy ± 0.2 % Brix and ± 0.10 g/100 mL, respectively. The vitamin C content of the fruit was tested according to the description of Kampfenkel et al. (1995), which is based on the reduction of Fe3 to Fe2 by ascorbate and the spectrofluorometric detection (525 nm) of Fe2 involved with 2,2′-dipyridyl. Each treatment was performed with three replicates.
EC50 practical value (mg/L)
SR
5.76 800.0 215.98 337.84 142.45 627.35
157.0 104.4 299.2 229.6
1.38 3.25 0.48 2.73
2.7. Fungal ITS sequencing and data analysis Samples of flesh were collected from treated and control jujube fruit after storage for 35 d, and 10 g of the samples was used to extract DNA using the DNA extraction kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. DNA integrity, concentration and purity were monitored on 1% agarose gels. ITS genes of distinct regions were amplified using specific primer ITS5-1737 F with 12 bp barcode, and the PCR products were utilized to generate sequencing libraries using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (Thermo Fisher Scientific, USA) following the manufacturer's recommendations, and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, USA) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an IlluminaHiseq2500 platform in Guangdong Magigene Biotechnology Co., Ltd. (Magigene, Guangdong). Sequences analysis was performed using search software (V1, http://www.drive5.com/usearch/). Sequences with ≥97% similarity were assigned to the same OTU (operational taxonomic unit). For each representative sequence, the Unite (http://unite.ut.ee/index.php) database was used to annotate taxonomic information (set the confidence threshold to default to ≥0.5). The indices of alpha diversity, including observed species, Chao1, Shannon, Simpson, and dominance, were calculated using QIIME (V1.9.1) and displayed with R software (V2.15.3) (http://qiime.org/scripts/alpha_diversity.html). 2.8. Statistical analysis Statistical analysis was performed by using GraphPad Prism 5. Data were compared with a one- or two-way ANOVA. * P < 0.05 was considered significant.
Fig. 1. Effect of time on the mean particle size distribution and mean zeta potential of the formulation containing natamycin and carboxymethyl chitosan. Data are shown as the mean values ± standard deviation (SD), and the different letters (a–c) indicate that they are significantly different at p < 0.05.
3. Results and discussion it was used as an individual treatment. For instance, when the mixture has a ratio value of 500:1 (CMCS : NATA), the EC50 of the mixture was reduced to 229.6 mg/L, comparable to that of the EC50 of CMCS (800 mg/L). According to Wadley's method, there are clear interactions between NATA and CMCS; synergistic interactions were found with the ratios of 1:100 and 1:500, but the ratios 1:300 and 1:50 had antagonistic and additive interactions, respectively. Similar to our findings, synergistic effects of chitosan or oligochitosan combined with grape seed extract (Bautista-Baños et al., 2003), ethanol (Romanazzi et al., 2007), silicon (Yang et al., 2010) or ε-poly-l-lysine (Sun et al., 2018)
3.1. Bioactivity of NATA and CMCS against A. alternara Generally, mixtures of chemical fungicides have two advantages: one is to broaden the antimicrobial spectrum of the product, and the other is to minimize the selection pressure for the development of resistant strains. Our results (Table 1) indicated that A. alternara was sensitive to both NATA and CMCS but that the treatments varied in their effectiveness, with EC50 values of 5.76 mg/L and 800 mg/L, respectively. When CMCS was mixed with NATA within the ratios from 50:1 to 500 : 1, the amount of CMCS was considerably lower than when 3
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Fig. 2. Effects of postharvest natamycin and carboxymethyl chitosan (1:100 wt%) on vitamin C content (a), soluble solids content (b), firmness (c) and titratable acidity (d) of the fresh jujube fruit during storage. Bars represent the standard deviation, and asterisk indicates a significant difference (* p < 0.05, ** P < 0.01 and *** P < 0.001).
advantages in comparison to chitosan, such as reduced viscosity, enhanced biocompatibility and water solubility, as well as better moisture retention, film forming and emulsion stabilizing characteristics (Shariatinia, 2018). Therefore, we believe that the different modes of action of NATA and chitosan against fungi, in addition to the excellent physical properties of CMCS, might be the reasons for the synergistic effects, but these combined effects are dose dependent.
3.2. Physical characterization of the formulation Measuring particle size distributions in formulations and understanding how they affect the emulsifiers or coating agents can be critical to the success of their biological activity (Wedel et al., 2018). It is known that the optimum particle size for suspension concentrates is 5 μm or less. The particle size analysis showed that the diameter of the NATA suspension concentrates was within 100 nm (unpublished data), whereas after blending with CMCS, the particle size of the formulations increased to a mean diameter of 966.4 ± 142.2 nm (Fig. 1). The formulations were further evaluated by analysis of the particle aging period. The results showed that the particle size was significantly increased to a mean diameter of 1736.9 ± 135.5 nm and 2542.3 ± 226.7 after the preparation of 30 d and 60 d (Fig. 1), respectively, suggesting that particle aggregation occurs in the formulation during long-term storage. Rampino et al. (2013) reported that nanoparticle suspensions containing mannitol and polyethylene-glycol tended to have an increasing particle size, especially at low concentrations (Rampino et al., 2013). In addition, individual nanoparticles may interact to form aggregates in a nanoparticle-rich solution, and the water combined with sugar may exert mechanical forces on the nanoparticles, resulting in their fusion (Cesàro et al., 2008). Although the size of the formulation increased with increasing storage time, the zeta potential of the prepared formulations ranged from 31.42 to 37.74 mV (Fig. 1), indicating the great physical stability of the prepared formulations.
Fig. 3. Effects of postharvest treatment of natamycin and carboxymethyl chitosan (1:100 wt %) on the decay rate of the fresh jujube fruit for 40 d storage at 10 °C.
have been reported. NATA binds to cell membrane sterols that cause the repression of ergosterol biosynthesis in fungi (te Welscher et al., 2008), while chitosan appears to rely on electrostatic interactions between positive charges of chitosan and the negative charges of phospholipids in the fungal plasma membrane (Palma-Guerrero et al., 2009). CMCS is produced by carboxymethylated chitosan, and it has several practical 4
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titratable acidity and vitamin C (Hong et al., 2012). Zhu et al. (2008) reported that chitosan coating on postharvest mango led to an inhibition of the decline in the respiration rate, fruit weight, and titratable acidity and delayed the increase in total soluble solids during storage (Zhu et al., 2008). Moreover, our results suggested that the application of the formulation suppressed the respiration rate and ethylene production (Fig. 4). The respiration rate varied among the different time points, but the pattern of change was consistent with ethylene production (Fig. 4). Recently, jujube fruit was characterized as being a nonclimacteric fruit during ripening, but ethylene is still necessary to maintain normal ripening (Zhang et al., 2018). Indeed, the chitosan coating provides a semipermeable film on the fruit surface, which modifies the internal atmosphere by decreasing oxygen levels and/or elevating carbon dioxide levels, which has two functions. One function is to suppress the fruit respiration level, resulting in a slowing of the synthesis and use of metabolites, such as the hydrolysis of carbohydrates to sugars, which is consistent with the low content of total soluble solids. The other function is to decrease metabolic activity, including ethylene production, which delays fruit ripening and senescence processes.
3.4. Microbiologic population analysis The amplification and high-throughput sequencing of the fungal nuclear internal transcribed spacer (ITS) region can now enable identification and relative quantification of fungal community members, with well-accepted experimental settings, providing new insights into fungal community ecology (Lindahl et al., 2013). In the current study, the raw data were recovered with a total number of 205,181 and 328,641 tags from control and treated samples, respectively. After quality optimization of the data, 201,529 and 322,462 tags were filtered from raw data (Tale S1). A large number of fungal OTUs were identified and classified (Table S2), and the fungal α-diversity was further assessed by targeting ITS2 gene libraries, as shown in Table S3. These OTUs spanned 3466 phyla, 3428 classes, 3392 orders, 3281 families, and 3260 genera in the control group but just 605 phyla, 601 classes, 537 orders, 484 families, and 464 genera in the treated group, suggesting that the formulation can significantly protect the jujube fruit with low fungal colonization. The most abundant OTU was assigned to the known genus Ophiocordyceps, followed by Alternaria and others including Verticillium and Aspergillus (Fig. 5), and the relative abundances of the genera Alternaria and Ophiocordyceps were much higher in the control group than in the treated group (Fig. S1). Consistent with the above results, the formulation should likely play a critical role in the growth inhibition of the plant pathogens, including A. alternara and Penicillium expansum; these pathogens are main causes of postharvest diseases of jujube fruit (Tian et al., 2005). In addition to the direct antimicrobial activity of NATA and CMCS, we believe that the formulation treatment can induce plant resistance to pathogens because chitosan induced jujube disease resistance through the upregulation of defense-related enzymes, including peroxidase and β-1,3-glucanase (Yan et al., 2012). Ophiocordyceps is a widespread genus that contains approximately 140 species that grow on insects (Baral, 2017). As a typical example, O. sinensis is a well-known medicinal fungus that parasitizes larvae of the genus Thitarodes to form fruiting bodies used as traditional Chinese medicine (Chioza and Ohga, 2014). Indeed, the substantial merit of Ophiocordyceps spp. in terms of medicinal benefits has largely been reported (Wang et al., 2015; Prathumpai and Kocharin, 2016), which is in accordance with the health-promoting effects of jujube fruit for the liver, kidneys and immune system. Hence, we infer that the large amount of Ophiocordyceps spp. within fresh jujube fruit may be one of the reasons for the fruits’ nutritive value. This is the first report that the genus Ophiocordyceps lives within fresh jujube fruit, but the detailed function must be further investigated.
Fig. 4. Effects of postharvest treatment of natamycin and carboxymethyl chitosan (1:100 wt %) on the respiration rate and ethylene production of fresh jujube fruit during storage. Bars represent the standard deviation, and the asterisk indicates a significant difference (* p < 0.05, ** P < 0.01 and *** P < 0.001).
3.3. Effect of the formulation on the shelf life of fresh jujube fruit To further understand the biological effect of the above-mentioned formulations, an in vivo test with a NATA:CMCS ratio of 1:100, which showed the highest synergism, was carried out on fresh jujube fruit during 40 d of storage at 10 °C. The results in this study clearly revealed that the formulation significantly reduced postharvest natural decay, promoted fruit firmness, and delayed loss of titratable acidity and vitamin C as storage time increased (Fig. 2), as well as the decay index, was reduced 23.8% in the treated samples (Fig. 3). Firmness is one of the most important features used to indicate the quality of fruit during the ripening process. As shown in Fig. 2, the initial firmness values (0–10 d) were similar between the control and treated samples; during days 10–20, the treated group showed slower changes of firmness compared to the control group; however, after 20 d of storage, the fruits dramatically lost firmness in the control group. The results indicate that the formulation with the CMCS coating may be functional in decreasing water and weight loss, maintaining the firmness of the fresh jujube fruit. Many studies have shown that chitosan, with its filmogenic properties, has beneficial effects on fruit firmness (Romanazzi et al., 2017). In addition, the formulation provided effective protection decline of vitamin C and titratable acidity during storage, while the increase in soluble solids was delayed in the treated group. Consistent with our results, for the 12-day storage of guava, the application of 2.0% chitosan significantly reduced the firmness and weight loss, delayed changes in the soluble solids content, and retarded the loss of 5
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Fig. 5. Heat map analysis of the relative abundance of the top 30 fungi between the treated and control samples at genus levels. The box with a redder color indicates that the fungal abundance in the sample is relatively higher.
4. Conclusion
for social development of Xinjiang production and Construction Corps (No. 20l6AD030) and Pearl River Nova Program of Guangzhou (No. 201710010135) for financial support for this research project. V.K.G. would like to acknowledge funding from the EU 7th Framework Programme for research, technological development and demonstration activities under grant agreement No. 621364 (TUTIC-Green), that provided support to make this work.
The development of food protecting films and coatings may provide an alternative approach to fruit in storing, transporting, and selling. The current study is an important complementary modification for understanding the individual and combined antifungal activity of NATA and CMCS against A. alternara; the synergistic interactions of NATA and CMCS were first confirmed with dose dependence experiments. All of these results have consistently indicated that the formulation incorporating NATA and CMCS possesses effective antimicrobial activity and helps to retain the nutritional quality of fresh jujube fruit and prolong its shelf life. This study suggested that the mixtures of NATA and CMCS represent an effective alternative to conventional chemical methods for slowing the postharvest decay of fresh jujube fruit, perhaps also including other horticultural products.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.postharvbio.2019.05. 020. References Amoucha, Y.S., Cohen, Y., 1988. Synergism in fungicide mixtures against Pseudoperonospora Cubensis. Phytoparasitica 16, 337. Baral, B., 2017. Entomopathogenicity and biological attributes of himalayan treasured fungus ophiocordyceps sinensis (Yarsagumba). J. Fungi Basel (Basel) 3, 1. Bautista-Baños, S., Hernandez-Lopez, M., Bosquez-Molina, E., Wilson, C.L., 2003. Effects of chitosan and plant extracts on growth of Colletotrichum gloeosporioides, anthracnose levels and quality of papaya fruit. Crop Prot. 22, 1087–1092. Cesàro, A., De Giacomo, O., Sussich, F., 2008. Water interplay in trehalose polymorphism. Food Chem. 106, 1318–1328. Chioza, A., Ohga, S., 2014. A review on fungal isolates reported as anamorphs of Ophiocordyceps sinensis. J. Mycol. 2014, 1–5. Cong, F., Zhang, Y., Dong, W., 2007. Use of surface coatings with natamycin to improve the storability of Hami melon at ambient temperature. Postharv. Biol. Technol. 46,
Conflict of interest None Acknowledgments The authors would like to thank the Scientific and Technology Service Net Project of Chinese Academy of Sciences (No. KFJEW-STS085), Guangdong Provincial Agricultural Department Project for Rural Revitalization (No. 2018LM2177). Scientific and technological projects 6
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Synergistic interaction of natamycin with carboxymethyl chitosan for controlling Alternata alternara, a cause of black spot rot in postharvest jujube fruit
T
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Liang Gonga,b,1, Zhiyong Zhaoc,1, Chunxiao Yina, Vijai Kumar Guptad, Xianhui Zhangc, , Yueming Jianga,b a Key Laboratory of Plant Resource Conservation and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China b Key Laboratory of Post-Harvest Handling of Fruits, Ministry of Agriculture, Guangzhou 510650, China c Xinjiang Academy of Agricultural and Reclamation Science, Shihezi 832000, China d Department of Chemistry and Biotechnology, ERA Chair of Green Chemistry, Tallinn University of Technology, Tallinn 12618, Estonia
A R T I C LE I N FO
A B S T R A C T
Keywords: Jujube fruit Natamycin Carboxymethyl chitosan Alternata alternara ITS sequencing
Black spot rot of jujube, caused by Alternata alternara, is an important disease affecting jujube tree and fruit production. In this study, the mixtures of natamycin (NATA) and carboxymethyl chitosan (CMCS) were investigated for their combined and/or individual bioactivity against A. alternara and the quality maintenance of postharvest jujube fruit. The combined effectiveness between NATA and CMCS was calculated by Wadley’s method, and the results indicated synergistic interactions (SR ≥ 1.5) between NATA and CMCS up to the ratios of 1: 100 and 1: 500. Then, an emulsion was developed by mixing NATA and CMCS and was characterized by determining the particle size and the zeta potential. The results suggested that the emulsion has great physical stability after storage for 60 d at 25 °C. Furthermore, the emulsion was applied to the postharvest treatment of fresh jujube fruit. The results indicated that the treatment significantly decreased the fruit respiration rate and ethylene production, reduced postharvest natural decay, promoted fruit firmness, and delayed loss of titratable acidity and vitamin C during the storage 40 d at 10 °C, maintaining a better quality of the jujube fruit. Finally, high-throughput sequencing was used to determine the sequences of the ITS2 gene in ITS5-1737F variable regions between the treated and control jujube fruit. The results showed that the emulsion reduced the entire fungal counts and altered the absolute abundance of plant pathogens, including Alternaria species, in the jujube fruit. Taken together, these results suggested that the formulation incorporating NATA and CMCS represents an alternative to control postharvest diseases and to improve the shelf life quality of jujube fruit.
1. Introduction Black spot rot caused by Alternata alternara is an important disease of postharvest jujube fruit. A. alternara is a low-temperature tolerant pathogen that can germinate and grow even in −2 °C conditions; it can infect wide-ranging plant hosts and lead to serious damage on the surface of the jujube fruit (Yuan et al., 2019), causing dark brown lesions. In addition, A. alternara can produce mycotoxins including tenuazonic acid, alternariol methylether and alternariol, which can lead to food safety issues and threaten human and animal health (De Berardis et al., 2018; Hu et al., 2017). Hence, the discovery of an effective method for the control of A. alternata is crucial to the sustainable
development of the jujube industry. Instead of using chemical fungicides, scientists would prefer to create environmentally acceptable alternatives that are safe in food. Li et al. (2018) reported that ethyl p-coumarate exerts antifungal activity against A. alternata with a half-inhibition concentration of 176.8 μg/ mL. Antagonistic yeast, mainly including Aureobasidium pullulans, was found to reduce A. alternata infection and tenuazonic acid production in grapes (Prendes et al., 2018). Induced resistance was activated by γaminobutyric acid in tomato, which could reduce the infection of A. alternata (Yang et al., 2017). In addition, food additives were generally recognized as safe substances and hence widely utilized for control of A. alternata in postharvest fruits and vegetables (Rodriguez-Garcia et al.,
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Corresponding authors. E-mail address: [email protected] (X. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.postharvbio.2019.05.020 Received 22 January 2019; Received in revised form 22 May 2019; Accepted 24 May 2019 Available online 05 July 2019 0925-5214/ © 2019 Published by Elsevier B.V.
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2.2. In vitro experiments
2016; Fagundes et al., 2013). Natamycin (NATA) is a polyene macrolide antibiotic that is produced by fermentation of Streptomyces spp. In China, NATA may be applied to cheese, meat and fruit juice with amounts not exceeding 300 μg/mL. It has been reported that NATA combined with azithromycin had a synergistic effect against Aspergillus flavus and Fusarium solani in clinical trials (Guo et al., 2018). Sradhanjali et al. (2018) also demonstrated the occurrence of synergy between NATA and voriconazole against clinical isolates, including Fusarium, Candida, Aspergillus and Curvularia spp. (Sradhanjali et al., 2018). Chemical fungicides, such as fludioxonil or cyprodinil, and dip treatments of transplants with NATA were highly effective for managing crown rot of strawberry caused by Colletotrichum acutatum (Haack et al., 2018). However, the use of NATA as a food additive is strictly regulated on the basis of the Chinese national standard (GB2760-2014). To improve the efficiency of treatment, we have to optimize the components of the formulation in the finished product. Chitosan is another widely used food additive and a suitable alternative to synthetic fungicides for treating postharvest fruits and vegetables because of its good antimicrobial properties and induced resistance for sustainable crop protection (Romanazzi et al., 2017). It has been reported that chitosan-based edible coatings on postharvest fruits and vegetables can control moisture transfer, inhibit the respiration rate and oxidation processes, and ultimately extend shelf life (Kerch, 2015). According to a database search, the combined use of NATA with chitosan in an edible coating could extend the shelf life of Hami melon, causing decreases in weight loss and decay (Cong et al., 2007). The effects of vacuum packaging with chitosan and NATA on phyllo pastry quality were distinct, with significant reductions in microbial species populations and an extension of shelf life eleven days (Tsiraki et al., 2018). Tsiraki et al. (2017) reported that the application of chitosan and NATA on a dough-based wheat product could result in a doubling of the shelf life and allow the product to retain excellent sensorial characteristics (Tsiraki et al., 2017). However, the interaction of chitosan and NATA against A. alternata in postharvest fresh jujube fruit has never been reported. Chinese winter jujube (Ziziphus jujuba Mill. cv. Dongzao) is one of the most upscale fresh foods in China, and it has high nutritional value with amino acids, triterpene acids, multiplex vitamins and trace elements (Shi et al., 2018). A. alternara can seriously affect the yield and quality of fresh jujube, and its infection may occur both at harvest and postharvest stages. In this paper, possible synergistic interactions of NATA and carboxymethyl chitosan against A. alternara were investigated according to Wadley’s method. A formulation containing a suitable ratio of NATA and carboxymethyl chitosan (CMCS) was developed and utilized to maintain the nutritional quality features of the postharvest jujube fruit, including firmness, total soluble solids, titratable acidity and vitamin C. In addition, metagenomic studies of the fruit material were performed to investigate the changes in fungal populations after the treatment. This study provides a promising formulation for protecting jujube fruit during postharvest storage.
The individual and combinative antifungal activity of NATA and CMCS were assessed by the radial growth test on PDA according to our previous report (Gong et al., 2016). In brief, five concentration series were assigned for each individual and combined substance, and each one was performed with three biological repeats. After incubation for 7 d, the diameter of the mycelial growth was measured with a ruler (mm), and the percentage of growth inhibition (I) was calculated as follows: I (%) = [(C − T)/(C − C0)] × 100. T indicates the mycelial diameter of the treated group; C indicates the mycelial diameter of the control group; C0 indicates the initial diameter of the fungal agar discs (5 mm). The results of the interactions between NATA (A) and CMCS (B) were calculated based on Wadley methods (Amoucha and Cohen, 1988), which is applied for estimating the optimum ratio of the components in mixtures to achieve the highest control effectiveness. The procedures of this method are shown as follows, firstly the EC50 (median effect concentration) practical values of the mixtures and single components were calculated by using the GraphPad Prism 5 software with the above mentioned I value, and then compared with the EC50 theoretical value as follows:
a b ⎞ + EC50 theoreticalvalue = (a+b)/⎜⎛ ⎟ EC EC (A)50 (B)50 ⎠ ⎝ a and b are the ratios of the components in the mixture; Finally, the synergistic ratio (SR) was calculated and evaluated as follows: SR = EC50 theoretical value / EC50 practical value; SR ≥ 1.5 means synergism; SR ≤ 0.5 means antagonism; and 05 < SR < 1.5 means an additive response. 2.3. Emulsion preparation and characterization analysis NATA (5%, w/v) suspension concentrate was prepared according to our patent description (Application No. 201910064513.1); then, the emulsion was obtained by dissolving each substance in deionized water to the final formulation of CMCS (1000 mg/L) and NATA (10 mg/L). The zeta potential and mean particle diameter of the emulsion were determined by zeta potential analyzer (Brookhaven Instruments Ltd., Texas, USA). Emulsion samples were diluted 1:10 using deionized water. Samples were measured at day 1, day 30 and day 60 after the preparation. The data were reported according to the result of three independent replicates. 2.4. In vivo experiments experiments The fresh winter jujube (Z. jujuba Mill. cv. Dongzao) were purchased from Shangdong Jinan, China in October 2017, and those were harvested at commercial maturity stage, with less than 25% red color on the skin. The fruits were further selected with uniformity in size, color, as well as no mechanical damage and fungal infection; after being washed with clean water and air-dried, the fruit were dipped in the above-mentioned final formulation for 5 min. The group that was only treated with water was used as a negative control. After air drying, the treated fruit was stored for 40 d at 10 °C and 85 to 95% relative humidity. The experiment was performed in three replicates, with 100 jujube fruits per replicate. The decay index of the jujube fruits after the postharvest storage was calculated according to our previous report (Gong et al., 2016).
2. Material and methods 2.1. Chemical agents and fungal isolates NATA (wt % > 95%) was purchased from Fruida medicinal Co. (Shangdong, China). CMCS was obtained from Yuanyebio Co., Ltd. (Shanghai, China); the powder contained 80% carboxylated chitosan. All the other chemicals were of analytical grade and were obtained from Guangzhou Chemical Reagent Co., Ltd. (Guangzhou, China). Alternata alternara was derived from a single spore isolated from Xinjiang jujubes, which was further identified according to its TEF1-α sequence (Kahl et al., 2015). The pathogens were cultured on potato dextrose agar (PDA) medium with a regular photoperiod of 12 h light/ 12 h dark at 25 °C.
2.5. Determination of ethylene production and respiration rate of the fruit For each treatment, 500 g of the fruit was sealed in 5 L gas-tight jars at 25 °C. After 2 h, 1 mL gas was removed from the headspace using a syringe and injected into a gas chromatograph (SQ-206, Beijing, China) 2
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equipped with an activated alumina column and a flame ionization detector for ethylene determination and a thermal conductivity detector for CO2 determination. The rates of ethylene production and respiration were calculated based on the report (Hua et al., 2014). Three replications for each treatment were performed.
Table 1 Efficacy of natamycin and carboxymethyl chitosan for controlling mycelial growth of Alternata alternara on potato dextrose agar. Compounds and ratios
Regression equation of virulence (y=)
R2
natamycin (A) carboxymethyl chitosan (B) A : B = 1 : 50 A : B = 1 : 100 A : B = 1 : 300 A : B = 1 : 500
0.1351X+86.55 42.15X+40.77
0.9643 0.8783
1.000X+64.17 0.01191X+83.30 0.1673X+55 1.000X+53.67
0.9221 0.9916 0.8073 0.81
EC50 theoretical value
2.6. Fruit quality determination Firmness, soluble solids content, titratable acidity and vitamin C content of the fruit were determined during the storage process. Flesh firmness was determined on the position near the equator of the fruit using a handheld fruit firmness tester (GY4, China), which was equipped with a cylindrical plunger 7.9 mm in diameter. Soluble solids content (SSC) and titratable acidity (TA) were determined by a digital refractometer PAL-BX/ACID1 (ATAGO, Japan) with the measurement accuracy ± 0.2 % Brix and ± 0.10 g/100 mL, respectively. The vitamin C content of the fruit was tested according to the description of Kampfenkel et al. (1995), which is based on the reduction of Fe3 to Fe2 by ascorbate and the spectrofluorometric detection (525 nm) of Fe2 involved with 2,2′-dipyridyl. Each treatment was performed with three replicates.
EC50 practical value (mg/L)
SR
5.76 800.0 215.98 337.84 142.45 627.35
157.0 104.4 299.2 229.6
1.38 3.25 0.48 2.73
2.7. Fungal ITS sequencing and data analysis Samples of flesh were collected from treated and control jujube fruit after storage for 35 d, and 10 g of the samples was used to extract DNA using the DNA extraction kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. DNA integrity, concentration and purity were monitored on 1% agarose gels. ITS genes of distinct regions were amplified using specific primer ITS5-1737 F with 12 bp barcode, and the PCR products were utilized to generate sequencing libraries using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (Thermo Fisher Scientific, USA) following the manufacturer's recommendations, and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, USA) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an IlluminaHiseq2500 platform in Guangdong Magigene Biotechnology Co., Ltd. (Magigene, Guangdong). Sequences analysis was performed using search software (V1, http://www.drive5.com/usearch/). Sequences with ≥97% similarity were assigned to the same OTU (operational taxonomic unit). For each representative sequence, the Unite (http://unite.ut.ee/index.php) database was used to annotate taxonomic information (set the confidence threshold to default to ≥0.5). The indices of alpha diversity, including observed species, Chao1, Shannon, Simpson, and dominance, were calculated using QIIME (V1.9.1) and displayed with R software (V2.15.3) (http://qiime.org/scripts/alpha_diversity.html). 2.8. Statistical analysis Statistical analysis was performed by using GraphPad Prism 5. Data were compared with a one- or two-way ANOVA. * P < 0.05 was considered significant.
Fig. 1. Effect of time on the mean particle size distribution and mean zeta potential of the formulation containing natamycin and carboxymethyl chitosan. Data are shown as the mean values ± standard deviation (SD), and the different letters (a–c) indicate that they are significantly different at p < 0.05.
3. Results and discussion it was used as an individual treatment. For instance, when the mixture has a ratio value of 500:1 (CMCS : NATA), the EC50 of the mixture was reduced to 229.6 mg/L, comparable to that of the EC50 of CMCS (800 mg/L). According to Wadley's method, there are clear interactions between NATA and CMCS; synergistic interactions were found with the ratios of 1:100 and 1:500, but the ratios 1:300 and 1:50 had antagonistic and additive interactions, respectively. Similar to our findings, synergistic effects of chitosan or oligochitosan combined with grape seed extract (Bautista-Baños et al., 2003), ethanol (Romanazzi et al., 2007), silicon (Yang et al., 2010) or ε-poly-l-lysine (Sun et al., 2018)
3.1. Bioactivity of NATA and CMCS against A. alternara Generally, mixtures of chemical fungicides have two advantages: one is to broaden the antimicrobial spectrum of the product, and the other is to minimize the selection pressure for the development of resistant strains. Our results (Table 1) indicated that A. alternara was sensitive to both NATA and CMCS but that the treatments varied in their effectiveness, with EC50 values of 5.76 mg/L and 800 mg/L, respectively. When CMCS was mixed with NATA within the ratios from 50:1 to 500 : 1, the amount of CMCS was considerably lower than when 3
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Fig. 2. Effects of postharvest natamycin and carboxymethyl chitosan (1:100 wt%) on vitamin C content (a), soluble solids content (b), firmness (c) and titratable acidity (d) of the fresh jujube fruit during storage. Bars represent the standard deviation, and asterisk indicates a significant difference (* p < 0.05, ** P < 0.01 and *** P < 0.001).
advantages in comparison to chitosan, such as reduced viscosity, enhanced biocompatibility and water solubility, as well as better moisture retention, film forming and emulsion stabilizing characteristics (Shariatinia, 2018). Therefore, we believe that the different modes of action of NATA and chitosan against fungi, in addition to the excellent physical properties of CMCS, might be the reasons for the synergistic effects, but these combined effects are dose dependent.
3.2. Physical characterization of the formulation Measuring particle size distributions in formulations and understanding how they affect the emulsifiers or coating agents can be critical to the success of their biological activity (Wedel et al., 2018). It is known that the optimum particle size for suspension concentrates is 5 μm or less. The particle size analysis showed that the diameter of the NATA suspension concentrates was within 100 nm (unpublished data), whereas after blending with CMCS, the particle size of the formulations increased to a mean diameter of 966.4 ± 142.2 nm (Fig. 1). The formulations were further evaluated by analysis of the particle aging period. The results showed that the particle size was significantly increased to a mean diameter of 1736.9 ± 135.5 nm and 2542.3 ± 226.7 after the preparation of 30 d and 60 d (Fig. 1), respectively, suggesting that particle aggregation occurs in the formulation during long-term storage. Rampino et al. (2013) reported that nanoparticle suspensions containing mannitol and polyethylene-glycol tended to have an increasing particle size, especially at low concentrations (Rampino et al., 2013). In addition, individual nanoparticles may interact to form aggregates in a nanoparticle-rich solution, and the water combined with sugar may exert mechanical forces on the nanoparticles, resulting in their fusion (Cesàro et al., 2008). Although the size of the formulation increased with increasing storage time, the zeta potential of the prepared formulations ranged from 31.42 to 37.74 mV (Fig. 1), indicating the great physical stability of the prepared formulations.
Fig. 3. Effects of postharvest treatment of natamycin and carboxymethyl chitosan (1:100 wt %) on the decay rate of the fresh jujube fruit for 40 d storage at 10 °C.
have been reported. NATA binds to cell membrane sterols that cause the repression of ergosterol biosynthesis in fungi (te Welscher et al., 2008), while chitosan appears to rely on electrostatic interactions between positive charges of chitosan and the negative charges of phospholipids in the fungal plasma membrane (Palma-Guerrero et al., 2009). CMCS is produced by carboxymethylated chitosan, and it has several practical 4
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titratable acidity and vitamin C (Hong et al., 2012). Zhu et al. (2008) reported that chitosan coating on postharvest mango led to an inhibition of the decline in the respiration rate, fruit weight, and titratable acidity and delayed the increase in total soluble solids during storage (Zhu et al., 2008). Moreover, our results suggested that the application of the formulation suppressed the respiration rate and ethylene production (Fig. 4). The respiration rate varied among the different time points, but the pattern of change was consistent with ethylene production (Fig. 4). Recently, jujube fruit was characterized as being a nonclimacteric fruit during ripening, but ethylene is still necessary to maintain normal ripening (Zhang et al., 2018). Indeed, the chitosan coating provides a semipermeable film on the fruit surface, which modifies the internal atmosphere by decreasing oxygen levels and/or elevating carbon dioxide levels, which has two functions. One function is to suppress the fruit respiration level, resulting in a slowing of the synthesis and use of metabolites, such as the hydrolysis of carbohydrates to sugars, which is consistent with the low content of total soluble solids. The other function is to decrease metabolic activity, including ethylene production, which delays fruit ripening and senescence processes.
3.4. Microbiologic population analysis The amplification and high-throughput sequencing of the fungal nuclear internal transcribed spacer (ITS) region can now enable identification and relative quantification of fungal community members, with well-accepted experimental settings, providing new insights into fungal community ecology (Lindahl et al., 2013). In the current study, the raw data were recovered with a total number of 205,181 and 328,641 tags from control and treated samples, respectively. After quality optimization of the data, 201,529 and 322,462 tags were filtered from raw data (Tale S1). A large number of fungal OTUs were identified and classified (Table S2), and the fungal α-diversity was further assessed by targeting ITS2 gene libraries, as shown in Table S3. These OTUs spanned 3466 phyla, 3428 classes, 3392 orders, 3281 families, and 3260 genera in the control group but just 605 phyla, 601 classes, 537 orders, 484 families, and 464 genera in the treated group, suggesting that the formulation can significantly protect the jujube fruit with low fungal colonization. The most abundant OTU was assigned to the known genus Ophiocordyceps, followed by Alternaria and others including Verticillium and Aspergillus (Fig. 5), and the relative abundances of the genera Alternaria and Ophiocordyceps were much higher in the control group than in the treated group (Fig. S1). Consistent with the above results, the formulation should likely play a critical role in the growth inhibition of the plant pathogens, including A. alternara and Penicillium expansum; these pathogens are main causes of postharvest diseases of jujube fruit (Tian et al., 2005). In addition to the direct antimicrobial activity of NATA and CMCS, we believe that the formulation treatment can induce plant resistance to pathogens because chitosan induced jujube disease resistance through the upregulation of defense-related enzymes, including peroxidase and β-1,3-glucanase (Yan et al., 2012). Ophiocordyceps is a widespread genus that contains approximately 140 species that grow on insects (Baral, 2017). As a typical example, O. sinensis is a well-known medicinal fungus that parasitizes larvae of the genus Thitarodes to form fruiting bodies used as traditional Chinese medicine (Chioza and Ohga, 2014). Indeed, the substantial merit of Ophiocordyceps spp. in terms of medicinal benefits has largely been reported (Wang et al., 2015; Prathumpai and Kocharin, 2016), which is in accordance with the health-promoting effects of jujube fruit for the liver, kidneys and immune system. Hence, we infer that the large amount of Ophiocordyceps spp. within fresh jujube fruit may be one of the reasons for the fruits’ nutritive value. This is the first report that the genus Ophiocordyceps lives within fresh jujube fruit, but the detailed function must be further investigated.
Fig. 4. Effects of postharvest treatment of natamycin and carboxymethyl chitosan (1:100 wt %) on the respiration rate and ethylene production of fresh jujube fruit during storage. Bars represent the standard deviation, and the asterisk indicates a significant difference (* p < 0.05, ** P < 0.01 and *** P < 0.001).
3.3. Effect of the formulation on the shelf life of fresh jujube fruit To further understand the biological effect of the above-mentioned formulations, an in vivo test with a NATA:CMCS ratio of 1:100, which showed the highest synergism, was carried out on fresh jujube fruit during 40 d of storage at 10 °C. The results in this study clearly revealed that the formulation significantly reduced postharvest natural decay, promoted fruit firmness, and delayed loss of titratable acidity and vitamin C as storage time increased (Fig. 2), as well as the decay index, was reduced 23.8% in the treated samples (Fig. 3). Firmness is one of the most important features used to indicate the quality of fruit during the ripening process. As shown in Fig. 2, the initial firmness values (0–10 d) were similar between the control and treated samples; during days 10–20, the treated group showed slower changes of firmness compared to the control group; however, after 20 d of storage, the fruits dramatically lost firmness in the control group. The results indicate that the formulation with the CMCS coating may be functional in decreasing water and weight loss, maintaining the firmness of the fresh jujube fruit. Many studies have shown that chitosan, with its filmogenic properties, has beneficial effects on fruit firmness (Romanazzi et al., 2017). In addition, the formulation provided effective protection decline of vitamin C and titratable acidity during storage, while the increase in soluble solids was delayed in the treated group. Consistent with our results, for the 12-day storage of guava, the application of 2.0% chitosan significantly reduced the firmness and weight loss, delayed changes in the soluble solids content, and retarded the loss of 5
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Fig. 5. Heat map analysis of the relative abundance of the top 30 fungi between the treated and control samples at genus levels. The box with a redder color indicates that the fungal abundance in the sample is relatively higher.
4. Conclusion
for social development of Xinjiang production and Construction Corps (No. 20l6AD030) and Pearl River Nova Program of Guangzhou (No. 201710010135) for financial support for this research project. V.K.G. would like to acknowledge funding from the EU 7th Framework Programme for research, technological development and demonstration activities under grant agreement No. 621364 (TUTIC-Green), that provided support to make this work.
The development of food protecting films and coatings may provide an alternative approach to fruit in storing, transporting, and selling. The current study is an important complementary modification for understanding the individual and combined antifungal activity of NATA and CMCS against A. alternara; the synergistic interactions of NATA and CMCS were first confirmed with dose dependence experiments. All of these results have consistently indicated that the formulation incorporating NATA and CMCS possesses effective antimicrobial activity and helps to retain the nutritional quality of fresh jujube fruit and prolong its shelf life. This study suggested that the mixtures of NATA and CMCS represent an effective alternative to conventional chemical methods for slowing the postharvest decay of fresh jujube fruit, perhaps also including other horticultural products.
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Conflict of interest None Acknowledgments The authors would like to thank the Scientific and Technology Service Net Project of Chinese Academy of Sciences (No. KFJEW-STS085), Guangdong Provincial Agricultural Department Project for Rural Revitalization (No. 2018LM2177). Scientific and technological projects 6
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