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Impact of carboxymethyl cellulose coating enriched with extract of Impatiens balsamina stems on preservation of ‘Newhall’ navel orange
Scientia Horticulturae 160 (2013) 44–48
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Impact of carboxymethyl cellulose coating enriched with extract of Impatiens balsamina stems on preservation of ‘Newhall’ navel orange Rong Zeng a,∗ , Ashan Zhang b , Jinyin Chen c , Yongqi Fu c a
Department of Food Science, Foshan University, Foshan, 528000, PR China Administration Office of Western Eco-protection Forest, Yinchuan 750000, PR China Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables, Jiangxi Agricultural University, Nanchang 330045, PR China b c
a r t i c l e
i n f o
Article history: Received 8 February 2013 Received in revised form 10 May 2013 Accepted 16 May 2013 Keywords: Fruit quality Postharvest physiology Decay rate Weight loss Film Cold storage
a b s t r a c t A new approach to the control of postharvest decay, while maintaining fruit quality, has been implemented by the application of extract of Impatiens balsamina L. stems, a commonly used traditional Chinese medicine, amended coatings to ‘Newhall’ navel orange (Citrus sinensis L., Osbeck). After harvest, navel oranges were dipped in amended coating and then the samples were stored at 5 ◦ C and 90–95% RH after being dried naturally. The data suggested that the coating treatment, respectively, reduced the decay rate and weight loss of fruit from 10.2% to 6.1% and from 6.33% to 2.91% after 100 days storage, with none deleterious effects on fruit quality such as soluble solids content (SSC), titratable acidity (TA) and asorbic acid content (AsA). Moreover, the activities of scavenger antioxidant and defense enzymes, including peroxidase (POD), superoxide dismutase (SOD), chitinase (CHI) and -1,3-glucanase (GLU), were also increased by the coating treatment. It indicates that the film may be an effective and safe alternative preservative for navel orange. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Fresh navel orange is one of the consumers’ most favorite fruit for its highly nutritional value. However, injuries sustained by navel orange fruits during harvest allow the entry of pathogens, including Penicillium italicum and Penicillium digitatum, which are the causal agents of blue mold and green mold respectively. These two pathogens are considered the cause of most of the serious postharvest losses of citrus fruit (Porat et al., 2000). Synthetic fungicides such as prochloraz, imazalil and pyrimethanil are generally used in packhouse against postharvest pathogens of fruit and vegetable (Hamed et al., 2012; Sharma et al., 2009). Nevertheless, it has become the global trend to replace synthetic fungicides with safer and biodegradable alternatives to reduce the decay loss (Bosquez-Molina et al., 2010; Cerqueira et al., 2009; Xing et al., 2011). Natural derived antimicrobial agents from plant origin have been reported as a novel and safe alternative and supplement to control postharvest diseases and prolong shelf life (Lee et al., 2007; Mosqueda-Melgar et al., 2008).
∗ Corresponding author at: College of Food and Horticultural Science, Foshan University, Foshan City 528000, Guangdong Province, PR China. Tel.: +86 757 85592528; fax: +86 757 85592529. E-mail addresses: [email protected], [email protected] (R. Zeng). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.05.015
In recent years, we have screened 96 herb extracts for antifungal activity against P. digitatum and P. italicum and demonstrated that the Impatiens balsamina extract (IBE) was able to significantly inhibit both molds in vitro. The stems of I. balsamina have been widely used in traditional Chinese medicine to treat carbuncle, rheumatism, fractures, traumatic swelling and aches, fingernail inflammation, etc. Moreover, in some regions of China, people ingest this plant as a vegetable or anti-cancer herb, although there are currently no reports confirming the efficacy of this practice (Ding et al., 2008). The objective of this work is to evaluate the influences of IBE amended coating on ‘Newhall’ navel orange during storage at 5 ◦ C. 2. Materials and methods 2.1. Preparation of amended coating 2.1.1. Preparation of IBE The stems of I. balsamina (origin: Anhui province, China) were purchased from a traditional Chinese medicine store. The sample were grounded into fine powder (less than 20 mesh) after being dried below 50 ◦ C. A mixture of 200 g sample was extracted with 2 L 95% (v/v) ethanol at 30 ◦ C with ultrasonic assisted extraction (40 kHz) for 6 h, and then was filtered. The supernatants were collected and concentrated in a rotary evaporation at 35 ◦ C and stored at 4 ◦ C. Solution of 1 g/ml (raw herb/solvent: w/v) was made in
R. Zeng et al. / Scientia Horticulturae 160 (2013) 44–48
sterile water, to which 0.1% Tween80 could be added to enhance the solubility of the extracts. 2.1.2. Preparation of coating Carboxymethyl cellulose (CMC) solution (1.5%, w/v) was prepared by dissolving 1.5 g of CMC powder in 100 mL of 0.05% citric acid, 0.5% sucrose esters, 0.5% glycerin and 2% calcium propionate in distilled water, with agitation for 8 h. All the film reagents are food grade. Put 5 ml IBE solution into the coating, with agitation for 1 h. 2.2. Fruit preparation ‘Newhall’ navel oranges were harvested at commercially mature stage (19th December, 2011) from Chang-an Orchard in Jiangxi province, China. Fruits were uniformed in terms of shape, size, color and free of any damage or visual defects. They were washed with tap water and air-dried, then dipped by hand into IBE amended coating while the control group was treated without coating. All fruits were air-dried, packed into perforated, low-density polyethylene bags (d = 0.04 mm) and stored at 5 ◦ C. Each treatment comprised six replicate bags, each containing 50 fruits. Samples were taken initially and at 10-day intervals during storage for quality parameters and other analysis. 2.3. Evaluation of quality and physiological parameters 2.3.1. Quality measurement Decay incidence was expressed by the percentage of fruits indicating fungal infection. Weight loss was calculated on the basis of a comparison of initial and final weights. Flesh tissues from 10 navel oranges were homogenized in a waring blender and centrifuged at 8000 rpm for 10 min at 4 ◦ C. The supernatant phase was collected to analyze soluble solids content (SSC), titratable acidity (TA) and asorbic acid content (AsA). SSC expressed in ◦ Brix was assayed by a RA-250WE digital brix-meter (KYOTO, Tokyo, Japan) at a temperature of 20 ± 0.5 ◦ C. TA expressed as percentage of citric acid was determined by the titration using a standard solution of sodium hydroxide (0.1 M). AsA was assayed by the titration using a solution of 2,6-dichlorophenolindo-phenol and the value was expressed in mg/100 ml pulp juice. 2.3.2. Physiological parameters evaluation Activities of peroxidase (POD) and superoxide dismutase (SOD) were determined by following the methods of Prochazkova et al. (2001). Pulp tissue (1 g) was homogenized in 10 ml PBS (25 mmol/L, pH 7.8), containing 0.8 g/L PVPP and 1 mmol/L EDTA, then centrifuged at 12 000 rpm for 15 min at 4 ◦ C. The resulting supernatants were used directly for POD and SOD assays. For POD determination, 0.5 ml of enzyme extract was incubated in 2 ml buffered substrate (100 mmol/L sodium phosphate, pH6.4 and 25 mmol/L guaiacol) for 5 min at 30 ◦ C and the increasing absorbance measured at 470 nm every 30 s for 180 s after adding 0.2 ml H2 O2 (0.5 mol/L). POD activity was expressed as unit/g FW, one unit was defined as an increase of 1 absorbance at OD 470 nm per min. For SOD determination, the reaction mixture (3 ml) contained 50 mmol/L sodium phosphate buffer (pH 7.8), 130 mmol/L methionine, 750 mol/L nitro-blue tetrazolium, 100 mol/L EDTA-Na2 , 20 mol/L riboflavin and 0.1 ml of the enzyme extract. The mixtures were illuminated by light (4000 lx) for 20 min at 30 ◦ C and the absorbance was then determined at 560 nm. Identical solutions held in the dark served as blanks. One unit of SOD was defined as the amount of enzyme with gave half-maximal inhibition and the SOD activity was expressed as unit/g FW.
45
A weighed portion of pericarp tissue was grounded in liquid nitrogen and homogenized in 0.1 M sodium citrate buffer, pH 5.0, at a ratio of 1:2 (w/v) with a pestle and mortar. The homogenate was centrifuged at 10,000 rpm for 5 min at 4 ◦ C, then the supernatant was desalted by dialysis overnight at 4 ◦ C and used as crude enzyme preparation. Chitinase (CHI, EC 3.2.1.14) activity was colorimetric assayed following Mauch et al. (1984) with some modifications. An aliquot (0.5 ml) of the crude enzyme preparation was mixed with 0.5 ml 10 g/L colloidal chitin (Sigma), 0.5 ml 50 mmol/L sodium phosphate buffer (pH 6.4) and incubated in a water bath at 37 ◦ C for 1 h. Then, 0.1 mL 20 g/L desalted snailase (Sigma) was added, and the mixture was incubated at 37 ◦ C for another 1 h. The reaction was stopped by addition of 0.2 ml of 0.6 M potassium tetraborate and boiling for 5 min. After rapid cooling, 2 ml of 100 g/L 4-(dimethylamino) benzaldehyde reagent diluted with glacial acetic acid (1:5 v/v) was added and the mixture was incubated at 37 ◦ C for 20 min, and then absorbance was measured at 585 nm. CHI activity was calculated for an enzyme concentration approaching zero using standard curves and expressed as unit/g FW. The amount of enzyme producing 1 nmol N-acetyl-d-glucosamine per hour under these assay conditions was defined as one unit. -1,3-glucanase (Gülc¸in et al., 2004) activity was assayed by measuring the reducing release of sugar from laminarin as the substrate as described by (Zheng et al., 2011) with minor modifications. Desalted enzyme extract (400 l) was mixed with 100 l laminarin (0.4 g/L), and the mixture was incubated in a shaking water bath at 37 ◦ C for 1 h. The reaction was stopped by adding 1.5 ml DNS reagent and boiling for 5 min. The mixture was diluted to 25 ml and absorbance at 540 nm was determined. Enzyme activity is expressed as unit/g FW, where a unit is defined as the formation of 1 nmol glucose equivalents released from laminarin per hour under these assay conditions. 2.4. Statistical analysis All statistical analysis was performed using SPSS 11.0 and Excel (2007). The statistical significance was applied at the level P < 0.05. When the analysis was statistically significant, Ducan’s multiple range test was applied to the separate mean values. Each experiment had three replicates and whenever similar results were obtained, the experiments were conducted a fourth time. 3. Results 3.1. Decay rate and weight loss Fruits of control treatment began to become infected with pathogens 50 days after storage while clear signs of pathogens stasis being found 70 days after storage on those with amended coating treatment (Fig. 1A). After been stored for 100 days, fruits treated by amended coating exhibited significantly lower decay incidence than the control group at the level of P < 0.05 (6.1% and 10.2%, respectively). Meanwhile, the amended coating group displayed less moisture loss than the control treatment, but the values did not differ significantly until 40 days after storage (Fig. 1B). The weight loss of the coating navel oranges and control group reached 2.91% and 6.33% respectively after 100 days storage. 3.2. Fruit quality As illustrated in Fig. 2A, the SSC increased continuously during the early stage of storage and declined slightly during the later storage days of both treatments. The SSC in the control navel oranges reached peak value (14.93 ± 0.12 ◦ Brix) 30 days after storage, which
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R. Zeng et al. / Scientia Horticulturae 160 (2013) 44–48
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Fig. 1. The decay rate (A) and weight loss (B) in the coating and control treatment navel oranges during cold storage (5 ◦ C). Bars indicate standard deviation of three replicates.
was the same as the coating group after stored for 40 days. The SSCs of coating treatment were significantly higher than the control from 40 to 70 days after storage, while there was little difference during the later storage stage. The levels of TA of navel oranges in both treatments decreased gradually with storage time (Fig. 2B). Fruits treated with coating tended to have higher TA content than those in the control group, but no significant difference was found between the two groups. Nevertheless, coating treatment had no negative impact on AsA and total sugar contents of navel orange (Fig. 2C and D).
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As revealed in Fig. 3A, similar trends occurred in the control and coating treated fruit. SOD activity in control group increased initially and reached peak value 20 days after storage, yet that in coating treated navel oranges reached peak value 40 days after storage and was significantly higher than that in control group since then. POD activity in coating fruits rose to two peaks at the 10th and 80th day respectively after storage, which were 34.75 unit g−1 FW and 28.64 unit g−1 FW respectively (Fig. 3B). In the control group,
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Fig. 2. Effects of coating on SSC (A), TA (B), the contents of ASA (C) and total sugar (D) in navel oranges during cold storage (5 ◦ C). Bars indicate standard deviation of three replicates.
R. Zeng et al. / Scientia Horticulturae 160 (2013) 44–48 24
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Fig. 3. Effects of coating on the acitivities of SOD (A), POD (B), GLU (C) and CHI (D) in navel oranges during storage (5 ◦ C). Bars indicate standard deviation of three replicates.
POD activity increased initially and then declined dramatically and was significantly lower than coating group. The results showed GLU activity changed gently in control fruits and reached the maximum level of 3.44 unit g−1 FW 20 days after storage, while the peak value of coating fruits were 6.94 unit g−1 FW (Fig. 3C). GLU activities of coating oranges were higher than those of untreated ones throughout the storage. We could observe that coating treatment notably enhanced the activity of CHI (Fig. 3D). CHI activity increased continuously in both groups and has value peaks at 80 days after storage, which in coating oranges was significantly higher than controlled ones. 4. Discussion The exploration of natural preservatives to improve safety and quality of fresh fruits and vegetables is attracting increasing interest as a result of the growing demand to reduce the use of chemical fungicides. The antimicrobial properties of components derived from many plant organs have been empirically recognized for centuries, but only came to scientific attention in the food industry in recent years (Zeng et al., 2012). However, the application of plant extracts in the packing house environment was previously found to be unable to reduce the incidence of P. digitatum in citrus fruit in vivo and caused severe rind damage (Plaza et al., 2004), which may be related with the plant extracts components and applied concentration. Our previous work has already demonstrated that IBE shows antimicrobial activity against P. italicum and P. digitatum in vitro. Data in this research shows that IBE amended with composite CMC film can significantly alleviate the fruit decay and weight loss with no negative effect on overall quality. Fruit quality refers to a range of properties that are related to flavor, appearance and nutrient contents in the case of navel orange. These parameters change during storage, the increase or reduction being related to desirable quality or quality loss (Serrano et al., 2008). We found that coating treatment with IBE had no
deleterious effects on fruit quality such as contents of SSC, TA and AsA compared with the control group. Activities of oxyradical detoxification and defense enzymes, including SOD, POD, CHT and GLU of the fruit were analyzed to observe the further effects of coating treatment on postharvest physiological change. SOD and POD are critical enzymes in plant tissue and many effective supplements can increase activities of scavenger antioxidant enzymes to reduce the acceleration of the ripening process, and in turn prolong fruit shelf-life (Zeng et al., 2010). Both of the SOD and POD activities of two groups showed a trend of rising during the early storage stage and then declined, and the treated group changed more gently than the control group. This phenomenon may be probably due to the accumulation of O•2− in cells of untreated fruit during the early storage stage which induced the higher SOD activity to protect the fruit tissue from injury. The level of SOD and POD activities of coating fruits was more stable, which showed the fruits had more capacity to remain the balance between O•2 − generation and the capacity to eliminate it. Some inducing agents can develop a systemic increase in resistance in the plant against future attack by a variety of microbial pathogens, which is called induced systemic resistance response involving the de novo production of the pathogenesis-related (PR) proteins (Jaiti et al., 2009; Sparla et al., 2004). Chitinase and 1,3-glucanase play important roles in plant disease resistance system for their capacity to hydrolysis chitin and -1,3-glucan in the cell walls of the fungal pathogens so as to protect the host (Martin, 2001; Schlumbaum et al., 1986). Literature data indicate that induced systemic resistance can be elicited by exogenous application of various chemical and biological compounds. Our experiments showed that coating treatment increased CHI and GLU activities significantly (Nielsen et al., 1994; Tripathi et al., 2010; Zhang et al., 2002). In conclusion, compared with the uncoated group, the amended coating exhibited a beneficial impact on the overall quality of navel
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orange by reducing the moisture loss and fruit spoilage, maintaining titratable acidity and ascorbic acid content. Moreover, the coating could effectively enhance the activities of SOD, POD, CHT and GLU. It indicates that the film may be an effective and safe alternative preservative for navel orange. Acknowledgements This work was financially supported by the National Science Foundation of China (Grant No. 31160343), the 12th Five-Year National Key Technologies R&D Program of China (Grant No. 2012BAD38B03-2), National Agricultural Achievements Transformation Fund (Grant No. 2011GB2C500017) and Jiangxi Provincial Education Development Fund for Transformation of the Scientific Research’s Achievements (111). References Bosquez-Molina, E., Jesús E.R.-d. Bautista-Banos, S., Verde-Calvo, J.R., Morales-López, J., 2010. Inhibitory effect of essential oils against Colletotrichum gloeosporioides and Rhizopus stolonifer in stored papaya fruit and their possible application in coatings. Postharvest Biol. Technol. 57, 132–137. Cerqueira, M.A., Lima, Á.M., Teixeira, J.A., Moreira, R.A., Vicente, A.A., 2009. Suitability of novel galactomannans as edible coatings for tropical fruits. J. Food Eng. 94, 372–378. Ding, Z.S., Jiang, F.S., Chen, N.P., Lv, G.Y., Zhu, C.G., 2008. Isolation and identification of an anti-tumor component from leaves of Impatiens balsamina. Molecules 13, 220–229. Gülc¸in, Ì., Güngör S¸at, I˙ ., Beydemir, S¸., Elmastas¸, M., I˙ rfan Küfrevioˇglu, Ö., 2004. Comparison of antioxidant activity of clove (Eugenia caryophylata Thunb) buds and lavender (Lavandula stoechas L.). Food Chem. 87, 393–400. Hamed, S.F., Sadek, Z., Edris, A., 2012. Antioxidant and antimicrobial activities of clove bud essential oil and eugenol nanoparticles in alcohol-free microemulsion. J. Oleo. Sci. 61, 641–648. Jaiti, F., Verdeil, J.L., El Hadrami, I., 2009. Effect of jasmonic acid on the induction of polyphenoloxidase and peroxidase activities in relation to date palm resistance against Fusarium oxysporum f. sp. albedinis. Physiol. Mol. Plant Pathol. 74, 84–90. Lee, S.H., Chang, K.S., Su, M.S., Huang, Y.S., Jang, H.-D., 2007. Effects of some Chinese medicinal plant extracts on five different fungi. Food Control 18, 1547–1554. Martin, H., 2001. Induced systemic resistance (ISR) against pathogens–a promising field for ecological research. Persp. Plant Ecol. Evol. System. 4, 65–79.
Mauch, F., Hadwiger, L.A., Boller, T., 1984. Ethylene: Symptom, not signal for the induction of chitinase and, -1,3-glucanase in pea pods by pathogens and elicitors. Plant Physiol. 76, 607–611. Mosqueda-Melgar, J., Raybaudi-Massilia, R.M., Martín-Belloso, O., 2008. Combination of high-intensity pulsed electric fields with natural antimicrobials to inactivate pathogenic microorganisms and extend the shelf-life of melon and watermelon juices. Food Microbiol. 25, 479–491. Nielsen, K.K., Bojsen, K., Collinge, D.B., Mikkelsen, J.D., 1994. Induced resistance in sugar beet against Cercospora beticola: induction by dichloroisonicotinic acid is independent of chitinase and -1,3-glucanase transcript accumulation. Physiol. Mol. Plant Pathol. 45, 89–99. Plaza, P., Torres, R., Usall, J., Lamarca, N., Vinas, I., 2004. Evaluation of the potential of commercial post-harvest application of essential oils to control citrus decay. J. Horticult. Sci. Biotechnol. 79, 935–940. Porat, R., Daus, A., Weiss, B., Cohen, L., Fallik, E., 2000. Reduction of postharvest decay in organic citrus fruit by a short hot water brushing treatment. Postharvest Biol. Technol. 18, 151–157. Prochazkova, D., Sairam, R.K., Srivastava, G.C., Singh, D.V., 2001. Oxidative stress and antioxidant activity as the basis of senescence in maize leaves. Plant Science 161, 765–771. Schlumbaum, A., Mauch, F., Vögeli, U., Boller, T., 1986. Plant chitinases are potent inhibitors of fungal growth. Nature 324, 365–367. Serrano, M., Martínez-Romero, D., Guillén, F., Valverde, J.M., Zapata, P.J., Castillo, S., Valero, D., 2008. The addition of essential oils to MAP as a tool to maintain the overall quality of fruits. Trends in Food Sci. Technol. 19, 464–471. Sharma, R.R., Singh, D., Singh, R., 2009. Biological control of postharvest diseases of fruits and vegetables by microbial antagonists: a review. Biol. Control 50, 205–221. Sparla, F., Rotino, L., Valgimigli, M.C., Pupillo, P., Trost, P., 2004. Systemic resistance induced by benzothiadiazole in pear inoculated with the agent of fire blight (Erwinia amylovora). Sci. Hortic. 101, 269–279. Tripathi, D., Jiang, Y.-L., Kumar, D., 2010. SABP2, a methyl salicylate esterase is required for the systemic acquired resistance induced by acibenzolar-S-methyl in plants. FEBS Lett. 584, 3458–3463. Xing, Y., Li, X.H., Xu, Q.L., Yun, J., Lu, Y., Tang, Y., 2011. Effects of chitosan coating enriched with cinnamon oil on qualitative properties of sweet pepper (Capsicum annuum L). Food Chem. 124, 1443–1450. Zeng, K., Deng, Y., Ming, J., Deng, L., 2010. Induction of disease resistance and ROS metabolism in navel oranges by chitosan. Sci. Hortic. 126, 223–228. Zeng, R., Zhang, A.S., Chen, J.Y., Fu, Y.Q., 2012. Postharvest quality and physiological responses of clove bud extract dip on ‘Newhall’ navel orange. Sci. Hortic. 138, 253–258. Zhang, S., Moyne, A.-L., Reddy, M.S., Kloepper, J.W., 2002. The role of salicylic acid in induced systemic resistance elicited by plant growth-promoting rhizobacteria against blue mold of tobacco. Biol. Control 25, 288–296. Zheng, Y., Shen, L., Yu, M.M., Fan, B., Zhao, D.Y., Liu, L.Y., Sheng, J., 2011. Nitric oxide synthase as a postharvest response in pathogen resistance of tomato fruit. Postharvest Biol. Technol. 60, 38–46.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Impact of carboxymethyl cellulose coating enriched with extract of Impatiens balsamina stems on preservation of ‘Newhall’ navel orange Rong Zeng a,∗ , Ashan Zhang b , Jinyin Chen c , Yongqi Fu c a
Department of Food Science, Foshan University, Foshan, 528000, PR China Administration Office of Western Eco-protection Forest, Yinchuan 750000, PR China Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables, Jiangxi Agricultural University, Nanchang 330045, PR China b c
a r t i c l e
i n f o
Article history: Received 8 February 2013 Received in revised form 10 May 2013 Accepted 16 May 2013 Keywords: Fruit quality Postharvest physiology Decay rate Weight loss Film Cold storage
a b s t r a c t A new approach to the control of postharvest decay, while maintaining fruit quality, has been implemented by the application of extract of Impatiens balsamina L. stems, a commonly used traditional Chinese medicine, amended coatings to ‘Newhall’ navel orange (Citrus sinensis L., Osbeck). After harvest, navel oranges were dipped in amended coating and then the samples were stored at 5 ◦ C and 90–95% RH after being dried naturally. The data suggested that the coating treatment, respectively, reduced the decay rate and weight loss of fruit from 10.2% to 6.1% and from 6.33% to 2.91% after 100 days storage, with none deleterious effects on fruit quality such as soluble solids content (SSC), titratable acidity (TA) and asorbic acid content (AsA). Moreover, the activities of scavenger antioxidant and defense enzymes, including peroxidase (POD), superoxide dismutase (SOD), chitinase (CHI) and -1,3-glucanase (GLU), were also increased by the coating treatment. It indicates that the film may be an effective and safe alternative preservative for navel orange. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Fresh navel orange is one of the consumers’ most favorite fruit for its highly nutritional value. However, injuries sustained by navel orange fruits during harvest allow the entry of pathogens, including Penicillium italicum and Penicillium digitatum, which are the causal agents of blue mold and green mold respectively. These two pathogens are considered the cause of most of the serious postharvest losses of citrus fruit (Porat et al., 2000). Synthetic fungicides such as prochloraz, imazalil and pyrimethanil are generally used in packhouse against postharvest pathogens of fruit and vegetable (Hamed et al., 2012; Sharma et al., 2009). Nevertheless, it has become the global trend to replace synthetic fungicides with safer and biodegradable alternatives to reduce the decay loss (Bosquez-Molina et al., 2010; Cerqueira et al., 2009; Xing et al., 2011). Natural derived antimicrobial agents from plant origin have been reported as a novel and safe alternative and supplement to control postharvest diseases and prolong shelf life (Lee et al., 2007; Mosqueda-Melgar et al., 2008).
∗ Corresponding author at: College of Food and Horticultural Science, Foshan University, Foshan City 528000, Guangdong Province, PR China. Tel.: +86 757 85592528; fax: +86 757 85592529. E-mail addresses: [email protected], [email protected] (R. Zeng). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.05.015
In recent years, we have screened 96 herb extracts for antifungal activity against P. digitatum and P. italicum and demonstrated that the Impatiens balsamina extract (IBE) was able to significantly inhibit both molds in vitro. The stems of I. balsamina have been widely used in traditional Chinese medicine to treat carbuncle, rheumatism, fractures, traumatic swelling and aches, fingernail inflammation, etc. Moreover, in some regions of China, people ingest this plant as a vegetable or anti-cancer herb, although there are currently no reports confirming the efficacy of this practice (Ding et al., 2008). The objective of this work is to evaluate the influences of IBE amended coating on ‘Newhall’ navel orange during storage at 5 ◦ C. 2. Materials and methods 2.1. Preparation of amended coating 2.1.1. Preparation of IBE The stems of I. balsamina (origin: Anhui province, China) were purchased from a traditional Chinese medicine store. The sample were grounded into fine powder (less than 20 mesh) after being dried below 50 ◦ C. A mixture of 200 g sample was extracted with 2 L 95% (v/v) ethanol at 30 ◦ C with ultrasonic assisted extraction (40 kHz) for 6 h, and then was filtered. The supernatants were collected and concentrated in a rotary evaporation at 35 ◦ C and stored at 4 ◦ C. Solution of 1 g/ml (raw herb/solvent: w/v) was made in
R. Zeng et al. / Scientia Horticulturae 160 (2013) 44–48
sterile water, to which 0.1% Tween80 could be added to enhance the solubility of the extracts. 2.1.2. Preparation of coating Carboxymethyl cellulose (CMC) solution (1.5%, w/v) was prepared by dissolving 1.5 g of CMC powder in 100 mL of 0.05% citric acid, 0.5% sucrose esters, 0.5% glycerin and 2% calcium propionate in distilled water, with agitation for 8 h. All the film reagents are food grade. Put 5 ml IBE solution into the coating, with agitation for 1 h. 2.2. Fruit preparation ‘Newhall’ navel oranges were harvested at commercially mature stage (19th December, 2011) from Chang-an Orchard in Jiangxi province, China. Fruits were uniformed in terms of shape, size, color and free of any damage or visual defects. They were washed with tap water and air-dried, then dipped by hand into IBE amended coating while the control group was treated without coating. All fruits were air-dried, packed into perforated, low-density polyethylene bags (d = 0.04 mm) and stored at 5 ◦ C. Each treatment comprised six replicate bags, each containing 50 fruits. Samples were taken initially and at 10-day intervals during storage for quality parameters and other analysis. 2.3. Evaluation of quality and physiological parameters 2.3.1. Quality measurement Decay incidence was expressed by the percentage of fruits indicating fungal infection. Weight loss was calculated on the basis of a comparison of initial and final weights. Flesh tissues from 10 navel oranges were homogenized in a waring blender and centrifuged at 8000 rpm for 10 min at 4 ◦ C. The supernatant phase was collected to analyze soluble solids content (SSC), titratable acidity (TA) and asorbic acid content (AsA). SSC expressed in ◦ Brix was assayed by a RA-250WE digital brix-meter (KYOTO, Tokyo, Japan) at a temperature of 20 ± 0.5 ◦ C. TA expressed as percentage of citric acid was determined by the titration using a standard solution of sodium hydroxide (0.1 M). AsA was assayed by the titration using a solution of 2,6-dichlorophenolindo-phenol and the value was expressed in mg/100 ml pulp juice. 2.3.2. Physiological parameters evaluation Activities of peroxidase (POD) and superoxide dismutase (SOD) were determined by following the methods of Prochazkova et al. (2001). Pulp tissue (1 g) was homogenized in 10 ml PBS (25 mmol/L, pH 7.8), containing 0.8 g/L PVPP and 1 mmol/L EDTA, then centrifuged at 12 000 rpm for 15 min at 4 ◦ C. The resulting supernatants were used directly for POD and SOD assays. For POD determination, 0.5 ml of enzyme extract was incubated in 2 ml buffered substrate (100 mmol/L sodium phosphate, pH6.4 and 25 mmol/L guaiacol) for 5 min at 30 ◦ C and the increasing absorbance measured at 470 nm every 30 s for 180 s after adding 0.2 ml H2 O2 (0.5 mol/L). POD activity was expressed as unit/g FW, one unit was defined as an increase of 1 absorbance at OD 470 nm per min. For SOD determination, the reaction mixture (3 ml) contained 50 mmol/L sodium phosphate buffer (pH 7.8), 130 mmol/L methionine, 750 mol/L nitro-blue tetrazolium, 100 mol/L EDTA-Na2 , 20 mol/L riboflavin and 0.1 ml of the enzyme extract. The mixtures were illuminated by light (4000 lx) for 20 min at 30 ◦ C and the absorbance was then determined at 560 nm. Identical solutions held in the dark served as blanks. One unit of SOD was defined as the amount of enzyme with gave half-maximal inhibition and the SOD activity was expressed as unit/g FW.
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A weighed portion of pericarp tissue was grounded in liquid nitrogen and homogenized in 0.1 M sodium citrate buffer, pH 5.0, at a ratio of 1:2 (w/v) with a pestle and mortar. The homogenate was centrifuged at 10,000 rpm for 5 min at 4 ◦ C, then the supernatant was desalted by dialysis overnight at 4 ◦ C and used as crude enzyme preparation. Chitinase (CHI, EC 3.2.1.14) activity was colorimetric assayed following Mauch et al. (1984) with some modifications. An aliquot (0.5 ml) of the crude enzyme preparation was mixed with 0.5 ml 10 g/L colloidal chitin (Sigma), 0.5 ml 50 mmol/L sodium phosphate buffer (pH 6.4) and incubated in a water bath at 37 ◦ C for 1 h. Then, 0.1 mL 20 g/L desalted snailase (Sigma) was added, and the mixture was incubated at 37 ◦ C for another 1 h. The reaction was stopped by addition of 0.2 ml of 0.6 M potassium tetraborate and boiling for 5 min. After rapid cooling, 2 ml of 100 g/L 4-(dimethylamino) benzaldehyde reagent diluted with glacial acetic acid (1:5 v/v) was added and the mixture was incubated at 37 ◦ C for 20 min, and then absorbance was measured at 585 nm. CHI activity was calculated for an enzyme concentration approaching zero using standard curves and expressed as unit/g FW. The amount of enzyme producing 1 nmol N-acetyl-d-glucosamine per hour under these assay conditions was defined as one unit. -1,3-glucanase (Gülc¸in et al., 2004) activity was assayed by measuring the reducing release of sugar from laminarin as the substrate as described by (Zheng et al., 2011) with minor modifications. Desalted enzyme extract (400 l) was mixed with 100 l laminarin (0.4 g/L), and the mixture was incubated in a shaking water bath at 37 ◦ C for 1 h. The reaction was stopped by adding 1.5 ml DNS reagent and boiling for 5 min. The mixture was diluted to 25 ml and absorbance at 540 nm was determined. Enzyme activity is expressed as unit/g FW, where a unit is defined as the formation of 1 nmol glucose equivalents released from laminarin per hour under these assay conditions. 2.4. Statistical analysis All statistical analysis was performed using SPSS 11.0 and Excel (2007). The statistical significance was applied at the level P < 0.05. When the analysis was statistically significant, Ducan’s multiple range test was applied to the separate mean values. Each experiment had three replicates and whenever similar results were obtained, the experiments were conducted a fourth time. 3. Results 3.1. Decay rate and weight loss Fruits of control treatment began to become infected with pathogens 50 days after storage while clear signs of pathogens stasis being found 70 days after storage on those with amended coating treatment (Fig. 1A). After been stored for 100 days, fruits treated by amended coating exhibited significantly lower decay incidence than the control group at the level of P < 0.05 (6.1% and 10.2%, respectively). Meanwhile, the amended coating group displayed less moisture loss than the control treatment, but the values did not differ significantly until 40 days after storage (Fig. 1B). The weight loss of the coating navel oranges and control group reached 2.91% and 6.33% respectively after 100 days storage. 3.2. Fruit quality As illustrated in Fig. 2A, the SSC increased continuously during the early stage of storage and declined slightly during the later storage days of both treatments. The SSC in the control navel oranges reached peak value (14.93 ± 0.12 ◦ Brix) 30 days after storage, which
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Fig. 1. The decay rate (A) and weight loss (B) in the coating and control treatment navel oranges during cold storage (5 ◦ C). Bars indicate standard deviation of three replicates.
was the same as the coating group after stored for 40 days. The SSCs of coating treatment were significantly higher than the control from 40 to 70 days after storage, while there was little difference during the later storage stage. The levels of TA of navel oranges in both treatments decreased gradually with storage time (Fig. 2B). Fruits treated with coating tended to have higher TA content than those in the control group, but no significant difference was found between the two groups. Nevertheless, coating treatment had no negative impact on AsA and total sugar contents of navel orange (Fig. 2C and D).
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As revealed in Fig. 3A, similar trends occurred in the control and coating treated fruit. SOD activity in control group increased initially and reached peak value 20 days after storage, yet that in coating treated navel oranges reached peak value 40 days after storage and was significantly higher than that in control group since then. POD activity in coating fruits rose to two peaks at the 10th and 80th day respectively after storage, which were 34.75 unit g−1 FW and 28.64 unit g−1 FW respectively (Fig. 3B). In the control group,
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Fig. 2. Effects of coating on SSC (A), TA (B), the contents of ASA (C) and total sugar (D) in navel oranges during cold storage (5 ◦ C). Bars indicate standard deviation of three replicates.
R. Zeng et al. / Scientia Horticulturae 160 (2013) 44–48 24
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Fig. 3. Effects of coating on the acitivities of SOD (A), POD (B), GLU (C) and CHI (D) in navel oranges during storage (5 ◦ C). Bars indicate standard deviation of three replicates.
POD activity increased initially and then declined dramatically and was significantly lower than coating group. The results showed GLU activity changed gently in control fruits and reached the maximum level of 3.44 unit g−1 FW 20 days after storage, while the peak value of coating fruits were 6.94 unit g−1 FW (Fig. 3C). GLU activities of coating oranges were higher than those of untreated ones throughout the storage. We could observe that coating treatment notably enhanced the activity of CHI (Fig. 3D). CHI activity increased continuously in both groups and has value peaks at 80 days after storage, which in coating oranges was significantly higher than controlled ones. 4. Discussion The exploration of natural preservatives to improve safety and quality of fresh fruits and vegetables is attracting increasing interest as a result of the growing demand to reduce the use of chemical fungicides. The antimicrobial properties of components derived from many plant organs have been empirically recognized for centuries, but only came to scientific attention in the food industry in recent years (Zeng et al., 2012). However, the application of plant extracts in the packing house environment was previously found to be unable to reduce the incidence of P. digitatum in citrus fruit in vivo and caused severe rind damage (Plaza et al., 2004), which may be related with the plant extracts components and applied concentration. Our previous work has already demonstrated that IBE shows antimicrobial activity against P. italicum and P. digitatum in vitro. Data in this research shows that IBE amended with composite CMC film can significantly alleviate the fruit decay and weight loss with no negative effect on overall quality. Fruit quality refers to a range of properties that are related to flavor, appearance and nutrient contents in the case of navel orange. These parameters change during storage, the increase or reduction being related to desirable quality or quality loss (Serrano et al., 2008). We found that coating treatment with IBE had no
deleterious effects on fruit quality such as contents of SSC, TA and AsA compared with the control group. Activities of oxyradical detoxification and defense enzymes, including SOD, POD, CHT and GLU of the fruit were analyzed to observe the further effects of coating treatment on postharvest physiological change. SOD and POD are critical enzymes in plant tissue and many effective supplements can increase activities of scavenger antioxidant enzymes to reduce the acceleration of the ripening process, and in turn prolong fruit shelf-life (Zeng et al., 2010). Both of the SOD and POD activities of two groups showed a trend of rising during the early storage stage and then declined, and the treated group changed more gently than the control group. This phenomenon may be probably due to the accumulation of O•2− in cells of untreated fruit during the early storage stage which induced the higher SOD activity to protect the fruit tissue from injury. The level of SOD and POD activities of coating fruits was more stable, which showed the fruits had more capacity to remain the balance between O•2 − generation and the capacity to eliminate it. Some inducing agents can develop a systemic increase in resistance in the plant against future attack by a variety of microbial pathogens, which is called induced systemic resistance response involving the de novo production of the pathogenesis-related (PR) proteins (Jaiti et al., 2009; Sparla et al., 2004). Chitinase and 1,3-glucanase play important roles in plant disease resistance system for their capacity to hydrolysis chitin and -1,3-glucan in the cell walls of the fungal pathogens so as to protect the host (Martin, 2001; Schlumbaum et al., 1986). Literature data indicate that induced systemic resistance can be elicited by exogenous application of various chemical and biological compounds. Our experiments showed that coating treatment increased CHI and GLU activities significantly (Nielsen et al., 1994; Tripathi et al., 2010; Zhang et al., 2002). In conclusion, compared with the uncoated group, the amended coating exhibited a beneficial impact on the overall quality of navel
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orange by reducing the moisture loss and fruit spoilage, maintaining titratable acidity and ascorbic acid content. Moreover, the coating could effectively enhance the activities of SOD, POD, CHT and GLU. It indicates that the film may be an effective and safe alternative preservative for navel orange. Acknowledgements This work was financially supported by the National Science Foundation of China (Grant No. 31160343), the 12th Five-Year National Key Technologies R&D Program of China (Grant No. 2012BAD38B03-2), National Agricultural Achievements Transformation Fund (Grant No. 2011GB2C500017) and Jiangxi Provincial Education Development Fund for Transformation of the Scientific Research’s Achievements (111). References Bosquez-Molina, E., Jesús E.R.-d. Bautista-Banos, S., Verde-Calvo, J.R., Morales-López, J., 2010. Inhibitory effect of essential oils against Colletotrichum gloeosporioides and Rhizopus stolonifer in stored papaya fruit and their possible application in coatings. Postharvest Biol. Technol. 57, 132–137. Cerqueira, M.A., Lima, Á.M., Teixeira, J.A., Moreira, R.A., Vicente, A.A., 2009. Suitability of novel galactomannans as edible coatings for tropical fruits. J. Food Eng. 94, 372–378. Ding, Z.S., Jiang, F.S., Chen, N.P., Lv, G.Y., Zhu, C.G., 2008. Isolation and identification of an anti-tumor component from leaves of Impatiens balsamina. Molecules 13, 220–229. Gülc¸in, Ì., Güngör S¸at, I˙ ., Beydemir, S¸., Elmastas¸, M., I˙ rfan Küfrevioˇglu, Ö., 2004. Comparison of antioxidant activity of clove (Eugenia caryophylata Thunb) buds and lavender (Lavandula stoechas L.). Food Chem. 87, 393–400. Hamed, S.F., Sadek, Z., Edris, A., 2012. Antioxidant and antimicrobial activities of clove bud essential oil and eugenol nanoparticles in alcohol-free microemulsion. J. Oleo. Sci. 61, 641–648. Jaiti, F., Verdeil, J.L., El Hadrami, I., 2009. Effect of jasmonic acid on the induction of polyphenoloxidase and peroxidase activities in relation to date palm resistance against Fusarium oxysporum f. sp. albedinis. Physiol. Mol. Plant Pathol. 74, 84–90. Lee, S.H., Chang, K.S., Su, M.S., Huang, Y.S., Jang, H.-D., 2007. Effects of some Chinese medicinal plant extracts on five different fungi. Food Control 18, 1547–1554. Martin, H., 2001. Induced systemic resistance (ISR) against pathogens–a promising field for ecological research. Persp. Plant Ecol. Evol. System. 4, 65–79.
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