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WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland e-mail: info@world-food.net Journal of Food, Agriculture & Environment Vol.12 (2): 188-192. 2014 www.world-food.net Natamycin treatment to control postharvest mold development and improve storability of citrus fruits Bensu Yiğiter 1, Fatih Onay 1, Nese Basaran Akgul 2 and Pervin Basaran Akocak 1, 3 * Department of Food Engineering, Suleyman Demirel University, Isparta 32100, Turkey. Department of Food Engineering, Yildiz Technical University Esenler, Istanbul 34210, Turkey. 3 Department of Biofunctional Science and Technology, Sabbatical Leave Hiroshima University, Hiroshima 7398525, Japan. *e-mail: pervinbasaran@sdu.edu.tr 1 2 Received 25 February 2014, accepted 24 April 2014. Abstract Green and blue mold caused by Penicillium digitatum and Penicillium italicum, respectively, are the most harmful citrus fruit post-harvest pathogens. In this work, in vivo antifungal effect of Generally Recognised as Safe (GRAS) food additive natamycin pre-treatment was evaluated on artificially inoculated oranges and lemons for the control of green and blue mold during storage. Furthermore, the physico-chemical and other quality properties of the citrus fruits were evaluated after each treatment. Postharvest spray application of natamycin (NT) as low as 4.5 mg/ml on oranges infected by P. italicum significantly reduced the incidence of the disease from 90-100% in the control to 50% after 4 weeks. On infected lemons, the incidence of P. digitatum was significantly reduced by 5 mg/ml NT and resulted in 80% decay after 2 weeks, while control samples were fully spoiled. Various amounts of NT impregnated paper wrappers were tested to determine effectiveness in preserving the quality of the citrus fruits with respect to fruit deterioration. The 5 mg/ml NT impregnated paper wrap gave an improvement with the reduction of 40% of decay after 4 weeks of storage. In general, NT-treated paper wrap application was more effective than spray treatments in controlling both blue and green mold diseases on the surface citrus fruits and extending storage duration up to additional 2-3 weeks. External and internal physico-chemical quality parameters (decay incidence, colour development, loss of firmness, soluble solids and acidity) of fruits were evaluated. NT treatment may offer a combination mean to control postharvest fungal development of fresh citrus fruit. Key words: Citrus, postharvest, natamycin, shelf life, storage, fruit deterioration. Introduction Citrus products (orange, tangerines, grapefruit, lemons and limes) are major agricultural crops in Turkey, in terms of cultivated area (53,000 ha) and total annual production (1.7 million tons) 1. Filamentous green and blue moulds (Penicillium digitatum) and Penicillium italicum) are the devastating cause of spoilage and loss in quality and nutrition content as well as considerable economic losses during postharvest storage of citrus fruits 2. Conventionally, these pathogens are controlled by the application of synthetic fungicides (such as borax, sodium bicarbonate, sodium carbonate, imazalil, sodium ortho-phenyl phenate, thiabendazole, azoxystrobin, pyrimethanil or fludioxonil) during postharvest fruit handling 3-5. Nevertheless, a number of fungicides are no longer permitted for postharvest treatment and have been restricted or removed from the market altogether due to possible toxicological effects and consumer demands for healthier foods 6. Meanwhile, P. italicum has been reported to develop resistance to commonly used fungicides and therefore, higher amounts of treatments are acquired 7. Hence, due to limitations of the current antifungals, alternative, natural Generally Recognized as Safe (GRAS) fungicides to conventional fungicides are needed in order to substitute or combine in hurdle current control strategies and to lower the harmful environmental pollution burden 8. The antifungal natamycin (NT GRAS) is a commonly used organic food additive. It is a 26-member polyene macrolide antifungal commercially produced by the bacterium Streptomyces natalensis and other related Streptomyces species 9. NT has broad antifungal active against molds and yeast but not bacteria, because 188 polyenes macrolids have ring structures that interact with membrane sterols with high affinity, thus causing the alteration of membrane structure and leading to the leakage of cellular materials 10, 11 . NT shows activity over a wide pH range (2-7) and effective at low concentrations (as low as 20 mg/l) and it shows no toxic effects even when ingested as high as 500 mg/l 9, 12. In 2009, the European Food Safety Authority (EFSA) has published a favourable scientific opinion on the use of NT (E235) as a food additive and it is permitted to be used in soft drinks, surface treatment of various cheese and meat products 13. P. digitatum and P. italicum cause decay from surface-borne infections through injuries or micro wounds formed on the skin during harvest and fruit handling and therefore, unavoidable. Additionally, these fungi are able to infect stored fruits by mycelial spread from infected fruits to adjacent healthy fruit, causing ‘nests’ of decay in storage bins. Losses up to 30% of harvested citrus fruits when no postharvest fungicides were applied have been reported 14. Thin tissue paper wrapper for lemon and orange fruits is commonly used to wrap individual fruits, which are intended to be exported to abroad. The loss of weight in fruit during shipping or holding is reduced to a point, where it is most advantageous in maintaining the appearance and taste characteristics of the fruit. The porosity of the tissue paper wrappers permits the citrus fruit cells to “breath” without inhibiting the natural processes of living cells of the fruits, while controlling the development of mould on the surface during cold storage. Furthermore, tissue paper prevents formation of spoilage ‘pockets’ within the shipping Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 containers and fruits are longer protected from cross contamination from spoilage. To our knowledge, antifungal effect of NT has not been evaluated on citrus fruits. In this study, the effectiveness of NT to control green and blue mold on artificially inoculated citrus fruits was evaluated and its impact on decay control, physico-chemical and other quality properties of the citrus fruits and storability were assessed in conventional cold storage conditions. Materials and Methods Fungal strains and determination of minimum inhibitory concentration (MIC) with antifungal agar diffusion assay: The virulent isolates of P. digitatum L-1 and P. italicum P-1 were isolated and characterised 14 in our laboratory from decayed lemons and oranges, respectively. The antifungal activity of NT was determined using a modified version of two-layer agar-well diffusion method 15. Fungal inhibition concentration (0.05, 0.09, 0.19, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 mg/ml) in liquid conditions was determined in serial 2-fold dilutions of NT prepared in Potato Dextrose Broth. The experimental MIC was determined as the lowest concentration of 7 natamycin, at which no visible growth of the fungi on PDA punched agar was observed for all after 7 days incubation. Fruits and storage conditions: Fruits of two lemon cultivars (Citrus limon, Mayer and Molla memed) and one orange cultivar (Citrus sinensis, Washington Navel) of uniform size and appearance were obtained from commercial groves in Antalya area (Turkey) in late 2012. In order to minimise interference from natural infections, fruits were selected for absence of injuries and visible defects, washed with fresh water, air-dried and stored at 4°C. Antifungal treatments and fruit quality evaluation: Fresh, visually healthy fruits were surface cleaned with water, wounded and fungal inoculated as follows. Fruits were punctured 3 mm in flavedo with a sterile stainless steel probe (0.1 × 30 mm) at the equatorial and horizontal. Fungal isolates were grown on PDA for 1-2 weeks at 20-25°C and spores were harvested by rubbing a glass rod over the PDA plate and then suspended in a small volume of sterile peptone water (0.05% w/v). Fungal inoculum and suspension (106 spores/ml) were prepared as described earlier 14, and punctured fruits were immersed for 10 min in conidial suspension and air-dried at room temperature for 1-2 h before applying NT. Each fruit cultivar was separated into the following five sets with eight oranges in each set: Control fruits (wounded and no inoculation), infected control (wounded and inoculated with fungi), NT- treated with spraying and NT-treated paper wrapped (Fig. 1). Fruits were sprayed manually using plastic sprayers by about Fruits (washed air-dried) Tissue wrap Punctured Inoculated with 1-2 h conidial air dried suspension No treatment Spray treatment 0.125 mg/ml NT Spray treatment 1.25 mg/ml NT Dipped in Fruits 0.125 mg/ml NT 2-4 h wrapped Dipped in air dried individually 1.25 mg/ml NT Figure 1. Experimental scheme of NT fruit treatment. Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 1 ml (0.5 and 5 mg/ml NT)/fruit on all sides at low pressure (15 cm above the fruit) and air-dried for 2 h at RT. For paper wrapping, commercial citrus tissue paper wrappers were impregnated with NT by dipping into NT solution (0.5 and 5 mg/ml) for 1 min for full absorbance. Then, papers were air-dried and used for wrapping of individual fruits. Treated and control fruits were placed in covered plastic containers (each containing 8-10 fruits) and two boxes per each treatment. The boxes were stored at around 5°C, 90-95% RH for up to 8 weeks. Periodically, fruits were sampled and fungal development and fruit quality changes on their surfaces were visually and quantitatively analysed and physico-chemical and sensory fruit qualities were assessed. Microbiological quality analysis of natamycin-treated fruits: Fungal development during storage at 5°C was determined as follows: ten disks (r = 0.8 cm, h = 0.4 cm, 1 g) of two fruits per treatment surface were excised with a sterile steel instrument and immediately analysed for fungal count. The population of fungi was serially diluted in peptone water and enumerated periodically by plating on duplicate potato dextrose agar (PDA). Incidence of decay was expressed as the percentage of infected fruit caused by fungal pathogens. Determination of physico-chemical quality properties of natamycin-treated fruits: External and internal physico-chemical quality parameters (decay incidence, colour development, loss of firmness, soluble solids and acidity were determined at harvest (initial quality) and periodically after fungal inoculation for each set of fruit (control, infected and/or treated) at the end of each of the storage periods. The juice was extracted from individual fruit with a small laboratory hand reamer. The pH and titratable acidity (TA) were assessed in juice by titration. Percentage of soluble solids content (SSC) was determined with a digital refractometer (Atago Hand Refractometer N-1E, Tokyo, Japan). Skin colour was measured on opposite cheeks of citrus fruits using a Precise Colour Reader TCR 200 (Beijing, China) and expressed as a colour index, as described by Jimenez-Cuesta et al. 16. Firmness (elasticity and skin strength) of fruits per treatment was determined at the end of each storage period using a Texture Analyser Stable Micro System (Surrey, UK). Analysis of vitamin C and other organic acids: The organic acids and ascorbic acid content were determined via HPLC method by using HPLC-SPD-10Avp UV-VIS with a reverse phase column Prodigy ODS-2 (250 × 4.6 mm) and mobile phase consisting in methanol: 0.6% H3PO4 (5:95, v/v) (pH 2.5) at a flow rate of 0.8 ml/ min 14. Aliquot samples of juice per treatment were kept at -20°C until analysed. Determination of total and individual phenolic compounds: Total content of phenolic compounds were determined by the technique of Folin-Ciocalteu as described by Singleton et al. 17. Individual phenolic compounds (phenolic compounds (naringin, hesperidin, quercetin and naringenin) were determined by HPLC using a modified version of method of Caponio et al. 18. Prepared extract was injected into a Shimadzu series HPLC equipped with a vacuum degasser (DGU-14A), pump (LC-10ADvp) and a DAD detector (λmax = 278) were used for data collection and analysis. The column 189 temperature was 30°C and the flow rate was fixed at 0.8 ml/min. Statistical analysis: Each experiment was carried out at least in duplicate. The variance and regression analysis were conducted with SAS software (Version 8.0, 2000, Cary, NC, USA). Significance was assigned to comparisons with a P-value of less than 0.05 (P≤0.05). Groups detected as significantly different were marked in the tables with different letters. Table 1. P. italicum fungal development on chemically-treated oranges and lemons (Log CFU/g peel). Storage duration Week 1 Week 2 Week 3 Week 4 Week 6 Week 8 Week 1 Week 2 Week 3 Week 4 Week 5 Fruit sample Oranges Lemons Control contaminated 2.74±0.26 3.09±0.90 3.40±0.46 3.36±0.24 2.99±0.20 3.99±0.40 - 4.5 mg/ml NT wrap 2.20±0.73 2.82±0.25 2.31±0.64 2.92±0.94 3.08±0.67 3.72±0.59 2.15±0.30 2.22±0.40 3.00±0.56 3.75±0.67 3.78±0.11 0.5 mg/ml NT wrap 1.85±0.30 2.44±0.63 2.90±0.58 3.19±0.88 3.88±0.76 2.71±0.14 2.57±0.89 3.75±0.37 4.0±0.74 - 5 mg/ml NT spray 2.51±0.11 2.63±0.47 2.90±0.60 3.08±0.67 3.81±0.19 3.82±0.18 2.11±0.56 2.60±0.51 3.47±0.09 3.85±0.93 3.75±0.91 0.5 mg/ml NT spray 2.78±0.18 3.10±0.11 3.18±0.68 3.26±0.74 4.08±0.08 2.35±0.32 2.69±0.50 3.51±0.15 - Results and Discussion Antifungal activity of natamycin in vitro: The inhibition No healthy fruits were left. effect of NT was investigated with PDA plate assay and tubes assay. Inhibition zones observed for both P. digitatum L-1 of blue mold caused by P. italicum P-1 on oranges was reduced and P. italicum P-1 with PDA plate assays showed that as low as by 30% after 21 days of storage at 5°C (Table 1). Comparable 0.192 mg/ml of NT fully inhibited fungal development and reduced results were obtained when the pathogen was P. digitatum, disease the proliferation of both fungi. EC50 concentrations of NT that incidence was reduced from 80-90% in the control to 40-50% in inhibited growth of P. digitatum L-1 and P. italicum P-1 were 0.192 the case of 5 mg/l NT. and 0.125 mg/ml on the PDA, respectively. In tubes, P. digitatum Citrus fruits are often treated with Na2CO3 before marketing to and P. italicum showed similar FIC behaviour with an average improve cleaning and to reduce postharvest rotting 3. Palou et al. 19 percentage inhibition of 64.5% at 1.25 mg/ml NT concentration. demonstrated that 3% Na2CO3 treatment for 150 s at 50°C inhibited NT showed more a fungistatic inhibition than fungicidal effect both green and blue molds of Clementine mandarins without (Fig. 2). The mechanism of microbial inactivation by NT is injuring the fruits. El-Mougy et al. 20 observed that a citric acid intimately related to the formation of pores in the microbial cell treatment also significantly reduced growth and spore production membrane 10. Depending on the type of the injury and surrounding of P. digitatum and P. italicum on citrus. More recently, environment, these injured fungal cultures have the potential to Montesinos-Herrero et al. 21 studied the effect of ammonia gas resuscitate and resume growth under favourable conditions. The fumigation on citrus fruits and the incidence of P. digitatum was inhibition effect at concentrations above 1.25 mg/ml of NT was reduced nearly 80% on oranges treated with 3000 ml/l ammonia. irreversible for 21 days of incubation. The antifungal effect of NT on few fruits were investigated in earlier studies, NT (20 mg/l) in bilayer films reported to decrease decay severity of both Alternaria alternate and Fusarium semitectum fungal pathogens during storage of Hami melons 22. Fruit surface blemishes and resulting deterioration arise from jostling in shipment and handling in the course of transporting. Therefore, tissue papers where the fruits are wrapped are commonly used for packaging. Tissue wrapper also protects individual fruit pieces against infection by mold from contiguous fruits, help to retain the luster and selling appearance of the fruit Figure 2. Two-layer agar-well diffusion inhibition measurements of P. and prolong natural fruit quality. These tissue papers are soft, but digitatum. (1: 12.288 mg/ml, 2: 6.144 mg/ml, 3: 3.072 mg/ml, 4: 1.536 mg/ml, 5: 0.768 mg/ strong enough to hold individual fruit pieces without inhibiting ml, 6: 0.384 mg/ml, 7: 0.192 mg/ml, 8: 0.096 mg/ml, 9: 0.048 mg/ml, 10: 0.024 mg/ml, 11: the natural processes of living cells of the fruits and its moisture 0.012 mg/ml and 12: 0.06 mg/ml). absorbance makes it possible to impregnate with a liquid. Antifungal effect of natamycin on wounded and pathogen- Furthermore, dipping or spraying of fruits in antifungal solutions inoculated fruits: Wounds encountered by citrus fruits during may cause partial inactivation of the antifungal compound, while harvest, distribution, storage or retail allow the entry of fungal paper can retain chemical content for longer periods. Here, our pathogens and unavoidable wound-induced fungal infections experiments have demonstrated that after 4 weeks of storage at shorten storage life. In this study, the ability of NT treatments to 4°C, the incidence of P. digitatum mold on lemons wrapped in protect artificially wound-inoculated and cold-stored lemon and tissue paper impregnated with approximately 5 mg/ml was about orange fruits from green mold P. digitatum and blue mold P. 40-50%, while non-treated lemon fruits were completely decayed italicum was investigated. Postharvest NT application effectively at the end of the 2 weeks of incubation period. On oranges, the controlled decay on fruit, but the effectiveness of NT was effectiveness of NT on P. italicum decay was lower. After dependent on the concentration used. For instance, after 6 weeks incubation for 6 weeks, the incidence of blue mold on oranges of incubation, the incidence of blue mold on artificially inoculated treated with 5 mg/ml NT was 80%, while non-treated oranges were all oranges treated with NT at 1.25 mg/ml was about 40%, while non- spoiled at the end of 5 weeks (Table 1). Montesinos-Herrero et al. 21 treated orange fruits completely decayed at the end of the 4 weeks also observed that antifungal ammonia treatment showed different of incubation period. The incidence of green mold caused by P. levels of effectiveness on lemons and oranges artificially digitatum L-1 on lemons with 5 mg/l NT spray treatment was contaminated with P. italicum. reduced by 15% after 14 days of incubation, while the incidence 190 Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 Effect of natamycin treatment on physico- Table 3. Firmness (elasticity (mm) and skin strength (g)) and colour changes of chemical citrus fruit quality properties: Quality natamycin-treated lemon fruits. Untreated 4.5 mg/ml 0.5 mg/ml 5 mg/ml 0.5 mg/ml parameters of control and treated fruits during Storage Firmness lemons NT wrap NT wrap NT spray NT spray storage are presented in Table 2. Brix range for period 3.79±0.21a 4.07±0.38a 4.01±0.11a 4.66±0.09a 3.37±0.27a Elasticity orange fruits was 8-10% and for lemons 7-9.20% Week 1 Skin strength 1395±86.1b 1250±101b 1565±150b 1062±96.2b 1177±49.3b and it was not influenced by the postharvest Colour index 4.3±0.08c 5.1±0.09c 4.8±0.09c 2.6±0.07c 2.4±0.08c a a a a 4.61±0.15 4.21±0.15 4.25±0.15 4.82±0.07 5.99±0.02a Elasticity treatment. The pH values of chemically-treated and b b b b b untreated oranges and lemons were changed from Week 2 Skin strength 1631±81.2c 1267±79.9c 1215±177c 1066±40.6c 1747±42.4c Colour index 3.4±0.09 2.3±0.08 6.3±0.07 3.0±0.07 1.9±0.09 3.2 to 4.2 and from 2.48 to 3.15, respectively. No Elasticity 4.35±0.17a 5.8±0.19a b b significant (P<0.05) changes were observed Week 3 Skin strength 1284±82.2 1285±69.9 Colour index 2.9±0.08c 4.6±0.09c between treatment and control samples during storage. Soluble solids content, pH and maturity index increases throughout the storage were in good agreement of NT with cell membranes 26 that may affect the fruit metabolic pathway and senescence. Firmness and skin elasticity of citrus with the progress of the ripening process and earlier studies. As shown in Table 2, the ascorbic acid content of control and fruits in both treated and untreated conditions are presented in NT-treated fruits after storing showed slight increases 12% and Table 3. Elasticity increased in both control and treated samples 18% for oranges and lemons, respectively. The ascorbic acid from 3.37 to 5.87 mm with storage period under refrigerated content of treated fruits was significantly (P< 0.05) higher than in conditions, while skin strength changes showed no significant control samples. This study demonstrated that NT enhanced delay pattern. in fruit deterioration and loss of ascorbic acid. The total Conclusions polyphenolic content in lemons and oranges was in accordance with earlier studies 23. In general, during the first week of storage Application of proper and effective treatment practices is essential a slight increase in total phenolic compound content was noted in for control of postharvest diseases of fruits, maintaining shelf-life all treatments and control samples tested. Later, the total phenolic and high fruit quality throughout commercial supply-chains for compound content decreased gradually throughout the storage weeks to months. Currently available commercial chemicals have period for all samples. Spray treatment caused 12% loss of the led to the rise of resistant strains of the pathogenic fungi. The phenolic compounds after 4 weeks, when compared with initial findings of this study suggest that NT, a natural antifungal values. Similar losses were observed for wrap treatments. The compound, is effective for inhibition of green and blue mold on content of hesperidin was significantly lower in treated fruits than the surface citrus fruits and extending storage duration up to in untreated fruits at the same incubation period. The highest additional 2-3 weeks. NT treatment imparts no foreign taste to content of hesperidin was of 248.3 in untreated oranges as fruits wrapped and may also offer a combination mean to control measured in week one and the lowest one of 203.4 in treated orange postharvest fungal development of fresh fruit and vegetables. samples (Table 2). The content of quercetin in lemons also showed Acknowledgements the same trend that untreated samples contained nearly twice as much quercetin than NT-treated fruit samples while naringenin The authors are grateful to the Suleyman Demirel University for content did not change throughout the storage for both fruit the financial support of graduate studies projects (3278-YL2-12 and 3287-YL2-12). samples (Table 2). Yellowing colour is considered to be one of the most important external factors of citrus fruit maturity and quality perception by References the consumers. As the citrus fruits mature, the yellow-coloured 1Secer, A. 2012. Dogu Akdeniz Kalkınma Ajansı Doğrudan Faaliyet xanthophylls and carotenes become more apparent as the amount Destek Programı Narenciye Sektör Raporu. http://www.dogaka.org.tr/ of the green chlorophyll is reduced 24. Skin colour index of treated Icerik/Dosya/www.dogaka.org.tr_216_OI7P02MP_Narenciye _Sektor_Raporu.pdf and untreated fruits did not show significant changes due to the 2 Sukorini, H., Sangchote, S. and Khewkhom, N. 2013. Control of treatment (Table 3). Elasticity or skin strength of citrus fruits is postharvest green mold of citrus fruit with yeasts, medicinal plants, quality criteria for the estimation of storage shelf life and the and their combination. Post. Biol. Technol. 79:24-31. 25 acceptability to the buyers . Earlier studies reported interactions Table 2. Citric acid, ascorbic acid, phenolic (naringin, hesperidin, quercetin and naringenin) content and sugar composition of lemon and orange fruits treated with NT. Chemical content Citric acid (ȝg/g) Ascorbic acid (ȝg/g) Naringin Hesperidin Quercetin Naringenin Glucose Fructose Untreated oranges W1 10094±184a 299.12 0.85±0.07 248.3± 24.4± 35.90± W4 12685±195a 336.40 0.70± 219.95± 17.4± 18.80± NT treated oranges (5 mg/ml spray) W1 W4 16410 13613 326.9 367.24 0.55± 0.75± 203.4± 181.8± 23.90± 19.60± 26.70± 22.80± Untreated lemons W1 45540 320.3 0.95± 135.7± 0.4± 0.25± 8.90± 9.60± W5 48800 383.8 0.8± 25.25± 0.2± 0.2± 6.80± 7.10± NT treated lemons (5 mg/ml spray) W1 W5 49450 56920 306.8 384.7 0.95± 1.0± 120.35± 33.35± 0.1± 0.1± 0.2± 0.2± 9.20± 14.30± 9.70± 14.80± *Fruit quality after 5 weeks of storage was not determined because the incidence of decay was too high. Means within rows for each treatment followed with the same letters are not significantly (P<0.05) different according to LSD test. Journal of Food, Agriculture & Environment, Vol.12 (2), April 2014 191 Eckert, J. W. and Eaks, I. L. 1989. 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