ANTIFUNGAL EFFECT OF A BACTERIOCIN OF BACILLUS METHYLOTROPHICUS BM47 AND ITS POTENTIAL APPLICATION AS A BIOPRESERVATIVE IN TRADITIONAL BULGARIAN YOGURT
Yulian Tumbarski*1, Velichka Yanakieva1, Radosveta Nikolova1, Gergana Mineva1, Ivelina Deseva2, Dasha Mihaylova3 and Ivan Ivanov4
Address(es): Yulian Tumbarski, DVM, PhD 1Department of Microbiology, University of Food Technologies, 26, Maritsa Blvd., 4002 Plovdiv, Bulgaria. 2Department of Analytical Chemistry and Physicochemistry, University of Food Technologies, 26, Maritsa Blvd., 4002 Plovdiv, Bulgaria. 3Department of Biotechnology, University of Food Technologies, 26, Maritsa Blvd., 4002 Plovdiv, Bulgaria. 4Department of Organic Chemistry and Inorganic Chemistry, University of Food Technologies, 26, Maritsa Blvd., 4002 Plovdiv, Bulgaria.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.1.659-662
ARTICLE INFO ABSTRACT
Received 7. 5. 2018 Bacteriocins are biologically active compounds of proteinaceous nature synthesized by a large number of microorganisms, including Revised 5. 6. 2018 members of bacterial genus Bacillus and lactic acid bacteria (LAB). The broad antimicrobial spectrum of bacteriocins against various Accepted 11. 6. 2018 spoilage and pathogenic microorganisms stimulated the research efforts for their investigation and potential application in different Published 1. 8. 2018 branches of food industry as natural preservatives. The promising antimicrobial activity of bacteriocins makes them suitable for application as biopreservatives and alternatives of chemical preservatives in dairy industry for production of fermented or non-fermented milk products. Therefore, the aim of the present study was to evaluate the antifungal effect of a bacteriocin isolated from Bacillus Regular article methylotrophicus strain BM47 and the possibilities for its application as a potential biopreservative of traditional Bulgarian yogurt. The results demonstrated that the addition of a purified bacteriocin in dose of 1 AU/mL of milk led to significant reduction of the fungal spores and mycelial growth of the indicator microorganism Penicillium sp. in the yogurt, without a change of its organoleptic properties, and biochemical and microbiological parameters during the 4-week period of storage.
Keywords: Bulgarian yogurt, bacteriocins, Bacillus methylotrophicus, food biopreservation
INTRODUCTION non-milk products, and also extends their shelf life by inhibition of saprophytic and pathogenic microorganisms (Cholakov et al., 2017). Yogurt (or yoghurt) is one of the most famous and widely spread traditional dairy Although significant antimicrobial activity of the lactic acid produced at high products in Bulgaria, produced by bacterial fermentation of cow’s, buffalo’s, amounts during the milk fermentation and suppressive effect of the low pH, sheep’s and goat’s milk. Yogurt is known on the Balkans since ancient times, when numerous studies revealed the occurrence of foodborne pathogens, which may the Thracians have obtained it from self-fermented sheep’s milk. Nowadays, it survive the pasteurization or to enter the pasteurized milk via contamination or takes an important role in the diet of the Bulgarians due to its excellent nutritional experimental inoculation. The different pathogens can survive in yogurt for a characteristics and beneficial effects on human health. Besides the rich chemical various period of time, which for Listeria monocytogenes is limited between 9 and composition, including all natural nutritional constituents and significant amounts 15 h (Schaack and Marth, 1988) to >6 days (Ikonomov and Todorov, 1967); up of calcium in a bioavailable form, yogurt possesses many therapeutic effects on to 16 h for Escherichia coli O157:H7 of storage at 4-22ºC (Bachrouri et al., 2002); lactose intolerance and some gastrointestinal disorders such as diarrhea, from 2 to 4 days for Brucella spp. (Falenski et al., 2010); more than one week of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), and weight storage at 5°C for Yersinia enterocolitica (Ahmed et al., 1986); between 2 days at control (McKinley, 2005). Bulgarian yogurt it is often recommended as food for 4°C (Pazakova et al., 1997) and 10 days of storage at room temperature (22°C) improving of the metabolism, reducing of the cholesterol and even as an anti- for Staphylococcus aureus (Benkerroum et al., 2002); over 43 days of storage at carcinogenic agent (Fikiin et al., 1997). Many recent studies have demonstrated temperatures between 4 and 25℃ for Salmonella enterica serovar typhimurium that yogurt exhibits positive effects on the gut microbiota and is associated with a (Álvarez-Ordóñez et al., 2013). reduced risk for gastrointestinal diseases, cardiovascular diseases, type 2 diabetes, The raw milk for production of yogurt is often a vector for spoilage and toxigenic allergies, respiratory diseases, pregnancy outcomes, as well as for improving of microorganisms such as yeasts and fungi, which is serious problem nowadays due dental and bone health (Fisberg and Machado, 2015). to the resistance of fungal spores to exposure of high temperature and low pH. The The traditional Bulgarian yogurt, known also as “kiselo mlyako”, is a coagulated most commonly detected fungi in raw milk belong to the genera Penicillium, milk product, characterized by smooth texture, thick consistency and pleasant sour Geotrichum, Aspergillus, Mucor and Fusarium (Quigley et al., 2013) that renders flavor. The uniqueness of Bulgarian yogurt is famous worldwide and it is attributed the milk unsuitable either for direct consumption or for subsequent processing. In to the country’s microclimate and the specific fermentation by symbiotic cultures addition, it is estimated that between 5 and 10% of the world’s food production is of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. lost due to spoilage caused by fungal contamination (Pitt and Hocking, 2009). thermophilus (Tropcheva et al., 2014a). These starter bacteria are known to Therefore, the aim of the present study was to determine the antifungal activity produce a wide spectrum of volatile organic aroma compounds such as against Penicillium sp. of a bacteriocin isolated from Bacillus methylotrophicus acetaldehyde, diacetyl, volatile fatty acids that determine the specific flavour of BM47 and to evaluate the possibilities for its application as a potential Bulgarian yogurt (Beshkova et al., 1998). Another compound, produced by the biopreservative of traditional Bulgarian yogurt. starter microorganisms, which imparts the distinctive flavour of yogurt and its related products, is the lactic acid. Lactic acid is a major end product during the fermentation of carbohydrates by the lactic acid bacteria (LAB). The rapid fermentation process leads to improving of the organoleptic qualities of milk- and
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MATERIAL AND METHODS to 1.6×107 cfu/mL then a working dilution with spore concentration of 1.6×105 cfu/mL was prepared. Materials Minimal inhibitory concentration (MIC) of purified bacteriocin (PB) and Bacteriocin determination of the arbitrary units (AU)
Purified by fast protein liquid chromatography (FPLC) and lyophilized substance MIC of the purified bacteriocin of B. methylotrophicus BM47 was determined by containing a protein of intermediate molecular size (19578 Da) was used. This the conventional method according to Tumbarski et al. (2017). Series of two-fold bacteriocin was synthesized by Bacillus methylotrophicus strain BM47, isolated dilutions of the bacteriocin ranging from 10.0 to 0.079 mg/mL were prepared. from natural thermal spring water in Haskovo region, Bulgaria. Duplicate samples of each dilution were pipetted in quantity of 60 μL into wells cut in a preliminarily inoculated with the test microorganism LBG-agar medium. Indicator microorganism The Petri dishes were incubated at 30ºC for 48 h. The MIC value was determined as the lowest concentration of PB inhibiting completely the growth of the test The fungus Penicillium sp. (our isolate) from the collection of the Department of microorganism around the agar well. The calculation of arbitrary units (AU) for Microbiology at University of Food Technologies, Plovdiv, Bulgaria, was selected application in milk was based on the obtained MIC value. as indicator microorganism. Experimental procedure Culture media The traditional Bulgarian yogurt was prepared in our laboratory according to the Malt extract agar (MEA) Bulgarian State Standard (BSS 12:2010). The homogenized and pasteurized raw This medium was used for cultivation of the test fungus. MEA was prepared by cow’s milk was heated at 45-46°C and transferred in quantity of 100 mL into sterile the following prescription: 20 g malt extract, 20 g dextrose, 6 g peptone and 15 g plastic cups with lids. Then 1% of fresh symbiotic starter culture MZ2 (1.0×108 agar dissolved in 1L of deionized water. The final pH was corrected to 5.5 and the cfu/mL) was added to the cups and mixed. The cups were separated in four groups medium was sterilized by autoclaving at 121°C for 15 min. – the first group was kept as a control; the second group was treated with PB (1 AU/mL); the third group was inoculated with spore suspension of Penicillium sp., Luria-Bertani glucose agar (LBG) providing around 2.5×102 - 5.0×102 cfu/mL of milk; the fourth group was treated This medium was used for determination of MIC. LBG-agar was prepared by the with PB and inoculated with spore suspension of Penicillium sp. (in the same following prescription: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 10 g glucose amounts). For each day of the testing (1-st, 7-th, 14-th, 21-st and 28-th) separate and 15 g agar dissolved in 1 L of deionized water. The final pH was adjusted to 7.5 samples were provided. All milk samples were incubated at 44°C ± 2°C for 2.5– and the medium was autoclaved at 121°C for 20 min. 3.5 h. After coagulation, all samples were cooled and stored at 4°C. The morphological, biochemical and microbiological changes as well as the antifungal Chloramphenicol glucose agar (CGA) effect of PB in yogurt were monitored once a week, for 4 weeks. CGA is a selective medium for enumeration of yeasts and fungi, prepared according to the manufacturer’s (Scharlab S.L., Spain) prescription: 20 g dextrose, Enumeration of characteristic microorganisms 5 g yeast extract, 0.1 g chloramphenicol and 15 g agar dissolved in 1L of deionized water. The final pH was corrected to 6.6 and the medium was sterilized in autoclave Colony-count technique for lactobacilli and lactic streptococci was implemented at 121°C for 15 min. on MRS-agar and M17-agar respectively, at 37°C according to the Bulgarian State Standard BSS ISO 7889:2005. Man, Rogosa and Sharpe agar (MRS) MRS-agar is a selective medium for cultivation and enumeration of lactobacilli, Enumeration of yeasts and/or fungi in milk prepared according to the manufacturer’s (Merck, Germany) prescription: peptone proteose 10 g, meat extract 8 g, yeast extract 4 g, D(+)-glucose 20 g, sodium acetate Colony-count technique for yeasts and/or fungi in milk was implemented on CGA 5 g, triammonium citrate 2 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, at 25°C according to the Bulgarian State Standard BSS ISO 6611:2006. dipotassium phosphate 2 g, polysorbate 80 - 1 g and agar 14 g dissolved in 1L of deionized water. The final pH was corrected to 6.2 and the medium was autoclaved Determination of titratable acidity at 121°C for 15 min. Determination of titratable acidity was implemented according to the Bulgarian M17 agar State Standard BSS 1111:1980. The titratable acidity was determined by titration M17-agar is a selective medium for cultivation and enumeration of lactic of each sample with 0.1 N NaOH using phenolphthalein as an indicator until the streptococci, prepared according to the manufacturer’s (Merck, Germany) appearance of a pale pink colour persisting over 1 min. One Torner degree (°T) prescription: casein enzymic hydrolysate 2.5 g, peptic digest of animal tissue 2.5 corresponds to 1 mL of 0.1 N NaOH, needed for neutralisation of an equivalent g, papaic digest of soyaben meal 5 g, yeast extract 2.5 g, beef extract 5 g, ascorbic amount of organic acid, contained in 100 mL of milk. The results were calculated acid 0.5 g, magnesium sulphate 0.25 g, lactose 5 g, disodium--glycerophosphate as mean value of three consecutive experiments. 19 g and agar 10 g. The final pH was adjusted to 7.1 and the medium was sterilized by autoclaving at 121°C for 15 min. RESULTS AND DISCUSSION
Starter culture After estimation of MIC of PB of B. methylotrophicus BM47 against the indicator microorganism Penicillium sp. (2.5 mg/mL), the arbitrary units (AU) for Symbiotic starter culture MZ2, containing Lactobacillus delbrueckii subsp. application in milk were calculated to the amount of PB in 60 μL (0.15 mg) and bulgaricus LBG MZ (NBIMCC 3600) and Streptococcus salivarius subsp. this dose was accepted as equivalent to 100 AU. To provide the desirable thermophilus 35 (NBIMCC 3597) in ratio 1:2 was used. The starter culture MZ2 concentration of 1 AU/mL, in the experimental procedure 0.15 mg of PB was was kindly provided by Prof. Zapryana Denkova from the Department of added in 100 mL of milk. Microbiology at University of Food Technologies, Plovdiv, Bulgaria. During the 4-week period of storage under refrigeration conditions (4°C), the microbiological parameters – number and ratio of lactic acid bacteria – LAB Milk (Lactobacillus bulgaricus and Streptococcus thermophilus), presence of yeasts and fungi, as well as the titratable acidity of four samples - control, yogurt with addition Fresh, homogenized and pasteurized cow’s milk with pH 6.7 and titratable acidity of bacteriocin (sample 1), yogurt inoculated with Penicillium sp. (sample 2) and of 16.5°T was used. The milk was delivered by BCC Handel Ltd., town of Elena, yogurt with addition of bacteriocin and Penicillium sp. (sample 3) were monitored. Bulgaria. The antifungal effect of the purified bacteriocin on the test microorganism Penicillium sp. in the artificially contaminated samples also was observed. The Methods results are summarized in Table 1. As seen from the results presented in Table 1, during the first day of storage, the Preparation of spore suspension titratable acidity values and microbiological parameters of all yogurt samples were in compliance with the BSS 12:2010. The bacteriocin did not inhibit the indicator The test fungus Penicillium sp. was grown on MEA at 30ºC for 7 days or until microorganism Penicillium sp. in yogurt with addition of PB (sample 3) and it sporulation. The inoculum was prepared by addition of 5 mL of sterile 0.5% NaCl concentration was equal (2.3×102 cfu/g) to the yogurt without PB (sample 2). into the tube. After vigorous shaking the inoculum was filtered and replaced in Experimental results obtained on the 7-th day of monitoring period showed that another tube before use. The number of fungal spores was determined using a Lactobacillus bulgaricus and Streptococcus thermophilus retained a high Thoma’s haemocytometer. Final spore concentration in the inoculum was adjusted concentration of viable cells, indicating that bacteriocin did not inhibit LAB and did not affect their normal ratio (1:5), which was relatively constant until the end
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of the second week. The titratable acidity of all samples increased slightly, except reduced its concentration from 3.7×102 cfu/g (sample 2) to 10 cfu/g in the yogurt a control sample in which the increase in acidic degrees was most pronounced. A treated with bacteriocin (sample 3) (Fig. 1). weak inhibition of the test microorganism Penicillium sp. in the yogurt with The results of the microbiological analyses demonstrated on the 28-th day of addition of bacteriocin (sample 3) was observed. storage, the number of LAB decreased with 2-3 log units and their ratio changed On the 14-th and 21-st days of observation period, an insignificant decrease of the to 1:7 with a prevalence of S. thermophilus due to the faster death of Lb. bulgaricus viable lactobacilli and streptococci in the control yogurt sample and bacteriocin- cells in the end of storage of fermented milk products. Titratable acidity of all treated sample (sample 1) was detected, which maintained a relatively high samples at the end of monitoring period reached values between 130°T and concentration of viable cells and stable ratio (1:5 – 1:6). Experimental data showed 134.6°T, which was in the allowable limit of 150°T according to the BSS 12:2010. that the titratable acidity of the control yogurt sample reached 130°T on the 21-st The same trend of effective inhibition of the test fungus Penicillium sp. in the day, while the acidity of sample with addition of bacteriocin (sample 1) remained yogurt with bacteriocin (sample 3) was observed. relatively low - 110.8°T. It was found that bacteriocin of B. methylotrophicus BM47 inhibited effectively the indicator fungus Penicillium sp. and significantly
Table 1 Effect of PB of B. methylotrophicus BM47 on some microbiological and physicochemical parameters of Bulgarian yogurt Samples 1 2 3 Day Parameter Yogurt (control) Yogurt + Yogurt + PB + Yogurt + PB Penicillium sp. Penicillium sp. LB (cfu/g) 1.0×108 1.0×108 n. a. n. a. ST (cfu/g) 5.0×108 5.0×108 n. a. n. a. 1 Yeasts (cfu/g) <10 <10 <10 <10 Fungi (cfu/g) <10 <10 2.3×102 2.3×102 TA (°Т) 90.6 ± 0.04* 90.6 ± 0.04 90.6 ± 0.04 90.6 ± 0.04 LB (cfu/g) 9.0×107 6.6×107 n. a. n. a. ST (cfu/g) 4.5×108 3.3×108 n. a. n. a. 7 Yeasts (cfu/g) <10 <10 <10 <10 Fungi (cfu/g) <10 <10 2.5×102 1.5×102 TA (°Т) 99.9 ± 0.05 92.2 ± 0.06 94.1 ± 0.06 93.4 ± 0.05 LB (cfu/g) 4.0×107 3.0×107 n. a. n. a. ST (cfu/g) 2.0×108 1.5×108 n. a. n. a. 14 Yeasts (cfu/g) <10 <10 <10 <10 Fungi (cfu/g) <10 <10 2.8×102 60 TA (°Т) 129.3 ± 0.06 106.7 ± 0.06 110.7 ± 0.05 108.7 ± 0.05 LB (cfu/g) 2.4×106 1.7×106 n. a. n. a. ST (cfu/g) 1.5×107 1.0×107 n. a. n. a. 21 Yeasts (cfu/g) <10 <10 <10 <10 Fungi (cfu/g) <10 <10 3.7×102 10 TA (°Т) 130.0 ± 0.1 110.8 ± 0.1 120.5 ± 0.1 126.0 ± 0.1 LB (cfu/g) 1.5×105 1.2×105 n. a. n. a. ST (cfu/g) 1.0×106 1.0×106 n. a. n. a. 28 Yeasts (cfu/g) <10 <10 <10 <10 Fungi (cfu/g) <10 <10 3.7×102 10 TA (°Т) 134.6 ± 0.1 133.3 ± 0.1 129.7 ± 0.1 130.0 ± 0.1 Legend: PB – purified bacteriocin; LB - Lactobacillus bulgaricus; ST - Streptococcus thermophilus; TA - titratable acidity; n. a. – not applicable; * - ± SD – standard deviation (n=3).
Yeasts and fungi as a side microflora or contamination in all samples until the end of storage period were not detected. It was found that bacteriocin of B. The significant antifungal activity of the bacteriocin of B. methylotrophicus BM47 methylotrophicus BM47 did not affect the growth of LAB and did not change the was demonstrated in our previous study, in which besides on Penicillium sp. this organoleptic properties (colour, smell and consistency) of yogurt. Therefore, the bacteriocin showed inhibitory effect on fungi from the genera Aspergillus sp. and results of this study revealed the potential possibilities for application of Fusarium sp. with the same values of MIC – 2.5 mg/mL, and revealed promising bacteriocin of B. methylotrophicus BM47 as an agent for a biopreservation of potential as biopreservative of raw cow’s milk (Tumbarski et al., 2018). Bulgarian yogurt. Filamentous fungi are among the main spoilage microorganisms of different stored foods such as fermented dairy products (cheese and yogurt), bread, stored crops and animal food. Presently, the contamination of food with various fungal species is a serious problem due to three main reasons: a) alteration of the texture profile and external aspect of affected products; b) production of mycotoxins, conidia, mycelia and spores during fungal growth, which is related to some health risks such as allergic conditions; c) significant economic loses for the food industry. These problems could be overcome by adoption of different advanced approaches that prevent the growth of spoilage microorganisms and extend the shelf-life of milk products. The shelf-life of various cultured milk products could be enhanced by application of a single or a combination of the following biopreservation techniques: a) use of suitable starter cultures containing lactobacilli with proven antifungal activity - Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus coryniformis subsp. coryniformis, Lactobacillus sanfrancisensis, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus lactis subsp. cremoris (Tropcheva et al., 2014b; Salas et al., 2017); b) application of natural antimicrobial compounds such as bacteriocins (nisin) or bacteriocin-like substances (BLIS) from various bacterial species/strains (MicrogardTM, Lacticin 3147, Leucocin F10 and pediocin AcH); c) use of non-toxic and generally recognised as safe (GRAS) antifungal antibiotics obtained from different microorganisms such as Natamycin from Streptomyces natelensis (Sarkar, 2006; Hasan et al., 2008). Figure 1 Antifungal effect of the PB of B. methylotrophicus BM47 in yogurt on Some recent studies concerning the bioprotection of food described the the 21-st day of storage – sample inoculated with Penicillium sp. (left); sample possibilities for application of bacteriocins against different spoilage inoculated with Penicillium sp. and addition of PB in dose 1AU/mL (right) microorganisms and their effectiveness as biopreservatives of yogurt. Misirlilar et
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al. (2012) reported the inhibitory effect of nisin (produced by Lactococcus lactis and Penicillium roquefortii. International Journal of Food Science and subsp. lactis) on undesirable yeasts and fungi, and its application as GRAS agent Technology, 43, 2026-2032. https://doi.org/10.1111/j.1365-2621.2008.01816.x for extension of shelf-life without changes of the sensory properties of yogurt. The Ikonomov, L., Todorov, D. (1967). Microbiological studies on the pasteurization application of nisin against spoilage microorganisms in fermented dairy products of ewes' milk. III: Resistance of some pathogenic bacteria. Vet. Med. Nauki, Sof. is exploited also by other authors. Gupta and Prasad (1989) found that nisin at a 4, 99-108. (Dairy Sci. Abstr. 27(2), 82. concentration of 25 RU/g enhanced the shelf-life of stirred yogurt from 5–10 days Kumar, R., Sarkar, S. and Misra, A.K. (1998). Effect of nisin on the quality of dahi. at 7°C without significant change in its sensory properties such as flavor, body, Journal of Dairying Food and Home Sciences, 17, 13-16. texture and consistency. Nisin and nisin producing microorganisms were also McKinley, M. C. (2005). The nutrition and health benefits of yoghurt. found effective in biopreservation of dahi (traditional fermented milk product International Journal of Dairy Technology, 58(1), 1-12. typical for South Asian countries) during storage at 25°C (Rajmohan and Prasad, https://doi.org/10.1111/j.1471-0307.2005.00180.x 1995) and addition of nisin in dahi at a dose of 15 RU/g retained all its desirable Misirlilar, F., Kinik, Ö., Yerlikaya, O. (2012). Effect of protective culture and characteristics up to 35 days at 15±1°C (Kumar et al., 1998). biopreservatives on strained yoghurt quality. African Journal of Microbiology Research, 6(22), 4696-4701. https://doi.org/10.5897/AJMR11.1319 CONCLUSION Pazakova, J., Turek, P., Laciakova, A. (1997). The survival of Staphylococcus aureus during the fermentation and storage of yoghurt. Journal of Applied This is the first report performing the application of a bacteriocin synthesized by Microbiology, 82(5), 659-662. https://doi.org/10.1111/j.1365- B. methylotrophicus BM47 as an antifungal agent in biopreservation of traditional 2672.1997.tb03599.x Bulgarian yogurt. The addition of bacteriocin in milk for production of yogurt led Pitt, J. I., Hocking, A. D. (2009). Fungi and food spoilage. Third edition. Springer, to effective inhibition and reduction of the number of test microorganism ISBN 978-0-387-92207-2. Penicillium sp. in experimentally contaminated yogurt samples during four weeks Quigley, L., O’Sullivan, O., Stanton, C., Beresford, T. P., Ross, R. P., Fitzgerald, of storage at 4°C, without alteration of their organoleptic characteristics. G. F., Cotter, P. D. (2013). The complex microbiota of raw milk. FEMS Therefore, the bacteriocin isolated from B. methylotrophicus BM47 could be Microbiology Reviews, 37, 664–698. https://doi.org/10.1111/1574-6976.12030 considered as a potential agent for biopreservation of Bulgarian yogurt and suitable Rajmohan, S., Prasad, V. (1995). Effect of nisin on the chemical changes in dahi. for application in the dairy industry against spoilage microorganisms of fungal Indian Journal of Dairy Science, 48, 633. origin. Salas, M. L., Mounier, J., Valence, F., Coton, M., Thierry, A., Coton E. (2017). Antifungal Microbial Agents for Food Biopreservation - A Review. Acknowledgements: This work was financed by Fund „Science“ of University Microorganisms, 5(3), 37. https://doi.org/10.3390/microorganisms5030037 of Food Technologies, Plovdiv, Bulgaria (Grant 06/16-H). Sarkar, S. (2006). Shelf-life extension of cultured milk products. Nutrition and The authors declare that no conflict of interest exists. Food Science, 36(1), 24-31. https://doi.org/10.1108/00346650610642160 REFERENCES Schaack, M. M., Marth E. H. (1988). Behavior of Listeria monocytogenes in Skim Milk and in Yogurt Mix during Fermentation by Thermophilic Lactic Acid Ahmed, A. H., Moustafa, M., El-Bassiony, T. A. (1986). Growth and survival of Bacteria. Journal of Food Protection, 51(8), 607-614. Yersinia enterocolitica in yogurt. Journal of Food Protection, 49, 983-985. Tropcheva, R., Georgieva, R., Paskov, V., Karsheva, M., Danova, S. (2014a). Álvarez-Ordóñez, A., Valdés, L., Bernardo, A., Prieto, M., López M. (2013). Sensory properties of Bulgarian yogurts, supplemented with lactobacilli as Survival of acid adapted and non-acid adapted Salmonella typhimurium in probiotic adjuncts. Journal of Texture Studies, 45(3), 187-194. pasteurized orange juice and yogurt under different storage temperatures. Food https://doi.org/10.1111/jtxs.12065 Science and Technology International, 19(5), 407-414. Tropcheva, R., Nikolova, D., Evstatieva, Y., Danova S. (2014b). Antifungal https://doi.org/10.1177/1082013212455343 activity and identification of Lactobacilli, isolated from traditional dairy product Bachrouri, M., Quinto, E. J., Mora M. T. (2002). Survival of Escherichia coli “katak”. Anaerobe, 28, 78-84. http://dx.doi.org/10.1016/j.anaerobe.2014.05.010 O157:H7 During Storage of Yogurt at Different Temperatures. Journal of Food Tumbarski, Y., Lincheva, V., Petkova, N., Nikolova, R., Vrancheva, R., Ivanov I. Science, 67(5), 1899-1903. https://doi.org/10.1111/j.1365-2621.2002.tb08743.x (2017). Antimicrobial activity of extract from aerial parts of potentilla (Potentilla Benkerroum, N., Oubel, H., Mimoun, L. B. (2002). Behavior of Listeria reptans L.). Industrial Technologies, 4(1), 37-43. monocytogenes and Staphylococcus aureus in Yogurt Fermented with a Tumbarski, Y., Yanakieva, V., Nikolova, R., Mineva, G., Deseva, I., Mihaylova, Bacteriocin-Producing Thermophilic Starter. Journal of Food Protection, 65(5), D., Ivanov I. (2018). Application of a bacteriocin isolated from Bacillus 799–805. methylotrophicus BM47 as a biopreservative in raw cow’s milk. Industrial Beshkova, D., Simova, E., Frengova, G., Simov, Z. (1998). Production of flavour Technologies, vol. 5(1), in press. compounds by yogurt starter cultures. Journal of Industrial Microbiology and Biotechnology, 20, 180–186. https://doi.org/10.1038/sj.jim.2900504 Bulgarian State Standard BSS ISO 7889:2005. Yogurt - Enumeration of characteristic microorganisms - Colony-count technique at 37 degrees C. Bulgarian State Standard BSS 12:2010. Bulgarian Yogurt. Bulgarian State Standard BSS ISO 6611:2006. Milk and milk products - Enumeration of colony-forming units of yeasts and/or moulds - Colony-count technique at 25 degrees C. Bulgarian State Standard BSS 1111:1980. Milk and milk products – Determination of acidity. Cholakov, R., Tumbarski, Y., Yanakieva, V., Dobrev, I., Salim, Y., Denkova, Z. (2017). Antimicrobial activity of Leuconostoc lactis strain BT17, isolated from a spontaneously fermented cereal beverage (boza). Journal of microbiology, biotechnology and food sciences, 7(1), 47– 49. http://doi.org/10.15414/jmbfs.2017.7.1.47-49 Falenski, A., Mayer-Scholl, A., Filter, M., Göllner, C., Appel, B., Nöckler, K. (2010). Survival of Brucella spp. in mineral water, milk and yogurt. International Journal of Food Microbiology, 145(1), 326-330. https://doi.org/10.1016/j.ijfoodmicro.2010.11.033 Fikiin, K. A., Fikiin A. G., Russell, S. L., Fitt, P.W. (1997). Shelf-life extension of Bulgarian yoghurt by using a novel environment-friendly aircycle integrated system for thermal and refrigerated processing. In: Shelf-Life Prediction for Improved Safety and Quality of Foods –Proceedings of EU COPERNICUS Workshop, Project CIPA-CT94-0120, Wageningen (The Netherlands), 159-168. https://doi.org/10.13140/RG.2.1.5152.9447/1 Fisberg, M., Machado, R. (2015). History of yogurt and current patterns of consumption. Nutrition Reviews, 73(S1), 4–7. https://doi.org/10.1093/nutrit/nuv020 Gupta, R. K., Prasad, D. N. (1989). Incorporation of nisin in stirred yoghurt. II. Effect on biochemical activities during storage. Cultured Dairy Products Journal, 24, 9–10. Hasan, T., Eroglu, E., Soyer, F., Ozen, B. (2008). Antifungal activity of biopolymers containing natamycin and rosemary extract against Aspergillus niger
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INACTIVATION OF PISTACHIO CONTAMINANT ASPERGILLUS FLAVUS BY ATMOSPHERIC PRESSURE CAPACITIVE COUPLED PLASMA (AP-CCP)
Mohammad Aminroosta Tasouji1, Amir Hossein Ghorashi*2, Mohammad Taghi Hamedmoosavian1 , Mahdi Bakhshzad Mahmoudi2
Address(es): 1 Chemical Eng. Group, Faculty of Eng., Ferdowsi University, Mashhad, Iran. 2 Faculty of Plasma & Nuclear Fusion, Nuclear Science & Technology Institute, Tehran, Iran.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.1.668-671
ARTICLE INFO ABSTRACT
Received 15. 11. 2017 Aspergillus flavus toxin contamination of pistachio nuts is a serious problem in terms of health and food security. In this paper, in order Revised 29. 5. 2018 to increase the nut shelf life by eliminating or reducing the amount of toxin-producing fungus Aspergillus flavus, pistachios were irradiated Accepted 13. 6. 2018 with cold plasma, using Atmospheric Pressure Capacitive Coupled Plasma (AP-CCP) generating device using Argon gas. The advantages Published 1. 8. 2018 of this device are both its mobility, adjoining a nuts carrying container for thorough irradiation of the nuts and that the device can be designed in the form of an array of torches for treatment of large quantities of nuts in open air as against in a vaccum chamber. In this way, the sample of nuts are firstly soaked with fungus and then irradiated in different conditions in terms of power, pressure and time. Regular article The analysis of experimental results indicates 4 log (66.6%) fungus reductions. This pathogen reduction was observed in 100 Watts, at atmospheric pressure and irradiation duration of 10 minutes.
Keywords: Pistachio, Aspergillus flavus, Atmospheric pressure plasma, Plasma Treatment
INTRODUCTION having high-energy electrons (Petitpas et al., 2007). When the collision is low (cold plasma) the electron temperature is few thousand Kelvin degrees while the Mycotoxin is literally formed from two Greek words “Myco” and “toxin”. temperature of ions and approximate temperature of plasma is only about room Mycotoxins are a group of toxic compounds naturally produced by several species temperature (Lieberman et al., 2005). Non-equilibrium atmospheric pressure of fungi, mainly by Aspergillus flavus and Aspergillus parasitiscus. Aflatoxins are plasmas have potential for biomedical applications because they are non-thermal the most significant mycotoxins because of their toxic cancer-causing nature, and non-toxic. They can provide reasonable reactivity with a low gas temperature among which, the aflatoxins B1, B2, G1 and G2 are known as the most important at atmospheric pressure. The devices producing plasma are easy to construct types (Arrus et al., 2005); due to their high risks of contaminating agricultural because they do not need any vacuum system and can be operated with various products, they are considered as potential threat to human health and livelihoods. feeding gases under a wide range of driving frequencies. Atmospheric pressure Aspergillus favours pollution and products containing fat and high carbohydrates plasma jet devices generate plasma in surrounding air, so they are suitable for like oil seeds and nuts (Martins et al., 2008). Therefore, pistachios, peanuts, corn, direct treatment of targets of any size. Factors affecting the formation of plasma wheat, rice, almonds and figs are the main hosts of this fungus, where the are mainly: Plasma forming gas (Argon, Helium, Nitrogen, Methane, Sulfur contamination risks of unroasted nuts are much higher than the roasted. Pistachio hexafluoride etc.), gas flow rate, electric and magnetic fields, shape and is among the most expensive nuts and many people prefer to consume it fresh. arrangement of electrodes and finally, the kind of plasma generating device (Roth, Moreover, pistachio as an export product is the source of foreign currency income 2000). Aspergillus flavus and aflatoxin reduction/elimination methods with cold for those countries cultivating it, like Iran, USA, Turkey, Syria, Greece, etc. For plasma are earlier reported (Gadri et al., 2000; Ohkawa et al., 2006; Lee et al., instance, Iran has 440 thousand hectares of pistachio orchards acreage and the 2006; Schneider et al., 2005; Park et al., 2007). The achieved results reported by economic value of its export to 66 countries is annually about one billion US the referred research works were all achieved either in longer test duration and/or Dollars. Whilst, stringent national and international regulatory for contamination using different methods and environments. It is worth noting that, P. Basaran et al. levels are imposed for these toxins. For instance, in United States, the products that reported some effective results on treatment of Aspergillus parasiticus from contain less than 20 ppb the total aflatoxin and 15 ppb aflatoxins B1 have sampled nut surface with low pressure cold plasma using air gases and Sulfur permissions of purchase, sale, import and export (Gourama et al., 1995). hexafluoride (SF6) (Moisan et al., 2002; Basaran et al., 2008). In order to suppress fungal growth and reduce mycotoxin formation, many The main objective of the present research work was to explore possible remedies have so far been suggested i.e. chemical, physical, farming and biological applications of cold plasma, using Argon gas to inactivate/reduce Aspergillus methodological remedies. Although these methods can reduce surface fungal flavus on pistachios as a sample representative of varieties of nuts. The present contaminations, they often require either non-attainable equipment or expensive research has achieved good results by changing the plasma forming gas to Argon, chemicals and may also cause unacceptable detrimental effect to edible materials. using AP-CCP generating device for the first time. Moreover, Argon has its own Furthermore, some remedies may even cause excessive health hazards too. As advantages such as low cost, less power consumption, and high sterilization a result, some methods are considered impractical, costly, not completely effective, efficacy (Samarajeewa et al., 1991). Numerous data obtained for different input harmful, and time consuming for treatment of nuts in big scales. powers, temperatures and test time durations indicate the contribution of the Plasma is partially ionized gases also known as highly energized fourth state of present research study compared with previous reported works; by which the same matter that all or a significant parts of its atoms have lost one or more electrons results are reported with higher input power (300 W) and during longer time of 20 and are changed to positive ions. In other words, plasma is an electrically minutes. Besides the reason for avoiding the complexity of doing large number of conductive environment because of large number of positive and negative ions, tests on various types of nuts, this research was only conducted on pistachio nuts,
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because, pistachios are the most suitable substrate being contaminated by the cylinder, a glass made cylindrical movable container and the AP-CCP generating fungus and as mentioned above, pistachio is economically of utmost importance apparatus which works like a plasma torch (jet)). The AP-CCP apparatus consists for countries where it is cultivated such as authors’ native place. of two concentric electrodes through which the working gas flows: one was RF The sample pistachios used in this research were from Akbari brand which is powered and the other was grounded. In order to prevent arcing, a quartz tube was among the best quality and the most expensive one in Iran. The outer shell of the placed between the electrodes. By applying RF power to the inner electrode, the fruit is green and it turns to red while rippening. The weight of fresh fruit with gas discharge is ignited. The ionized gas exits through a nozzle by the flow rate of green shell is 48.41 grams. The wet green and bony shells are weighting 19.3 and 50 sccm. 29.8 grams, respectively. The dried fruit along with bony shell is 1.45 grams out The plasma jet was manually controlled to ensure irradiation of plasma all over the of which only 0.75 grams is pistachio brain. object (pistachios), placed in the container. The cable of RF power supply and Argon gas entered from the bottom end of the device. Once gas passing between MATERIALS AND METHODS the dielectrics inside the device, the ionization took place and the plasma was ejected from front end of the device. The experimental device is shown in Figure The main materials and instruments used in this research study are as followings: 2. Frequency of RF machine was 13.65 MHz and it could be set up at 50 ohm on
standby mode by using a matching network. The digital indicator showed the Pistachio samples; Ten unified pistachios for each test were used. variation in power supply. The pistachio irradiation was done very easily by direct Pistachios for each test were surface disinfested to eliminate possible exposure of plasma on the surface of samples in atmospheric pressure. The various growth of other microorganisms by rinsing them with alcohol. tests were performed with different input powers of 50, 75, 100 and 150 watts, Seven spores of Aspergillus flavus having 106 Colony Forming Unit per each one at time durations of 2, 6 and 10 minutes. However, the most favorable grams (6 logs CFU/gr.) provided by animal husbandry department of results, discussed in this paper, were obtained at 100 watts. To prepare and provide Tehran University. the samples for fungi counting and the final results of each test, the following steps Yeast Extract Glucose Chloramphenicol (YEGC) Agar product of were carefully taken: Merck KGaA, Germany, Code No. 1160000500. In order to obtain a more favorable result, the mixed microbial culture method was Autoclave 10 litters capacity. used. The colon growth of fungi was observed after the separation process. The Laboratory class N. colony growth, irradiation and counting process were repeated for each test. 0 Setting up sterilization till 134 C and setup time is 1 to 59 minutes. Incubator with capacity of 55 litters with temperature ranging of -10 to + 45 C0 trade mark PECO. Plates, plastic/glass containers for placing samples. AP-CCP generating device, which was made by authors at concerned laboratory in Plasma and Nuclear Fusion Department, Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran. Peptone water of 0.1%, product of Merck KGaA, Germany, Code No. 1072280500. Refrigerator, sampler calibrator, metallic sterilized clamps, balance, vibrator, distilled water, alcohol and basher.
Microbial Cultures Figure 2 AP-CCP generating device (experimental tool) Pure isolate of Aspergillus flavus provided by animal husbandry department of Tehran University, was grass cultured on YEGC Agar plate at Pastor specialized laboratory, Tehran. The plate were kept in an incubator at 37 C0 for 24 hours and 1,00E+07 6,60E+06 2,90E+06 then at 25 C0 for the next 48 hours. A total of 10 nuts were placed on 3 day-old 1,00E+06 3,50E+06 plate and gently rolled around to distribute spores uniformly. In order to increase 1,00E+05 the fungal contamination level, the sample nuts were incubated on 3 day-old YEGC plates for further 13 hours at 20 C0. Next, to eliminate excess moisture the samples 1,00E+04 7,50E+03 2,20E+03 were maintained under a laminar flow hood for 3 hours. 1,00E+03
Log(CFU/gr) 3,80E+02 1,00E+02 0 2 4 6 8 10 12 Time (min)
The Fungus control amount of irradiated Fungus
Figure 3 The rate of Fungi reduction by AP-CCP generating device at 100 watts
RESULTS AND DISCUSSION
After irradiation process with AP-CCP generating device, counting the irradiated sample fungus and also the control fungi count, the results as shown in Table 1 were obtained. Table 1 shows that pistachios have been affected by plasma, leading to a 3 log fungus reduction, while at 10 minutes irradiation time 4 logs of total fungus (6 logs) were reduced. The average of every three tests at three different times of 2, 6 and 10 minutes is, respectively, depicted in Figure 3. It is to be noted that within
Figure 1 Schematic setup of AP-CCP device: 1- Anode, 2- Body, 3- Cathode, 4- the laboratory set-ups available for this research, a number of tests were conducted Gas entrance with different input powers of 50, 75, 100, 125 and 150 Watts. At power inputs less than 100 Watts, no fungus reduction was observed even at longer irradiation Experimental Device and Process time and hence those results are not reported in this article. But at 150 Watts, the obtained results at lower time duration were not observably altered, while at 10 The schematic of AP-CCP generating device used in this study is shown in Figure minutes irradiation time the fungus was totally reduced (6 logs reduction), but 1 (It consists of a Radio Frequency (RF) power supply machine, Argon gas some burnings were observed on the sample shells and the handmade AP-CCP
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device was also heated up. Therefore, due to such uncertainties, the results for REFERENCES power input of 150 Watts are not included in this research report, too. However, it is expected that at large scale industrial application, where the systems can work Arrus, K., Blank, G., Abramson, D., Clear, R., & Holley, R. A. (2005). Aflatoxin automatically and probable human inaccuracies are removed, the observed damage production by Aspergillus flavus in Brazil nuts. Journal of Stored Products on the nut shells may not occur. Research, 41(5), 513-527.http://dx.doi.org/10.1016/j.jspr.2004.07.005 Martins, H. M., Almeida, I., Marques, M., & Bernardo, F. (2008). Interaction of Analysis Of Variance (ANOVA) test wild strains of Aspergilla with Aspergillus parasiticus ATCC15517 on aflatoxins production. International journal of molecular sciences, 9(3), 394-400. In order to perform the organoleptic analysis of plasma treated samples, a taste http://dx.doi.org/10.3390/ijms9030394 panel was organized to compare the sensory quality of pistachio samples before Gourama, H., & Bullerman, L. B. (1995). Aspergillus flavus and Aspergillus and after 10 minutes irradiation and for both power inputs of 100 and 150 watts, parasiticus: aflatoxigenic fungi of concern in foods and feeds: a review. Journal of separately. At each stage, a group of 10 experts having no allergic to pistachios Food protection, 58(12), 1395-1404. http://dx.doi.org/ 10.4315/0362-028X- were invited and samples including five pistachios of plasma treated were placed 58.12.1395 in dishes coded with random three digit numbers, and participants were asked to Petitpas, G., Rollier, J. D., Darmon, A., Gonzalez-Aguilar, J., Metkemeijer, R., & evaluate samples for color, odor, texture, overall appearance and acceptability, Fulcheri, L. (2007). A comparative study of non-thermal plasma assisted reforming using a 1–5 scale. Evaluations were made for each sample with respect to overall technologies. International Journal of Hydrogen Energy, 32(14), 2848-2867. acceptance and appearance. The sensory results were evaluated by two factors http://dx.doi.org/ 10.1016/j.ijhydene.2007.03.026 (panelist and product) ANOVA statistical method. At stage with 100 watts power Lieberman, M. A., & Lichtenberg, A. J. (2005). Principles of plasma discharges input, the sensory panelists observed no difference in overall appearance, color, and materials processing. John Wiley & Sons. http://dx.doi.org/ odor and texture but at stage with 150 watts the panelists observed slight alteration 10.1002/0471724254 only in texture of treated pistachios. Roth, J. R., Sherman, D. M., Gadri, R. B., Karakaya, F., Chen, Z., Montie, T. C.,. & Tsai, P. Y. (2000). A remote exposure reactor (RER) for plasma processing and Table 1 Fungi reduction by AP-CCP generating device at 100 watts sterilization by plasma active species at one atmosphere. IEEE Transactions on Time Plasma Science, 28(1), 56-63. http://dx.doi.org/ 10.1109/27.842864 Fngus control Irradiated sample (min) Gadri, R. B., Roth, J. R., Montie, T. C., Kelly-Wintenberg, K., Tsai, P. P. Y., 6 3 2 3.3 × 10 1.9 × 10 Helfritch, D. J., ... & Team, U. P. S. (2000). Sterilization and plasma processing of 6 3 2 3.9 × 10 2.2 × 10 room temperature surfaces with a one atmosphere uniform glow discharge plasma 6 3 2 3.4 × 10 2.5 × 10 (OAUGDP). Surface and Coatings Technology, 131(1), 528-541. 6 6.4 × 106 6.7 × 103 https://doi.org/10.1016/S0257-8972(00)00803-3 6 6.7 × 106 8.2 × 103 Ohkawa, H., Akitsu, T., Tsuji, M., Kimura, H., Kogoma, M., & Fukushima, K. 6 4.8 × 106 7.6 × 103 10 3.1 × 106 2.9 × 102 (2006). Pulse-modulated, high-frequency plasma sterilization at atmospheric- 10 3.3 × 106 4.4 × 102 pressure. Surface and coatings technology, 200(20), 5829-5835. 10 2.5 × 106 4.1 × 102 https://doi.org/10.1016/j.surfcoat.2005.08.124 Lee, K., Paek, K. H., Ju, W. T., & Lee, Y. (2006). Sterilization of bacteria, yeast, and bacterial endospores by atmospheric-pressure cold plasma using helium and CONCLUSION oxygen. The Journal of Microbiology, 44(3), 269-275. Park, B. J., Takatori, K., Sugita-Konishi, Y., Kim, I. H., Lee, M. H., Han, D. W., In comparison with other conventional methods such as: chemicals, physical and ... & Park, J. C. (2007). Degradation of mycotoxins using microwave-induced biological farming techniques (; Takahashi et al., 2002; Goryacheva et al., 2007; argon plasma at atmospheric pressure. Surface and Coatings Technology, 201(9), Rashid et al., 2008; Gupta et al., 2012; Hell et al., 2010; Tsitsigiannis et al., 5733-5737. https://doi.org/10.1016/j.surfcoat.2006.07.092 2012), and supporting the earlier research reports, the present research work proved Samarajeewa, U. (1991). In situ degradation of mycotoxins by physical methods. that using cold plasma is one of the best options for treatment, inactivation and/or Mycotoxins and Animal Foods, 785-796. acceptable reduction of fungus Aspergillus flavus amount from nut shells, Schneider, J., Baumgärtner, K. M., Feichtinger, J., Krüger, J., Muranyi, P., Schulz, preventing further toxin production in nuts; in general and particularly in A., ... & Schumacher, U. (2005). Investigation of the practicability of low-pressure pistachios. microwave plasmas in the sterilisation of food packaging materials at industrial In order to reduce the fungus Aspergillus flavus from pistachios, and for the first level. Surface and coatings technology, 200(1), 962-966. time an AP-CCP generating device was used in this research. Various tests were https://doi.org/10.1016/j.surfcoat.2005.01.114 carried out, using Argon gas as plasma forming gas in atmospheric pressure with Moisan, M., Barbeau, J., Crevier, M. C., Pelletier, J., Philip, N., & Saoudi, B. different input power and irradiation times of 2, 6 and 10 minutes respectively. The (2002). Plasma sterilization. Methods and mechanisms. Pure and applied acceptable amount of 4 log fungus reduction (about 67%) was obtained at power chemistry, 74(3), 349-358. http://dx.doi.org/10.1351/pac200274030349 input of 100 W for 10 minutes irradiation time with no alteration on the nut texture. Basaran, P., Basaran-Akgul, N., & Oksuz, L. (2008). Elimination of aspergillus Therefore, this amount of fungus reduction is considered as final and the confirmed parasiticus from nut surface with low pressure cold plasma (LPCP) treatment. Food result of this research report. It is to be mentioned that the same test with 150 W in Microbiology, 25(4), 626-632. http://dx.doi.org/10.1016/j.fm.2007.12.005 10 minutes was also carried out and the fungus was totally (100%) removed. But Uhm, H. S., Lim, J. P., & Li, S. Z. (2007). Sterilization of bacterial endospores by since the surface of pistachios, treated by plasma generated by power inputs above an atmospheric-pressure argon plasma jet. Applied physics letters, 90(26), 261501. 100 watts, were slightly altered in texture only and due to device heating; such http://dx.doi.org/10.1063/1.2747177 results are not declared as confirmed achievements of this research work. However, Gupta, M., Manisha, K., & Grover, R. (2012). Effect of various media types on the the authors believe that at big scale industrial application, where the devices would rate of growth of Aspergillus niger. Indian Journal of Fundamental and Applied easily bear higher input power, the present deficiency may not occur. Life Sciences, 2(2), 141-144. http://www.cibtech.org/jls.htm Nuts in general and particularly pistachios are of utmost economic importance in Takahashi, T., Chang, P. K., Matsushima, K., Yu, J., Abe, K., Bhatnagar, D., ... & view of earning foreign currencies from import/export of these energetic Koyama, Y. (2002). Nonfunctionality of Aspergillus sojae aflR in a strain of agricultural products. The prevailing fungus Aspergillus flavus is one of the most Aspergillus parasiticus with a disrupted aflR gene. Applied and environmental acute and the biggest threat to nuts health. The worldwide stringent imposed microbiology, 68(8), 3737-3743. http://dx.doi.org/10.1128/AEM.68.8.3737- regulations in this regard are some of unavoidable hazards in the exports/imports 3743.2002 of pistachios. To date, no remedies which could be applied in big industrial scale Rashid, M. A. L. I. H. A., Khalil, S. A. M. I. N. A., Ayub, N., Ahmed, W. A. S. E. has not been reported. Hence, it is hoped that the results of this research could E. M., & Khan, A. G. (2008). Categorization of Aspergillus flavus and Aspergillus encourage the related industries to use the developed method in order to solve this parasiticus isolates of stored wheat grains in to aflatoxinogenics and non- global problem and prevent the related annual economic losses. aflatoxinogenics. Pakistan Journal of Botany, 40(5), 2177-2192.
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acid by an isolate of Aspergillus flavus in surface agar culture. Applied and environmental microbiology, 63(3), 1048-1053. Goryacheva, I. Y., Saeger, S. D., Eremin, S. A., & Peteghem, C. V. (2007). Immunochemical methods for rapid mycotoxin detection: Evolution from single to multiple analyte screening: A review. Food additives and contaminants, 24(10), 1169-1183. http://dx.doi.org/10.1080/02652030701557179 Tsitsigiannis, D. I., Dimakopoulou, M., Antoniou, P. P., & Tjamos, E. C. (2012). Biological control strategies of mycotoxigenic fungi and associated mycotoxins in Mediterranean basin crops. Phytopathologia Mediterranea, 158-174. http://dx.doi.org/10.14601/Phytopathol_Mediterr-9497 Hell, K., Mutegi, C., & Fandohan, P. (2010). Aflatoxin control and prevention strategies in maize for Sub-Saharan Africa. Julius-Kühn-Archiv, (425), 534. http://dx.doi.org/10.5073/jka.20100425.388
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IDENTIFICATION OF LIPASE PRODUCING FUNGUS ISOLATED FROM DAIRY WASTE CONTAMINATED SOIL AND OPTIMIZATION OF CULTURE CONDITIONS FOR LIPASE PRODUCTION BY THE ISOLATED FUNGUS
Madonna Roy1, RupeshKumar2, Anand Ramteke2, Nandan Sit1,*
Address(es): 1 Department of Food Engineering and Technology, Tezpur University, Assam-784028, India. 2 Department of Molecular Biology and Biotechnology, Tezpur University, Assam-784028, India.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.1.698-704
ARTICLE INFO ABSTRACT
Received 6. 5. 2018 Fungal strain from dairy waste contaminated soil was isolated for lipase production. The isolated strain was checked for lipase Revised 25. 6. 2018 production and the species was identified morphologically by SEM and phylogenetically by NCBI-BLAST analysis. The culture Accepted 2. 7. 2018 conditions for production of lipase using the isolated fungus with mustard oil as substrate were optimized for obtaining maximum lipase Published 1. 8. 2018 activity and biomass by response surface methodology. Morphological identification and NCBI-BLAST analysis revealed the fungal strain to be of Aspergillus aculeatus. The optimum culture conditions were1% mustard oil concentration, incubation temperature of 35 ⁰C and incubation time of 214.74 h. The lipase activity and biomass at the optimized condition were 9.51 U/ml and 549.88 mg Regular article respectively. It can be concluded from the present study that the strain of the fungus Aspergillus aculeatus isolated from dairy waste contaminated soil can be potentially used to produce lipase.
Keywords: Dairy Waste; Fungus; Isolation; Identification; Lipase production; Optimization
INTRODUCTION 2006; Veerapagu et al., 2013), and few works on using response surface methodology for optimization (Salihu et al. 2011; Açıkel et al., 2010; Vargas et The demand for industrial enzymes, particularly of microbial origin, is ever al. 2008; Hosseinpour et al., 2012) are also reported, but most of the works were increasing owing to their applications in a wide variety of processes. In recent on optimizing solid state fermentation process. years lipases have gained importance over proteases and amylases, especially in The knowledge of new microorganisms capable of producing lipase, new the area of organic synthesis (Sivakumar, 2014). The enantioselective and supplements and substrates for lipase production along with knowledge of regioselective nature of lipases have been utilized for the resolution of chiral operational conditions will be very much helpful in achieving the best drugs, fat modification, synthesis of cocoa butter substituents, biofuels, and for combinations for high value lipase production and lower the production costs in synthesis of personal care products and flavour enhancers (Sundar and industrial processes (Falony et al., 2006; Salihu et al., 2011). In this regard, the Kumaresapillai, 2013). present study focussed on lipase producing microorganisms that would be Lipase or triacylglycerol acylhydrolases are hydrolytic enzymes which break naturally present in a particular environment, and dairy waste contaminated soil down triacylglycerides into free fatty acids and glycerols. Naturally, the would be one of them. As dairy wastes contain substantial amount of dairy fats, substrates of lipases are triacylglycerols which have very low solubility in water. therefore probability of having lipase producing fungi in this type of soil is high. In normal conditions lipases catalyse the hydrolysis of ester bonds and the Therefore, the objectives of this study are to isolate and identify a fugal species reaction takes place at the interface between an insoluble substrate phase and the from soil contaminated with dairy waste and to optimize the culture conditions aqueous phase in which the enzyme is dissolved. Under certain experimental for lipase production using the isolated fungus. conditions they are capable of reversing the reaction, e.g. such as in the absence of water. Esterification of fatty acids with glycerol and formation of glycerides MATERIALS AND METHODS takes place due to the reversed reaction. Lipases are ubiquitous enzymes which are widely distributed in plant, animal and microbial world. Among lipases of Soil sample collection plant, animal and microbial origin, it is the microbial lipases that finds immense application. Microbial lipase are preferred because they offer great variety of Soil sample contaminated with dairy waste was collected from near the dairy catalytic activities, high yields, ease of genetic manipulation, rapid growth on processing unit of “Spring Valley Bakery” in Tezpur town, Assam, India. The inexpensive media, regular supply and are not affected by seasonal fluctuations. samples were scooped with a clean spatula into airtight sterile plastics and Microbial lipases have also gained special industrial attention due to their labelled appropriately. They were then transported to the laboratory for analysis. stability, selectivity, and broad substrate specificity (Dutra et al., 2008; The isolation of the fungal cultures was carried out within 6 to 48 hours after Griebeler et al., 2011). sampling. Lipase-producing microorganisms have been found in diverse habitats such as industrial wastes, vegetable oil processing factories, dairies, soil contaminated Reagents and chemicals with oil, oilseeds, and decaying food, compost heaps, coal tips, and hot springs (Muthazhagan and Thangaraj, 2014). Fungi, yeast and bacteria are three main PCR buffer (Invitrogen, North America), dNTP mix (Invitrogen, North America), sources of microbial lipase for industrial production. Fungal lipases are MgCl2(Invitrogen, North America), Taq DNA Polymerase (Invitrogen, North particularly important in industrial applications (Nwuche and Ogbonna, 2011). America), DNA template, PCR water (HiMedia, Mumbai, India), ITS1-F and Earlier also researchers have isolated and identified various lipase producing ITS4-R primers (Biolinkk, New Delhi, India), Agarose (Invitrogen, North bacterial (Veerapagu et al., 2013; Rajeshkumar et al., 2013; Sirisha et al., America), 1x TAE Buffer (HiMedia, Mumbai, India), Electrophoretic System 2010) and fungal (Ülker et al., 2011; Fan et al., 2013; Griebeler et al., 2011; (Genei, Bangalore), Blue juice (Loading buffer, Invitrogen, North America), Colen et al., 2006; Nwuche and Ogbonna, 2011) strains from oil or lipid DNA Sample (PCR products), DNA Marker (100 bp ladder, Invitrogen, North contaminated soils and other sources for lipase production.The conditions for America). All other chemical used were of analytical grade or molecular biology lipase production have also been optimized (Sirisha et al., 2010; Falony et al., grade.
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Isolation of fungus Kumaresapillai, 2013). The carbon source glucose in the production medium was substituted by different concentrations of mustard oil as per design. The The fungal organism present in the soil sample was isolated by serial dilution and production medium was then incubated for different time and temperature as per agar plating and by inhibiting the growth of bacterial cells by adding 10% tartaric experimental design for lipase production. acid to PDA medium (Apinis, 1963). Molds and yeasts were identified and moulds were isolated from the PDA plate. The fungal isolate was then sub- Determination of lipase activity cultured to a new PDA plate and incubated at 27ºC for 24-48h. After 48 h of incubation the fungal isolates were stored in refrigerator. Lipase activity assay was done as per standard method by titrimetric using olive oil as a substrate (Jensen, 1983). Lipase activity was determined by incubating a Microscopic examination reaction mixture containing 5ml of olive oil emulsion, 2ml of 0.1 M phosphate buffer, pH 7.0 and 1.0 ml of the culture filtrate (lipase crude extract) at 37°C for The isolated fungal strain was observed under microscope at 40X magnification 30 min, with shaking of 130 rpm. After incubation, the reaction was stopped by after staining with lactophenol cotton blue (LPCB). the addition of 15ml of acetone-ethanol (1:1) and the liberated free fatty acids were titrated with 0.05N NaOH in the presence of phenolphthalein as indicator. Evaluation of lipase production One unit of lipase activity was calculated as the amount of enzyme liberating one micro mole of fatty acid per minute under standard assay conditions. Confirmation of lipase production by the isolated fungal strain was done using 푣표푙푢푚푒 표푓 푁푎푂퐻 푐표푛푠푢푚푒푑 𝑖푛 푚푙 × 푁표푟푚푎푙𝑖푡푦 표푓 푁푎푂퐻 two methods, by inoculating the microorganism in Tween 80 agar plate Lipase activity (U/ml/min) = containing phenol red indicator and in Tributyrin agar plates (Aoyama, 1988). 푡𝑖푚푒 표푓 𝑖푛푐푢푏푎푡𝑖표푛 𝑖푛 푚𝑖푛 × 푣표푙푢푚푒 표푓 푒푛푧푦푚푒 푠표푙푢푡𝑖표푛 𝑖푛 푚푙 × 1000
Identification of the fungal strain Determination of Biomass
Morphological characteristics by scanning electron microscope (SEM) For biomass determination, the culture supernatant was separated from the mycelium by filtration through an already weighed Whatmann filter paper Scanning electron microscopic (SEM) image of the isolated fungus was obtained (Sharma et al., 2001). The biomass was washed many times with distilled water at different magnification to study the morphological characteristics. Fresh and then dried at 80°C, for around 18-24 hours, and was weighed until constant culture was taken in an Eppendorf tube. PBS buffer (500µl) was added to the weight was obtained. culture. The Eppendorf tube was centrifuged at 8000rpm for 15min. This step was repeated three times. Small amount of the pellet was transferred to a new 푤푒𝑖푔ℎ푡 표푓 푓𝑖푙푡푒푟 푝푎푝푒푟 푤𝑖푡ℎ 푏𝑖표푚푎푠푠 − 푤푒𝑖푔ℎ푡 표푓 푒푚푝푡푦 푓𝑖푙푡푒푟 푝푎푝푒푟 Biomass (mg/ml) = Eppendorf tube. One (1) ml of 2.5% glutaraldehyde was added to the pellet and 푣표푙푢푚푒 표푓 푠푎푚푝푙푒 𝑖푛 푚푙 mixed. It was then incubated overnight at 4ºC. PBS buffer (500µl) was added to the culture and centrifuged at 8000rpm for 15min. The supernatant was Experimental design for optimization of culture conditions discarded. The pellet was washed with 1ml of 35%, 50%, 75%, 90%, 95%, 100% ethanol by centrifuging the sample at 3000rpm for 10min. After the last wash A Box-Behnken Design (BBD) with three numerical factors was employed to with 100% ethanol, 20µl of the sample was placed in a cover slip and allowed to design the experiments. The numerical factors were substrate i.e. mustard oil dry for the removal of ethanol. After dehydration, the specimens were mounted concentration (C), temperature of incubation (T) and incubation time (t). Mustard on the aluminium specimen holder by double-sided tape. The samples were oil was selected as a substrate as it is abundantly produced and consumed in the coated with platinum and examined under the microscope at an accelerating North-East region of India and may be useful for development interesterified voltage of 15 kV at different magnifications. products which might be useful for the people of this region. The substrate concentration was varied from 1 to5 %, incubation time was varied from 120 to Phylogenetic characterization 216 h and temperature was varied from 25 to 35 °C. A total of 17 experiments were performed (Table 1). Five experiments at the centre points of the design Genomic DNA was extracted with the established CTAB method (Wu et al., were performed to allow the estimation of pure error. All experiments were 2001). After isolation of DNA, quantification and analysis of quality of DNA carried out in a randomized order to minimize the effect of external factors. sample using NanoDrop spectrophotometer (NanoDrop 2000, Thermo Fisher, Response surfaces were generated to study the effect of interactions of various Wilmimgton, USA) was carried out. The ratio of the absorbance at 260 and culture conditions on lipase activity and biomass production. 280 nm (A260/280) was used to assess the purity of nucleic acids. For pure DNA, A260/280 is ~1.8 (Glasel, 1995). Data analysis and optimization The 18SrDNA was amplified by PCR using primer ITS1-F (5'- TCCGTAGGTGAACCTGCGG-3') and ITS4-R (5'- Design Expert version 8 was used for analysis of data for the responses and TCCTCCGCTTATTGATATGC-3'). Reaction mixture of 25 µl was composed of optimization. Experimental data were fitted to a second order polynomial model 2.5 µl 10x PCR buffer, 0.5 µl of 10 mMdNTP mix, 1.2 µl 25 mM MgCl2, 0.8 µl as follows: Taq DNA Polymerase, 1.2 µl Glycerol, 0.5 µl of Forward and Reverse Primers, 3 3 3 4.0 µl of DNA Template and PCR water. The PCR cycle parameters included 2 initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation Y o i X i ii X i ij X i X j (94°C, 30 s), annealing (56.5 °C, 30 s), extension (72°C, 30 s), and final i1 i1 i j1 extension at 72°C for 7 min (Veriti® Thermal Cycler, Applied Biosystems, Foster City, USA).The amplified product was electrophoresed on 2% agarose gel Where Y represents the response i.e. lipase activity or biomass, βo, is the constant, along with DNA molecular weight marker (Quick–loadTM100bp DNA ladder; βi, βii and βij are the regression coefficients and Xi and Xj are the independent Invitrogen, North America). Gel was stained with ethidium bromide and variables in coded values. visualized over Gel Documentation System. Sequencing of the purified DNA Significant terms in the model were found by analysis of variance (ANOVA). obtained from ITS-PCR was performed in Sequencer (Genetic Analyzer, Applied Model adequacy was checked by lack of fit test, R2, predicted R2, adequacy Biosystems) by Sanger sequencing method (Sanger and Coulson, 1975). The precision and predicted residual sum of squares (PRESS). A non-significant (p> nucleotide sequences were subjected to BLAST analysis on 0.05) lack of fit, predicted R2 comparable to fitted R2, low PRESS and adequacy (http://www.ncbi.nih.gov/ index.html). Phylogenetic trees were constructed and precision higher than 4, shows that the model fitted is adequate to predicting the fungal strain was identified. The sequences were submitted to Gene Bank at (Corzo et al. 2008; Mohapatra and Bal, 2010). NCBI database and accession numbers were obtained. The optimization of the lipase production was done using desirability function.
Lipase production RESULTS AND DISCUSSION
The identified fungal cultures maintained in PDA plates were grown in Microscopic Observation and confirmation of lipase production by the inoculation medium containing glucose (10g/l), peptone (20g/l), sodium chloride isolated fungus (5g/l) and yeast extract (5g/l) at pH 6.0. Cultures (72 h old) were prepared as spore suspension by adding 5ml sterile water. This 5ml of spore suspension was The isolated and sub-cultured fungus formed brown-black coloured colony and added with 45 ml of inoculum medium in 100 ml Erlenmeyer flasks. The total back spores were observed in the PDA plate. Lactophenol cotton blue slide contents were incubated in rotary shaker at 30ºC for 48 h. The prepared inoculum mounts of the isolated fungus was observed under phase contrast microscope. It (10%) were transferred to production medium which consists of inoculation is the most widely used method of staining and observing fungi. Hyphae and medium supplemented with salt solution containing ammonium sulphite (5 g/l), spores were clearly visible under the microscope (Figure 1). Conidiophores were sodium hydrogen phosphate (6 g/l), potassium hydrogen phosphate (2 g/l), seen which could mean that the isolated fungus might be ascomycetes. magnesium sulphate (3 g/l) and calcium chloride (3 g/l) (Sundar and
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Genomic DNA of the fungus was successfully isolated by the CTAB method. The isolation of the genomic DNA was confirmed by DNA quantification using NanoDrop. Quantification of the DNA was done to quantify and analyse the quality of DNA to ascertain the approximate quantity of DNA obtained and the suitability of DNA sample for further analysis. The ratio of the absorbance at 260 and 280 nm (A260/280) is used to assess the purity of nucleic acids. For pure DNA, A260/280 is approximately 1.8 (Glasel, 1995). From DNA quantification the pure DNA samples were selected. The rest of the samples were found to be contaminated with other molecules (i.e. proteins, organic compounds, other). Genomic DNA was successfully amplified by PCR using the ITS primer pair. PCR products generated from soil samples using the ITS primer pair were run on a 2% agarose gel. The size of the target amplicon of isolated fungus corresponds to 600bp as represented by the 100bp DNA ladder molecular size marker (Invitrogen, North America) (Figure 3).The species identity of the fungus was determined based on the alignment of their sequenced ITS regions with published sequences in the NCBI (National Center for Biotechnology Information) database. After sequencing, similarity search for the sequence data obtained was performed using BLAST (Basic Local Alignment Tool). BLAST result revealed the species identity of the unknown fungus to be Aspergillus aculeatus. The Figure 1 Microscopic view of the isolated fungus at 40X magnification BLAST search for homologues for the fungus isolated in the study yielded results that had an identity of 97% and query coverage of 92%. As inferred from the The isolated fungal strain was cultured in Tween80 agar plate containing phenol phylogenetic trees, the query fungus was very closely related to different red indicator as well as in tributyrin agar plate to see whether the strain produces Aspergillu saculeatus strains. Both the trees, Max likelihood (Figure 4a)) and lipase or not. Both the tests gave positive results for lipase production. A change Max parsimony (Figure 4b)) had a bootstrap value of 100% which indicates to in colouration from pink to yellow was observed in Tween80 agar plate whereas the high reproducibility of the tree. The tree can hence be considered consistent a clear zone of hydrolysis was seen in tributyrin agar plate confirming the over 100 iterations. Hence it can be concluded that the isolated fungi is a strain of production of lipase by the isolated fungal strain. Aspergillus aculeatus.On the basis of sequencing report obtained, the isolated fungus was characterized as Aspergillus aculeatus and was designated as strain Identification of the isolated fungus MRNS_TU (gb KR758756).
The SEM images of the hyphae along with the spores of the isolated fungal strain are shown in Figure 2. Observations of SEM images of the isolated fungi reveal swollen vesicle and ellipsoidal to subglobose or globose conidia which were found to resemble Aspergillus japonica as observed by Al-Musallam (1980).
Figure 2 SEM images of the isolated fungus with hyphae and spores at different magnification
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response variables, which is accounted for by the regression analysis (Mclaren et 1 2 3 4 5 6 7 8 M 9 10 11 al., 1977). When R2 approaches unity, the better the empirical model fits the actual data. Smaller values of R2 show less relevance of the dependent variables in the model and could not explain the variation. Further, a non- significant (p> 0.05) lack of fit, predicted R2 comparable to fitted R2, low PRESS and adequacy precision higher than 4, implies that the model fitted is adequate to predicting (Corzo et al., 2008). It can be observed from Table 2 that the probability (p) values of all the main 600bp factors, quadratic term for temperature (T2) and interaction of time and temperature i.e. T×t for the lipase activity were significant at p<0.05 and the model has non - significant lack of fit (p>0.05), which is good, and also the adequacy precision for the model was more than 4. The R2 value of the model was 0.99, whereas the adjusted R2 (0.98) and predicted R2 (0.96) were comparable indicating that the model fitted provided appropriate approximation of the true process.
Similarly, for the model of biomass production it was observed that the probability (p) values of incubation time and incubation temperature among the main factors, quadratic term for incubation time (t2) and temperature (T2) and interaction of time and temperature i.e. T×t for the lipase activity were significant at p<0.05 and the model has non - significant lack of fit (p>0.05). The adequacy Figure 3 The size of the amplified targetamplicon of extracted DNA as precision for the biomass model was more than 4. The R2 value of the model was represented in 1xTAE 2% agarose gel. Lane 1, 3, 5, 7, 9, 11 are positive i.e. 0.96, whereas the adjusted R2 (0.90) and predicted R2 (0.65). containing the amplified DNA and lane 2, 4, 6, 8, 10 are negative i.e. controls The final equations in terms of coded factors are as follows: without any sample. Lane M shows the 100bp DNA ladder molecular size marker Lipase activity,L = 6.26 + 1.71T+ 0.27t–0.14C+ 0.92T2 + 0.04t2 + 0.07C2 +0.19T × t + 0.05T×C+ 0.05t×C (a) Biomass, B = 584.00 + 79.88T + 58.13t + 4.75 C – 80.63T2 – 44.13t2 – 1.88C2 – 48.25T × t + 15.00T×C – 21.00t×C
Response surface plots were generated to study the effect of various parameters on the lipase activity and biomass production during fermentation. The effect of interaction of various factors on lipase activity is shown in Figure 5. Figure 5 a) shows the variation in lipase activity with time and temperature during fermentation. Variation in lipase activity could not be observed with time at lower temperature. But, as the temperature increased, slight variation in the lipase activity was seen with variation in time. The lipase activity was found to increase with increase in temperature. Initially the change was slower at lower temperatures, but as the temperature was increased the lipase activity increased at a faster rate. The increase in lipase activity at higher temperature could be due to the fact that at low temperature the growth of the microorganism was slower compared to that at higher temperature, thereby producing less lipase and when the temperature increased the microorganism could grow at a faster rate and (b) produced more lipase. Another reason could be that the optimum temperature for growth of the fungus and lipase production might be near 35 °C. Various authors have earlier reported that the activity of lipase produced by various fungal species was highest near 30 °C (Vargas et al., 2008; Kempka et al., 2007). Figure 5 b) shows the interaction of incubation time and concentration. It was observed that lipase activity decreased with increase in concentration of mustard oil, and the rate of decrease was higher when time of incubation was less. Lipase activity was found to increase with increase in time of incubation for all concentration of the substrate i.e. mustard oil. Lima et al. (2003) and Kamini et al. (1998) also reported a decrease in lipase activity with increased concentration of substrate during fermentation using Penicillium aurantiogriseum and Aspergillus niger respectively. The decrease in activity of lipase with increase in mustard oil concentration could be attributed to substrate inhibition at high concentration of substrate. When the time of incubation was more the effect of substrate concentration was less which can be explained by the fact that with time the microorganism might have developed some mechanism to overcome the Figure 4 The maximum likelihood (a) and maximum parsimony (b) phylogenetic inhibition caused by high substrate concentration. trees were constructed and bootstrapped (100 iterations) using robust From Figure 5 c) it can be observed that lipase activity increases with increase in phylogenetic analysis to represent the relationship between the isolated fungus temperature and was not much affected by change in concentration of the and respective species of the genus Aspergillusand related genera. Bootstrap substrate, although slight decrease in activity was observed with increase in value is noted on the branch and the scale bar represents nucleotide substitution concentration. The reason for such behaviour could be attributed to substrate per 100 nucleotide. inhibition at higher concentration and optimum temperature being near 35 °C, as
discussed earlier. Effect of culture conditions of Aspergillus aculeatus on the lipase activity and Figure 6 shows the effect of different culture conditions investigated on the cell biomass variation in biomass of A. aculeatus during lipase production. The effect of
interaction of time and temperature on growth of A. aculeatus is shown in Figure The values of lipase activity and cell biomass for different experimental 6 a). It was observed that when time of incubation was less biomass increased conditions are presented in Table 1. Second order polynomial equations were with increase in temperature. The rate of increase in biomass was initially more, obtained for lipase activity and cell biomass by multilinear regression analysis of and became almost constant with further increase in temperature. A similar the experimental data. To determine the significant effects of the process pattern was observed with time of incubation as well. At low incubation variables on responses, analysis of variance (ANOVA) was performed. temperature the rate of increase of biomass was more as compared to when the Regression coefficients of the different terms in the equations for lipase activity temperature of incubation was high. When the temperature or the time of and cell biomass in coded factors were obtained (Table 2). Model adequacy was incubation were high, it was found that initially the biomass increased with checked by lack of fit test and by considering fitted R2, predicted R2, PRESS and increase in incubation temperature and time up to a certain point, and then it adequacy precision. Coefficient of determination, R2, is defined as the ratio of the started decreasing with further increase in incubation temperature and time. explained variation to the total variation and is a measure of the degree of fit
(Haber and Runyon, 1977). It is also the proportion of the variability in the
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Table 1 Values of lipase activity and biomass for carious experimental conditions Concentration of Lipase Activity Biomass Sl. No. Temperature, T (°C) Time, t (h) mustard oil, C (%) (U/ml) (mg/ml) 1 25 120 3 5.32 270 2 25 168 1 5.83 432 3 25 168 5 5.41 434 4 25 216 3 5.58 466 5 30 120 1 6.33 457 6 30 120 5 5.98 486 7 30 168 3 6.33 554 8 30 168 3 6.41 581 9 30 168 3 6.39 566 10 30 168 3 6.2 595 11 30 168 3 5.98 624 12 30 216 1 6.67 632 13 30 216 5 6.52 577 14 35 120 3 8.48 549 15 35 168 1 9.01 539 16 35 168 5 8.78 601 17 35 216 3 9.52 552
Table 2 Analysis of variance (ANOVA) for the experimental data of lipase activity and biomass Lipase Activity Biomass Source Sum of Sum of F-Value p-value F-Value p-value Squares Squares Model 27.91 114.87 < 0.0001* 127801.4 17.87 0.0005* T 23.29 862.81 < 0.0001* 51040.13 64.23 < 0.0001* t 0.59 22.01 0.0022* 27028.13 34.01 0.0006* C 0.16 6.12 0.0425* 180.5 0.23 0.6482ns T×t 0.15 5.63 0.0493* 9312.25 11.72 0.0111* T×C 0.01 0.33 0.5812ns 900 1.13 0.3226ns t×C 0.01 0.37 0.5620ns 1764 2.22 0.1799ns T2 3.58 132.81 < 0.0001* 27370.1 34.44 0.0006* t2 0.01 0.25 0.6306ns 8197.96 10.32 0.0148* C2 0.02 0.82 0.3938ns 14.8026 0.02 0.8953ns Residual 0.19 5562.75
Lack of Fit 0.06 0.66 0.6175ns 2608.75 1.18 0.4230ns Pure Error 0.12 2954
Corr Total 28.10 133364
R2 0.99 0.96 Adjusted R2 0.98 0.91 Predicted R2 0.96 0.65 Adequacy Precision 32.05 15.70 *- Terms significant at p<0.05 ns- Terms not significant increase with increase in substrate concentration at higher temperature. This type Vargas et al. (2008) also observed a decrease in biomass during production of of behaviour shows that low temperature i.e. 25°C and high concentration of lipase by Penicillium simplicissimum. From these observations it can be inferred mustard oil is not favourable for growth of A. aculeatus. When the temperature that the temperature and time of incubation for achieving maximum biomass was was near 35°C, slight increase in biomass was observed with increase in substrate close to but less than 35°C and 216 h respectively. concentration. From this observation it can inferred that the optimum temperature The effect of interaction of concentration and time is depicted in Figure 6 b). for growth of A. aculeatus is near 35°C. The biomass was found to increase with increase in time of incubation. The rate of increase was more when time of incubation was less and gradually slowed as (a) the time was increased beyond a certain point, i.e. after a certain time the biomass did not increase and remained almost constant. The variation in biomass with time was observed to be more when the concentration of mustard oil i.e. substrate source was less as compared to when the substrate concentration was more. Higher variation in biomass with time at lower concentration of substrate could be attributed to the fact that when the substrate concentration was low, the broth 9.40 was dilute and the microorganism could not get sufficient time to reach the substrate at lower incubation time. But when the substrate concentration was high 8.40 it became easily accessible to the microorganism for growth and higher biomass 7.41 was achieved even at lesser incubation time. The biomass production was found 6.41 to increase with increase in concentration of substrate when the time of 5.41 incubation was less i.e. 120 h, but as the time of incubation was more, a decrease in biomass production was noticed with increase in concentration of substrate. A decrease in biomass production with substrate concentration at higher incubation activity, Lipase U/ml time might be due to the death of microorganisms at high substrate concentration. 216.0 When the time of incubation was sufficiently high, substrate inhibition may cause 35.0 192.0 death of the microorganism, thereby reducing the biomass. 32.5 From Figure 6 c) it can be observed that biomass increased with increase in 168.0 30.0 temperature till 32°C and then decreased with further increase in temperature for Time, h 144.0 27.5 all values of substrate concentration. It was also noticed that biomass production Temperature, °C decreased with increase in concentration of substrate at lower temperatures and 120.0 25.0
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(b) (b)
9.40 611.5 526.9 8.40 442.3 7.41 357.6 6.41 273.0 5.41
Biomass, mg/ml Biomass, mg/ml
Lipase activity, Lipase U/ml
5.00 5.00 216.00 216.0 4.00 4.00 192.00 192.0 3.00 168.00 3.00 168.0 Concentration, % 2.00 144.00 Time, h Concentration, % 2.00 144.0 Time, h 1.00 120.00 1.00 120.0 (c) (c)
611.5 9.40 526.9
8.40 442.3
7.41 357.6
6.41 273.0
5.41 Biomass, mg/ml Biomass, mg/ml
Lipase activity, Lipase U/ml
5.00 35.0 5.00 4.00 35.0 32.5 4.00 3.00 32.5 30.0 3.00 Concentration, % 2.00 27.5 30.0 Temperature, °C Concentration, % 2.00 27.5 1.00 25.0 Temperature, °C 1.00 25.0 Figure 6 Effect of interaction of (a) time and temperature, (b) concentration and
time and (c) concentration and temperature on biomass Figure 5 Effect of interaction of (a) time and temperature (b) concentration and time, and (c) concentration and temperature on lipase activity Optimization of culture conditions for production of lipase and validation
(a) Optimization of culture conditions for production of lipase was done numerically using desirability function that aimed at finding the values of process variables which would give maximum lipase activity and biomass. As the main aim of growing the microorganism was to produce lipase, therefore, lipase activity was given higher importance than biomass production while optimization. One solution with highest desirability was selected (Table 3). The optimum culture
611.5 condition with maximum lipase activity of 9.51 U/ml and biomass of 549.88 was obtained when mustard oil concentration was1%, temperature of incubation was 526.9 35 °C and incubation time was 214.74 h. For validation of optimization result 442.3 experiment were conducted at the optimum culture conditions. The experimental 357.6 values obtained for lipase activity and biomass were 9.33 U/ml and 496 mg. The 273.0 percentage error between the actual and predicetd values for lipase activity was 1.8% and for biomass it was 9.7% which are within the acceptable range (Qi et Biomass, mg/ml Biomass, mg/ml al., 2009).
216.00 35.0 192.00 32.5 168.00 30.0
Time, h 144.00 27.5 Temperature, °C 120.00 25.0
Table 3 Numerical solution of optimization and validation of optimization result Optimization result Experimental Result Temperatur Residual Percent error, % Time of Concentration (Predicted) (Actual) Sl. e of incubation, of mustard oil, Lipase Lipase Desirability No. incubation, Biomass, Biomass, Lipase Lipase h % Activity, Activity Biomass Biomass °C mg mg Activity Activity U/ml , U/ml 1. 35 214.74 1.00 9.51 549.88 9.33 496.00 0.18 53.88 1.8 9.7 0.91
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CONCLUSION Mohapatra, D., Bal, S., 2010. Optimization of polishing conditions for long grain basmati rice in a laboratory abrasive mill. Food Bioprocess Technol. 3, 466-472. From the present investigation it is concluded that the strain of the fungus https://doi.org/10.1007/s11947-009-0254-3 Aspergillus aculeatus isolated from dairy waste contaminated soil can be Muthazhagan, K., Thangaraj, M., 2014. Production and partial characterization of potentially used to produce lipase and mustard oil can be used as a source of lipase by Bacillus sp isolated from vellar estuary sediment. Int. J. Sci. Inv. Today. substrate for lipase production for food application and other industrial use. The 3, 639-653. results further revealed that although the biomass production decreases after a Nwuche, C. O., Ogbonna, J. C., 2011. Isolation of lipase producing fungi from certain time and temperature, lipase continues to be produced. This knowledge palm oil mill effluent (POME) dump sites at Nsukka. Braz. Arch.f Biol. can help us designing the fermentation process for lipase production in future Technol. 54, 113-116. http://dx.doi.org/10.1590/S1516-89132011000100015 using this isolated strain of fungus. Qi, B., Chen, X., Shen, F., Su, Y., Wan, Y., 2009. Optimization of enzymatic hydrolysis of wheat straw pretreated by alkaline peroxide using response surface Conflict of Interest methodology. Ind. Eng. Chem. Res. 48, 7346-7353. https://pubs.acs.org/doi/abs/10.1021/ie8016863 The authors confirm that no part of the manuscript have been plagiarized or self- Rajeshkumar, M. P., Mahendran, V. S., Balakrishnan, V., 2013. Isolation and plagiarized and declare no conflict of interest. identification of lipase producing organisms from diverse soil samples of Kolli hills. Int. J. Curr. Microbiol. App. Sci. 2, 205-210. REFERENCES Salihu, A., Alam, M. Z., AbdulKarim, M. I., Salleh, H. M., 2011. 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IN-SILICO STUDIES OF SOME NATURAL, SYNTHETIC AND SEMI-SYNTHETIC ANTIFUNGAL DRUGS FOR THEIR MULTI-TARGETING NATURE
Neelabh, Karuna Singh*
Address(es): Karuna Singh, Department of Zoology, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi-221005.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.1.711-716
ARTICLE INFO ABSTRACT
Received 6. 4. 2018 Research has shown that drugs or therapeutic agents which are directed at a particular target often undergo a reduction in efficacy, undesired safety profiles, compensatory and neutralizing effects, anti-target and counter target activities and also resistance against the Revised 14. 5. 2018 drug. Proper utilization of multiple targets can lead to a perfect blend between the efficacy and safety when compared against single- Accepted 3. 7. 2018 targeted drug design. The authors have utilized this concept in case of the antifungal drugs which generally act against one of the targets Published 1. 8. 2018 amongst chitinase, chitin synthase, 1, 3 beta glucan synthase and lanosterol 14 α-demethylase. Henceforth, the present study is an attempt to screen the known drugs for their multi-targeting nature, and to compare natural product based drugs with semi-synthetic and Regular article synthetic drugs in-silico. In the present study, eleven (7 synthetic and 4 natural) drugs namely Allosamidine, Methylxanthine, Acetozolamide, Nikkomycin Z, Polyoxin L, Caspofungin, Fluconazole, Argifin, Obovatol, Papulacandin and Ro-091470 have been chosen to study their effect against different targets. This exciting and unique in-silico study provides insight that some drugs can function equally good against all targets, while some have a better efficiency against a different target than the known one. All four studied natural product based drugs are found to be good at multi-targeting. All the drugs that were shown to have a good multi-targeting efficiency bind at the same region where the known drugs against that target bind. Furthermore, lanosterol 14 α-demethylase is found to be the best target amongst all the aforesaid fungal targets.
Keywords: Multi-targeting; In-silico; FireDock; Binding Energy; Drug designing
INTRODUCTION of the multi component extracts which are directed towards different targets (Csermely et al., 2005). Despite the mammoth strides taken in the screening of the drugs and rational drug designing, result in the form of number of successful drugs and discovery of novel targets is not satisfactory (Korcsmáros et al., 2007). Till date we have not been able to find tenable and broad spectrum therapeutic options against many life threatening diseases. The need of finding new therapeutic options intensifies more when it comes to fungi, because fungi are similar to humans and the drugs designed against them are likely to cause side-effects in human beings as well (Neelabh et al., 2018, Neelabh and Singh, 2018 ). For obtaining improved results in the field of drug development there is a need of modifying the approach we have towards drug designing or development. The goals or the procedure of drug development can be summarized in figure 1. In other words, it can be said that the chemical library of the potential therapeutic compounds is screened exhaustively through different computational as well as wet lab methods (Csermely et al., 2005). Furthermore, the best binder is obtained by evaluating the interaction between the ligand and the target (figure 2). But as already mentioned, this model lags far behind in the treatment of diseases such as cancer, AIDS, neurological, cardiovascular, mycoses etc. Failure in the treatment of life threatening diseases can be attributed to numerous reasons, primary amongst them is the “single-target” or “single-hit” against which the drug is being targeted. Unfortunately, this approach is not compatible with the modern day micro-organisms which have a capacity to fast become resistant to any specific drug. Moreover, studies on biological systems and the experience gained by clinical practices suggests that focussing on a single target might provide up regulation or down regulation of the target but might not be able to provide the desired result to the biologic body as a whole (figure 3a and figure 3b). Additionally, the Networking model says that it is much more fruitful to partially inhibit large number of small targets rather than completely inhibiting a single large target (Tang et al., 2013). Multi-targeting activity of the compounds can be easily seen in nature. For instance, snake venom and spider venoms utilize multiple targets in order to exert their effects. Similarly, plants use factors having effect on multiple targets that Figure 1 The goal of drug development defends them against any pathogenic attack. Traditional treatments also comprise
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Figure 2 The currently used model for drug development. The in-vitro and in- silico approaches utilize the chemical library and find suitable ligands for the target. Then through the various wet lab techniques the best binder to the target is determined.
Figure 3 a) Complete inactivation of the single target (regulator X) has been shown which is the current model of the single target drug design b) The partial inactivation of multiple targets (regulators X and Y), is the model that needs to be followed for producing efficient multi- target drugs.
Table 1 Drugs and their respective known targets Drugs tested Respective known targets Argifin(Natural) http://www.drugbank.ca/drugs/DB03632 Allosamidine Chitinase http://www.drugbank.ca/drugs/DB04628 PDB ID:2XVN Methylxanthine
http://www.chemspider.com/Chemical-Structure.72464.html Acetozolamide http://www.drugbank.ca/drugs/DB00819 Obovatol (Natural) http://chem.sis.nlm.nih.gov/chemidplus/rn/83864-78-2 Nikkomycin Z Chitin synthase http://www.chemicalbook.com/ChemicalProductProperty_EN_CB0767000.html Polyoxin L http://pubchem.ncbi.nlm.nih.gov/compound/100013#section=Top Papulacandin (Natural) http://pubchem.ncbi.nlm.nih.gov/compound/6450326 1,3 beta glucan synthase Caspofungin http://www.drugbank.ca/drugs/DB00520 Ro-091470 (Natural) http://www.guidechem.com/dictionary/en/135357-96-9.html Lanosterol 14 α-demethylase Fluconozole PDB ID: 4 LXJ http://www.drugbank.ca/drugs/DB00196
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The current manuscript focusses on the multi-targeted drug designing against FireDock mycological diseases. The authors have chosen 4 main target enzymes chitinase, chitin synthase, 1,3-beta-glucan synthase and lanosterol 14 α-demethylase which FireDock is an efficient and reliable method utilized for refinement and rescoring are main targets of currently available antifungals. Chitinase is a chief enzyme for rigid-body docking predictions. FireDock functions on the principle of which apart from fungal cell wall degradation and remodelling also performs “coarse refinement”. Coarse refinement comprises of side chain optimization and exogenous chitin decomposition in fungi (Hartl et al., 2012) while chitin rigid body minimization (Mashiach et al., 2008; Neelabh et al., 2015). synthase is another important enzyme which governs the synthesis of chitin in the fungal cell wall (Henar et al., 1998). 1,3-beta-glucan synthase is an enzyme RESULTS having multiple subunits and is responsible for fungal cell wall formation, division, septum deposition and ascospore wall assembly (Liu and Present manuscript is an attempt to screen the currently used antifungals for their Balasubramanium, 2001). Lanosterol 14 α-demethylase is a key enzyme that multi targeting efficiency. Due to the unavailability of the the structures for 1,3- mediates the conversion of lanosterol into ergosterol which is a key component of beta-glucan synthase and chitin synthase they have been modeled through Phyre2 the fungal cell membrane (Becher and Wirsel, 2012). web server and shown in figure 5a and figure 5b. The sequences utilized for the In the present manuscript the grouping of the drugs have been made in such a homology modeling have been mentioned in table 2. way that each group comprises of a natural antifungal and synthetic or semi synthetic antifungals (Table 1). In the first group of table 1, Argifin is the natural drug and all the other drugs in that group are non natural, synthetic or semi synthetic in nature. Similarly, Obovatol (Hwang et al., 2002), Papulacandin and Ro-091470 (Aoki et al., 1993) are the natural members of the groups having targets as chitin synthase, 1,3-beta- glucan synthase and lanosterol 14 α-demethylase respectively.
MATERIAL AND METHODS
The different fungal targets viz. chitinase, chitin synthase, 1,3-beta-glucan synthase and lanosterol 14 α-demethylase have been docked upon by different antifungals such as Argifin, Papulacandin, Polyoxin L, Obovatol, Allosamidine, Acetozolamide, Methylxanthine, Ro-091470, Fluconazole, Caspofungin and Nikkomycin. The authors could not find the suitable structures of 1,3-beta-glucan synthase and chitin synthase, therefore these structures were modelled through Phyre2 software and were utilized for docking. A brief outline for the above process has been provided in figure 4.
Figure 5a
Figure 5b
Figure 5 Modelled structures of a)1,3 beta glucan synthase b) Chitin synthase Figure 4 An outline of the methodology employed. both visualized by Discovery Studio visualization software. Further, validity of both the models was ascertained through RAMPAGE The authors have chosen natural, synthetic as well as semi-synthetic drugs that webserver and 93.4% residues were found in favoured region, 3.5% were found are specifically known to act on a particular target and further tested them for in allowed region and 3.2% in the outlier region for 1,3-beta-glucan synthase having multi targeting nature through FireDock which calculates the binding whereas 89.7% residues were found in favoured region, 7.6% were found in energy of the antifungal against the target. This binding energy is the indicator of allowed region and 2.7% in outlier region the affinity of antifungal towards its target. (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php). These structures have also been submitted to Protein model database (https://bioinformatics.cineca.it/PMDB/).
Table 2 Sequences utilized for 1,3-beta-glucan synthase and chitin synthase for homology modelling Target Sequence
GKSDNQNHAIIFYRGEYLQLIDANQDNYLEECLKIRNVLGEFFAKFPVAILGAREYIFSENIGILGDIAAGKEQTFGTLAARSLSYIGGKLHYGHPD FLNAIYMNTRGGVSKAQKGLHLNEDIFAGMLAFGRGGRIKHSEYYQCGKGRDLGFGTILNFQTKIGTGMGEQMLSREYYYLGTQLPIDRFLTFY 1,3 beta glucan synthase YGHPGFHINNILVMMSVQVFMLALVFLGTLNKQLTVSIFIVFWIAFVPLFVQELTERGTGRAILRLCKHFLSLSPVFEVFSTQIYMHSILNDLTFGG ARYIATGRGFATTRISFSILYSRFAGPSIYLGIRTLVILLFVTLTVWVPHLIYFWITVVGLCVA
VTDLSPYSYLPIDGEESLLRYVEEKKKLEEPHVFQLALGAYYNMRRTGQDQVIIASGPTGSGKSELKRLAIEAITQVSLANPGKKGSKIGLQVSSA EFILKCFGNAHTLSNDEASRFGTYTELQFNERGRLEGLKTIVYYFERSRVSQVPINGERNFHAFYYLVSGAPEEERNFLKLGDVSDYRYLNCRVR RVGVDDRHRYSQLRQAFKMIGISSRLIAQIFQLLASILHIGNLRFSPSDGIQEGASVINIDTLDTVAEFLGVHSESLAEIFSLKTILVRKEVCTTFLGP Chitin synthase EQAEQVRDELARTLYSLLFSWLNEHINTRLCKDSFGSFIALVDLPGIQRNSGSMGSLNSVDQFCLNFAAEKMHNWVLHRIHETTRQEAEAERLLI SRVPYFDNSECLGMLSNPRGGLISVIDDLSQKKRSESNLLESLGKRFHNHPSMSISPQGNRSSASFTINHYDGPVTYSTSNFLERNANETSTDIIQLL RGSSNPFIKGLFGMLVPVSEEGGEEASDFQVGGGNDESSPKELHCIAGQHWAAVDSLLKSFDQTQTWYIFALRPNDSQLPFQFDLRSMKQQVRS FGLVEMAQQLQTSWEVRLPHKEACERYNEELVYRGIPEGTGDVERLRDLKRLMSLNDADMGIGLQRVFLSNNLFHFLEDRLRAKEPGEQHAYE
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All the target proteins have an active site on which the drugs act and modify the function of the protein. The active site of the target proteins has been determined by the CASTp server and further docking of drugs on their respective known targets has been performed with the help of FireDock (figure 6).
Figure 7 Best docked structures. a) Ro-091470 binds 1,3-beta-glucan synthase where the drugs having the known activity (Caspofungin and Papulacandin) against this target bind b) Argifin binds to the same region or active site of lanosterol 14 α-demethylase where the drugs having the known activity (Fluconazole, Ro-091470) against the aforesaid target bind c) Papulacandin binds to the same region or active site of lanosterol 14 α-demethylase where the drugs having the known activity (Fluconazole, Ro-091470) against this target bind d) Polyoxin L binds to the same region or active site of 1,3-beta-glucan synthase where the drugs having the known activity (Papulacandin and Caspofungin) against the said target bind.
Binding energies of Obovatol, Nikkomycin and Polyoxin L have been obtained against their known target chitin synthase as well as other targets (Table 4).
Table 4 Results of Obovatol, Nikkomycin Z and Polyoxin L against chitin synthase (known target) and others obtained from FireDock.
Chitin 1,3 beta glucan Lanosterol 14 Drug synthase Chitinase synthase α-demethylase (Known) Obovatol -23.94 -27.70 -40.96 -43.63 Nikkomycin -21.86 -22.79 -58.83 -65.68 Z Polyoxin L -43.68 -38.71 -78.24 -67.27
This gives us an insight that Polyoxin L has the highest binding energy amongst Figure 6 Active sites of the target proteins and drugs docked at it. a) chitinase members of its group against their known target chitin synthase. Additionally, it docked with Argifin, Allosamidine, Methylxanthine, Acetozolamide b) chitin can be observed that Polyoxin L has high binding energies against all the other synthase docked with Obovatol, Nikkomycin and Polyoxin c) 1,3-beta-glucan targets as well, therefore, it can be assumed to have the highest multi-targeting synthase docked with Papulacandin and Caspofungin d) lanosterol 14 α- efficiency in its group. The highest binding energy of Polyoxin L is found against demethylase docked with Ro-091470 and Fluconazole. 1, 3 beta glucan synthase. On analysis of its best docked structure against 1,3- beta-glucan synthase (figure 7d) it can be concluded that it binds in the same area The binding energy of Argifin, Methylxanthine, Acetozolamide, and or the active site of the 1,3-beta-glucan synthase where all the other members of Allosamidine against chitinase has been depicted in table 3 which shows that the 1,3-beta-glucan synthase interacting drugs (Caspofungin and Papulacandin) Allosamidine has the highest binding energy against its known target chitinase. bind. On the other hand Argifin, a natural antifungal having the same target, has a On analysis of table 5, it portrays the binding energies of Caspofungin and greater multi-targeting efficiency than the other synthetic and semi synthetic Papulacandin against their known targets 1,3-beta-glucan synthase and other drugs in its group. Therefore, it could be a better drug in comparison to the others targets as well. Caspofungin has a higher binding energy than Papulacandin in its group. The highest binding energy of Argifin has been obtained against against their known target 1,3-beta-glucan synthase on the contrary, lanosterol 14 α-demethylase. Papulacandin has high binding energy against all the other targets. It is highest against lanosterol 14 α-demethylase. Table 3 Results of Argifin, Methylxanthine, Acetozolamide and Allosamidine against Chitinase (known target) and others obtained from FireDock. Table 5 Results of Caspofungin and Papulacandin against 1,3-beta-glucan synthase (known target) and others obtained from FireDock. 1,3 beta Chitinase Chitin Lanosterol 14 Drug glucan 1,3-beta- (Known) Synthase α-demethylase glucan Chitn Chitinase Lanosterol 14 synthase Drug synthase synthase α-demethylase Argifin -28.59 -25.53 -74.06 -74.74 (Known) Methylxanthine -25.42 -23.76 -50.05 -53.84 Caspofungin -117.59 -32.96 -42.12 -47.74 Acetozolamide -28.09 -21.56 -41.34 -37.92 Papulacandin -67.32 -37.88 -44.93 -61.54 Allosamidine -29.99 -23.89 -55.90 -63.88 On analysis of the best docked structure of Papulacandin against lanosterol 14 α- On examining the best docked structure of Argifin against lanosterol 14 α- demethylase (figure 7c) it can be said that Papulacandin binds to the same place demethylase it was observed that Argifin was also binding in the same area or like all the other drugs binding to lanosterol 14 α-demethylase in its group (Ro- active site where all the other known drugs against lanosterol 14 alpha 091470 and Fluconazole) and may exhibit activity against it in in-vivo and in- demethylase (Ro-091470 and Fluconazole) docked, and thus can be assumed to vitro studies. show activity against the aforesaid protein (figure 7b). On analysis of table 6 it becomes clear that Ro-091470 has a higher binding energy than fluconazole and also has the highest binding energy against all the other targets in its group and therefore might have a good multi-targeting efficiency. Ro-091470 has the highest binding energy against its known target
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lanosterol 14 α-demethylase which has already been analysed (figure 7d). The CONCLUSION second highest binding energy was found to be against 1,3 beta glucan synthase. Current manuscript is a unique in-silico study aimed to identify the multi- Table 6 Results of Fluconazole and Ro-091470 against lanosterol 14 α- targeting efficiency of some antifungals currently being used. Four natural demethylase (known target) and others obtained from FireDock. antifungals have also been screened for this approach of multi-targeting. It has Lanosterol 14 1,3 beta been found that most of the studied antifungals exhibit multi-targeting Chitin Chitinase Drug α-demethylase glucan efficiency but all of them have a higher binding affinity towards 1,3-beta-glucan synthase (Known) synthase synthase and lanosterol 14 α-demethylase. Moreover, amongst the natural Fluconazole -41.86 -20.83 -24.51 -36.40 antifungals, Argifin, Papulacandin and Ro-091470 have shown a high multi targeting efficiency and can be worked upon by the pharmaceutical companies by Ro-091470 -51.17 -29.70 -28.17 -42.64 using them as scaffolds and designing new antifungals or enhancing their other properties so that they can be used as suitable antifungals in future. However, On analysis of this docked structure it becomes clear that Ro-091470 binds to the more wet lab experiments need to be performed in order to ascertain the activity same region like all the other drugs binding to 1,3-beta-glucan synthase in its of these antifungals against different targets. group (Papulacandin and Caspofungin) as shown in figure 7a. In the current study, it is also clear that most of the antifungals studied have a Conflict of interest: Authors declare that there is no conflict of interest. higher binding energy against the targets lanosterol 14 α-demethylase and 1,3- beta-glucan synthase as compared to the chitin and chitin synthase. This indicates Acknowledgement: One of the authors Neelabh would like to thank Indian that lanosterol 14 α-demethylase and 1,3-beta-glucan synthase are comparatively Council of Medicla Research (ICMR) for providing Senior Research Fellowship. more suitable or better targets for the antifungals however, a wet lab validation is Additionally, the authors would like to thank Principal, Mahila Mahavidyalaya, required. Banaras Hindu University, Varanasi for providing the laboratory facilities.
DISCUSSION REFERENCES
Literature is full of reports containing information about drugs developed to aim Korcsmáros, T., Szalay, M. S., Böde, C., Kovács, I. A., & Csermely, P. (2007). at a single target. But according to the different studies conducted in the past, How to design multi-target drugs: target search options in cellular drugs which can aim at multiple targets are more succesful. Therefore, keeping networks. Expert opinion on drug discovery, 2(6), 799-808.doi: this concept in mind present manuscript tests the activity of some known https://doi.org/10.1517/17460441.2.6.799 antifungals against their targets and also computes their efficiency to be used for Csermely, P., Agoston, V., & Pongor, S. (2005). The efficiency of multi-target multi-targeting. Henceforth, in the present study, we have tested the activity of drugs: the network approach might help drug design. Trends in pharmacological some known antifungals against their targets and also computed their efficiency sciences, 26(4), 178-182.doi: https://doi.org/10.1016/j.tips.2005.02.007 to be used for multi-targeting. Tang, J., Karhinen, L., Xu, T., Szwajda, A., Yadav, B., Wennerberg, K., & Argifin, a natural compound was reported to have a docking score of -7.557 Aittokallio, T. (2013). Target inhibition networks: predicting selective Kj/mol against chitinase computed through the Glide software by Roy and combinations of druggable targets to block cancer survival pathways. PLoS Mukherjee (2015) which has a similar trend (with respect to negative binding computational biology, 9(9), e1003226.doi: energy) to the data reported here which is -28.59 Kcal/mol. The negative binding https://doi.org/10.1371/journal.pcbi.1003226 energy shows that the reaction is spontaneous and the complex formed is stable. Hartl, L., Zach, S., & Seidl-Seiboth, V. (2012). Fungal chitinases: diversity, In another study, the Glide score, of Allosamidine against fungal chitinase was mechanistic properties and biotechnological potential. Applied microbiology and found to be -9.94 by Jeyam et al., (2014). This is also supported by present biotechnology, 93(2), 533-543.doi: https://doi.org/10.1007/s00253-011-3723-3 study where we got the binding energy value as -29.99. There is no report on the Henar, M. V., Durán, A., & Roncero, C. (1999). Chitin synthases in yeast and determination of binding energy of Methylxanthin on fungal chitinase but fungi. Exs, 87, 55-69.doi: 10.1007/978-3-0348-8757-1_4 according to a study by Tsirilakis et al. (2012), Cryptococcus neoformans and Liu, J., & Balasubramanian, M. K. (2001). 1, 3-beta-Glucan synthase: a useful Aspergillus fumigatus have shown abnormal cell morphology on treatment with target for antifungal drugs. Current Drug Targets-Infectious Disorders, 1(2), 159- methylxanthin at a concentration between 0.5–10 mM. Schüttelkopf et al (2010) 169.doi: https://doi.org/10.2174/1568005014606107 reported that although Acetozolamide and its analogues are weak inhibitors of Becher, R., & Wirsel, S. G. (2012). Fungal cytochrome P450 sterol 14α- fungal chitinase yet it has high ligand efficiency and can be used as a therapeutic demethylase (CYP51) and azole resistance in plant and human pathogens. agent in the future. Similarly, Amanda et al., (2012) has also reported that Applied microbiology and biotechnology, 95(4), 825-840. doi: Acetozolamide affects the cell wall of Blastomyces dermatidis but could not https://doi.org/10.1007/s00253-012-4195-9 observe any change in its growth curve. These results are in unanimity with the Hwang, E. I., Kwon, B. M., Lee, S. H., Kim, N. R., Kang, T. H., Kim, Y. T., et results of the current study that Acetozolamide has a binding energy that is al. (2002). Obovatols, new chitin synthase 2 inhibitors of Saccharomyces comparatively lower than the binding energy it has against 1,3-beta-glucan cerevisiae from Magnolia obovata. Journal of Antimicrobial Chemotherapy, synthase and lanosterol 14 α-demethylase. 49(1), 95-101. doi: https://doi.org/10.1093/jac/49.1.95 Not much of the in-silico work has been published about chitin synthase. Aoki, Y., Yoshihara, F., Kondoh, M., Nakamura, Y., Nakayama, N., & Arisawa, Obovatol has been found to have a good activity as a chitin synthase 2 inhibitor M. (1993). Ro 09-1470 is a selective inhibitor of P-450 lanosterol C-14 against Saccharomyces cerevisiae (Hwang et al., 2002). In another study, demethylase of fungi. Antimicrobial agents and chemotherapy, 37(12), 2662- Nikkomycin has been shown to affect chitin synthase 1 and 3 but no effect was 2667. doi: http://10.1128/AAC.37.12.2662 found on chitin synthase 2 in case of Saccharomyces cerevisiae, however, in case Mashiach, E., Schneidman-Duhovny, D., Andrusier, N., Nussinov, R., & of Candida albicans, Nikkomycin has shown to inhibit all the three isozymes of Wolfson, H. J. (2008). FireDock: a web server for fast interaction refinement in chitin synthase (Kim et al., 2002). Studies conducted on the structure-activity molecular docking. Nucleic acids research, 36(suppl_2), W229-W232. doi: relationship (SAR) of polyoxins against chitin synthase suggests that Polyoxin L, https://doi.org/10.1093/nar/gkn186 has a weak inhibitory effect on chitin synthase most probable reason being Neelabh, Jeswara, K., Kumari, A., Singh, K. (2015) In-silico designing of NKK: absence of thymine which is assumed to be important for bioactivity (Li et al., A better ligand than Acyclovir against Herpes Simplex Virus. Indian J. Pharm 3: 2012). 48-55.doi not available Papulacandins (A-E), contain benzannulated spiroketal unit which is the active Neelabh & Singh, K. (2018) From Natural products to therapeutically important part of many biological compounds. These antifungals have been reportedly antifungals. Curent Trends in Biotechnology and Pharmacy, 12(2), 206-212. doi showing high specific activity against yeasts, but have shown inertness against not available filamentous fungi, bacteria and protozoa (Traxler et al., 1977). In this study, Neelabh, Tirkey, N.N, Singh, K. (2018) In-silico and in-vitro studies on fungal Firedock was used to determine the in-silico activity of Caspofungin against 1,3- chitinase as a target enzyme for antifungal activity of closantel. Journal of beta-glucan synthase and unlike the report of Jeyam et al., (2014) (-1.68 Microbiology, Biotechnology and Food Sciences, 7:459-453. doi: Kcal/mol), it was found to be -60.65 Kcal/mol. 10.15414/jmbfs.2018.7.5.449-453 Similarly, Ro-09-1470 showed high activity against Candida glabrata, Roy, D., & Mukherjee, K. (2015). Homology modeling and docking studies of Cryptococcus neoformans, Rhodotorula rubra, Trichosporon cutaneum, human chitotriosidase with its natural inhibitors. Journal of Proteins & Aureobasidium pullulans, Exophiala dermatitidis and Exophiala jeanselmei and Proteomics, 6(2).doi not available relatively less activity against Candida albicans, Candida tropicalis, Jeyam, M., Arangaraj, M., Ravikumar, P., & Shalini, G. (2014). Computational Saccharomyces cerevisiae (Aoki et al, 1992). It is also known to interact with analysis of phytocompounds with 1, 3-β-D-Glucan synthase for lanosterol 14 α-demethylase. The data presented in this manuscript supports the antidermatophytic activity. doi: 10.7324/JAPS.2014.40211 fact as the in-silico binding energy obtained is on the higher side i.e. -51.71 Tsirilakis, K., Kim, C., Vicencio, A. G., Andrade, C., Casadevall, A., & Kcal/mol. Vembu et al. (2014) has obtained a binding energy value -33.34 by Goldman, D. L. (2012). Methylxanthine inhibit fungal chitinases and exhibit performing docking of Fluconazole against lanosterol 14 α-demethylase by using antifungal activity. Mycopathologia, 173(2-3), 83-91. Glide software. Likewise, in the present study we have obtained the binding doi:https://doi.org/10.1007/s11046-011-9483-x energy value to be -41.86 Kcal/mol.
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Schüttelkopf, A. W., Gros, L., Blair, D. E., Frearson, J. A., van Aalten, D. M., & Gilbert, I. H. (2010). Acetazolamide-based fungal chitinase inhibitors. Bioorganic & medicinal chemistry, 18(23), 8334-8340. doi: https://doi.org/10.1016/j.bmc.2010.09.062 Searle, A. J., Winston, V., & Scalarone, G. M. (2012). Blastomyces dermatitidis: Chitinase Homology Model, in Silico Docking, and Inhibition Assay. Open Journal of Medical Microbiology, 2(1), 1. doi: http://dx.doi.org/10.4236/ojmm.2012.21001 Kim, M. K., Park, H. S., Kim, C. H., Park, H. M., & Choi, W. (2002). Inhibitory effect of nikkomycin Z on chitin synthases in Candida albicans. Yeast, 19(4), 341-349. doi: https://doi.org/10.1002/yea.837 Li, J., Li, L., Feng, C., Chen, Y., & Tan, H. (2012). Novel polyoxins generated by heterologously expressing polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes. Microbial cell factories, 11(1), 135. doi: https://doi.org/10.1186/1475-2859-11-135 Traxler, P., Gruner, J., & Auden, J. A. L. (1977). Papulacandins, a new family of antibiotics with antifungal activity. The Journal of antibiotics, 30(4), 289- 296.doi: https://doi.org/10.7164/antibiotics.30.289 Aoki, Y., Yamazaki, T., Kondoh, M., Sudoh, Y., Nakayama, N., Sekine, Y. & Arisawa, M. (1992). A new series of natural antifungals that inhibit P450 lanosterol C-14 demethylase. The Journal of antibiotics, 45(2), 160-170.doi: https://doi.org/10.7164/antibiotics.45.160 Vembu, S., Parasuraman, P., & Gopalakrishnan, M. (2014). Design, in silico molecular docking studies, synthesis, spectral characterization and in vitro antifungal evaluation of 1-(4-(1H-tetrazole-1-yl) phenyl)-3-arylprop-2-en-1-ones. Der Phar
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IN-VITRO ANTIBACTERIAL, PHYTOCHEMICAL, ANTIMYCOBACTERIAL ACTIVITIES AND GC-MS ANALYSES OF Bidens pilosa LEAF EXTRACT
Christiana Ajanaku*1, Johnbull Echeme2, Raphael Mordi1, Oladotun Bolade1, Stella Okoye1, Hassana Jonathan1, Oluwaseun Ejilude3
Address(es): 1Department of Chemistry, Covenant University, P.M.B. 1023, Ota, Ogun State, Nigeria. 2Department of Chemistry, Michael Okpara University of Agriculture, Umudike, Owerri, Imo State, Nigeria. 3Department of Medical Microbiology and Parasitology, Sacred Heart Hospitals, Lantoro, Abeokuta, Ogun State, Nigeria.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.1.721-725
ARTICLE INFO ABSTRACT
Received 29. 5. 2018 The phytochemical constituents, antimicrobial activity, anti-mycobacterial activity and gas chromatography-mass spectrometry (GC- Revised 3. 7. 2018 MS) analysis of the West African ecotype of Bidens pilosa was investigated for possible medicinal properties. The antimicrobial activity Accepted 3. 7. 2018 of the hexane, dichloromethane, ethyl acetate and methanol extracts from the leaf of Bidens pilosa was evaluated using agar dilution Published 1. 8. 2018 method. The qualitative and quantitative phytochemical screening was carried out according to standard procedures. Partitioned fractions of the methanolic extract was subjected to anti-mycobacterial bioassay. Different fractions of the leaf were subjected to GC- MS to ascertain the compounds present. The antimicrobial analysis revealed the methanolic fraction as having the highest number of Regular article activity against test organisms such as: Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans and Rhizopus sp. between 10 – 40 mm. The minimum inhibitory concentration showed the methanolic fraction to be active against Candida albicans and Rhizopus sp. at the concentration of 6.25 g/ml and 3.25 g/ml respectively. The phytochemical screening revealed the presence of alkaloids, cardiac glycosides and terpenoids in all the solvents. Tannin was present in all the solvent fractions except hexane fraction. Saponin was not found in any of the solvents. The hexane-methanol interface of the partitioned solvents was sensitive to the anti-mycobacterial activity while other solvents showed resistance. The GC-MS and the chromatogram gave insight into the volatile components of the leaf extract. The findings reveals Bidens pilosa as a medicinal plant with potentials for the treatment of tuberculosis.
Keywords: Phytochemicals, Bidens pilosa, antimicrobial, anti-mycobacterial, medicinal plants, infectious diseases, Tuberculosis
INTRODUCTION originated from South America, it is distributed widely in the subtropical and tropical regions. The Bidens pilosa is commonly called grab-a-leg, Spanish The use of alternative medicine, by developing countries is being fuelled by the needles, cobbler’s pegs, sticky beak, pitchforks, hairy tick weed, broomstick, concern of adverse effects of chemical drugs (WHO, 2002). Human health blackjack, demon spike grass or ghost needle weed (Bartolome et al., 2013). It is preservation greatly depends on the use of plants; which serves as sources of food a slender, yearly, erect, branching herb that grows up to 1.5 m average height and and medicines (Hamilton, 2004). It is widely reported that about 80% of the 2.0 m in good condition. It grows around maize, vegetables, pasture, sorghum, world population rely mostly on herbal medicines to meet their basic health needs coffee, cassava, cotton, coconut, tea, citrus, rubber, oil palm, papaya, tobacco and (Sofowora, 1996). The medicinal application of natural products has been found rice as a weed (Connelly, 2009). Studies have shown that Bidens pilosa is used useful since natural product has been discovered to have therapeutic and healing in traditional medicine, the herbal plant have numerous health benefits used in properties. Among the Nigerian natives, the ethno pharmacological use of plants the treatments of colds, flu, fever, wounds, jaundice, neuralgia, small pox, prevails. Some of the ailments which plants have been used to treat include: hepatitis, glandular sclerosis, snake bite, anaemia, colic, diuretic, conjunctivitis malaria, diarrhoea, burns, gonorrhoea, stomach disorders and other infectious and diarrhoea (Bartolome et al., 2013). Biochemical activities of the plant genus diseases (Aibinu et al., 2007). The use of fruits, for instance, soursop fruit was are directly related to the presence of secondary metabolites in the plants. been found to be effective in the destruction of cancer and is a thousand times (Adebayo et al, 2017). The focus of this paper was to investigate the more effective than chemotherapy. Other fruits include the use of garlic, onions, phytochemical, antibacterial, anti-mycobacterial and GC-MS analysis of the leaf turmeric, carrots, apples among others (Cooper, 2015). Current global trends in of Bidens pilosa plant which belongs to the family Asteraceae. drug synthesis have shown the increasing significance of plants in medicine. The potency of these plants is as a result of the presence of active components it MATERIALS AND METHODS contains. These compounds have specific roles it carries out on definite sites within the body system (Ijeh et al, 2004). The effect of drugs on the Collection of Bidens pilosa leaf physiological system is specific in action and dependent on the presence of bioactive molecules that are of plant origin (Nweze et al,. 2004). Such bioactive The leaf of Bidens pilosa free from infection was collected from Iyana Iyesi compounds sourced from plants contain antimicrobial and antioxidant properties. village of Ota, in Ogun state. The Local Government Area is Ado-odo Ota, is Infectious diseases like tuberculosis is caused by Mycobacterium tuberculosis located at 6o41′ N 3o41′ E to the north of the area. Plant samples was (Kumar et al., 2007). The development of multi-drug resistance has constituted a authenticated at the herbarium section of the Forest Research Institute of Nigeria growing threat to global TB program. Researchers have gone into solving with voucher No FHI 110016. The leaf part of the plant was used to prepare the problems of resistance to specific drugs such as pyrazinamide, isoniazid and PA- extract. The plant collected was rinsed with water to remove the dirt particles and 824. This has resulted in need for more drugs to combat multi-drug resistance to air dried. The dried leaf was hand pulverised, grinded to fine powder and stored tuberculosis (Olugbuyiro et al., 2009). till further use. Bidens pilosa is often considered as a weed plant and can be found in Japan, China, America and Africa. Although, it is commonly believed to have
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Extraction of Bidens pilosa leaf Test for Phenols
The fine powder of the Bidens pilosa leaf were dried and was extracted with To 1mL of the extract, 2 mL of distilled water followed by few drops of 10% different solvents of hexane, ethyl acetate, dichloromethane and methanol by cold ferric chloride was added. Formation of green colour indicated the presence of extraction at an average of 29 oC room temperature for 72 hours. The filtered phenols. portions were concentrated using a rotary evaporator and stored for further analysis. Test for Coumarins
Phytochemical screening of Bidens pilosa leaf To 1 mL of extract, 1ml of 10% Sodium hydroxide was added. Formation of yellow colour indicated the presence of coumarins. Phytochemical determination for the presence of reducing sugars, anthraquinones, terpenoids, saponins, alkaloids, oils and fats, flavonoids, tannins, Test for steroids and cardiac glycosides were carried out on all the extracts using standard qualitative methods as described by Sofowora (1996). The qualitative To 2 mL of the extract 5 mL of chloroform was added and filtered, 2 mL of phytochemical screening was carried out on each of the extracts obtained from acetic anhydride was added to 2 mL of filtrate with 2 mL of sulphuric acid. The hexane, ethyl acetate, dichloromethane and methanol solutions. colour changes from violet to blue or green this indicates the presence of steroids.
Test for Carbohydrates Test for Acids
To 2 mL of plant extracts, 1 mL of Molisch’s reagent and few drops of 1 mL of extract was treated with sodium bicarbonate solution. Formation of concentrated sulphuric acid was added. Purple colour formation indicated the effervescence indicates the presence of acids. presence of carbohydrates. Antibacterial activity bioassay of Bidens pilosa leaf Test for Tannins The extracts were evaluated for antibacterial activity against Bacillus spp, To 1 mL of plant extract, 2 mL of 5% ferric chloride was added. Formation of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus greenish black indicated the presence of tannins. varians, Serratia spp, and Aspergillus niger, using Sabouraud dextrose agar as the medium. Test for Saponins Determination of Minimum Inhibitory Concentration (MIC) To 2 mL of plant extract, 2 mL of distilled water was added and shaken in a graduated cylinder for 15 minutes lengthwise. Formation of 1cm layer of foam Minimum inhibitory concentration was carried out on the leaf of Bidens pilosa indicated the presence of saponins. using the method described by Mahesh and Satish (2008). The procedure, using nutrient agar as medium was carried out on the different fractions of B. pilosa Test for Flavonoids leaf that showed sensitivity against the growth of some selected organisms. These fractions were adjusted to 50, 25, 12.5, 6.25, 3.125 and 1.562 mg/ml 5 mL of dilute ammonia solution was added to 1 mL of the aqueous filtrate of concentrations by serial dilution method. The sterile nutrient agar plates were plant extract followed by addition of concentrated sulphuric acid. Appearance of seeded using swab sticks with the test organisms or isolates of 0.5% McFarland yellow coloration indicated the presence of flavonoids. standard. Sterile cork borer was used to bore wells of about 9 mm in diameter into the sterile nutrient agar plates. Sterile pipette of 1 mL were used to measure Test for Alkaloids 0.2 mL of each extract of different concentrations into the bored wells on the inoculated nutrient agar plates. The plates were observed for growth and death of To 2 mL of plant extract, 2ml of concentrated hydrochloric acid was added. Then test organisms after incubation at 37ºC for 24 hours. Gentamycin, with a few drops of Mayer’s reagent was added. Presence of green colour indicated the concentration of 10 µg was used as the standard antibiotic. presence of alkaloids. Anti-mycobacterial susceptibility test Test for Anthocyanin and Betacyanin The methanolic crude extract of the leaf part was partitioned into different To 2 mL of plant extract, 1 mL of 2M sodium hydroxide was added and heated solvents using: aqueous, chloroform, methanol and hexane. Each of the solvent for 5minutes at 100ºC. Formation of yellow colour indicated the presence of fractions were subjected to bio-assay against mycobacterial activity. Anti- betacyanin. mycobacterial susceptibility test was performed by proportion method as described by FMOH (2009). The Mycobacterium tuberculosis isolates (drug Test for Quinones susceptible and drug resistant) were tested against the partitioned fractions, rifampicin and levofloxacin (Sigma scientific laboratories USA). To 1 mL of extract, 1 mL of concentrated sulphuric acid was added. Formation of The proportion method was carried out from a subculture on Lowenstein-Jensen red colour indicated the presence of quinones. (L-J) medium. A representative sample of 5.0 mg to 10.0 mg from the sub-culture was obtained within 1 to 2 weeks after appearance of growth using a calibrated Test for Glycosides inoculating loop. Samples were placed into a sterile McCartney bottle (14 mL screw capped bottle) containing 1.0 mL distilled water and 10 glass beads. The To 2 mL of plant extract, 3 mL of chloroform and 10% ammonia solution was mixture was homogenized on a vortex mixer for 1 minute and the opacity of the added. Pink colour formation indicated the presence of glycosides. suspension was adjusted by the addition of sterile distilled water to a standard suspension containing 1 mg/mL of Mycobacterium tuberculosis isolates. Test for Cardiac glycosides Two serial dilutions were made from the suspension 10-2 mg/mL and 10-4 mg/mL using the calibrated inoculating loop and sterile McCartney vials containing 1.0 To 0.5 mL of extract, 2 mL of glacial acetic acid and few drops of 5% ferric mL of distilled water. 0.1 mL of 10-2 and 10-4 suspensions were inoculated onto 2 chloride were added. 1 mL of concentrated sulphuric acid was added. Brown ring slants of drug/extract free (control) medium. The suspensions were spread over formation at the interface indicated the presence of cardiac glycosides. the surface of the medium and kept at a slanting position with loosen caps. The seeded media were examined for contamination after 1 week .The slants were Test for Terpenoids incubated at 37oC . When the growth appeared on the control medium, the cap to the vial was tightened and incubation continued for 4 weeks. To 0.5 mL of extract, 2ml of chloroform was added and concentrated sulphuric When enough growth, more than 100 colonies for 10-2 suspension and more than acid was added carefully. Red brown colour formation at the interface indicated 50 colonies for 10-4 suspension, was observed on the drug/extract free medium at the presence of terpenoids. 4 weeks of incubation, the growth on all media was read. For strains showing drug susceptibility at 4 weeks, further reading was taken at 6 weeks. Quality Test for Triterpenoids control strains-H37RV was included in each batch of testing. The first reading of antimycobacterial susceptibility test result was done at 4 To 1.5 mL of extract, 1 mL of Libermann–Buchard Reagent (acetic anhydride + weeks (28 days) of incubation at 37oC. The growth on the drug/extract containing concentrated sulphuric acid) was added. Blue green colour formation indicated medium was compared with the growth on the drug/extract free medium at 10-4 the presence of triterpenoids. dilution. When the growth on the drug/extract containing medium was none or
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less than that of a drug/extract free medium at 10-4 dilution, the drug/extract was Table 1 Phytochemicals present in leaf fraction of Bidens pilosa with their classified as susceptible/sensitive. intensities The following formula was used to calculate the percent resistant: Intensity Ethyl Hexane Dichloromethane Methanol Number of colonies on drug containing medium Constituents Acetate %Resistance = Fraction Fraction Fraction Number of colonies on the drug free medium at 10−4dilution Fraction Carbohydrates +++ - - +++ The criteria for resistance is 1% of growth for all the drugs/extracts. No growth Tannins - +++ +++ +++ or less than 1% of colonies growing compared to the controls (Fujiki, 2001). Saponins - - - - Flavonoids +++ - - +++ Quantitative phytochemical evaluation Alkaloids +++ +++ +++ ++ Anthocyanin +++ - - - The total flavonoid in the Bidens pilosa leaf extracts was determined using the & Betacyanin aluminium chloride colorimetric assay in accordance with the method of Kalita Quinones +++ - - ++ et al., (2013) while the tannin and phenol contents determination was carried out Glycosides - - - +++ using Folin-ciocalteu’s spectrophotometric method of Nkafamiya (2006) and Cardiac +++ +++ +++ +++ Dewanto (2002) respectively. The alkaloid percentage was then estimated using Glycoside the Ladan et al., (2014) method while the total steroid content was measured Terpenoids +++ ++ +++ +++ spectrophotometrically by Lieberman-Burchard method of Sathishkumar and Triterpenoids +++ +++ +++ - Baskar (2014). Phenols - ++ +++ - Coumarins +++ ++ - - GC-MS analysis of Bidens pilosa leaf Steroids +++ +++ +++ - Acids - - - - GC-MS analysis of the leaf of Bidens pilosa extract was analysed using the Key: +++ = Intense; ++ = moderate; + = trace; - = not present equipment Agilent 7890A Gas Chromatography-Mass spectrometry system with Mass hunter acquisition software. The equipment has a HP-5MS ultra inert Phenols are present in the dichloromethane and ethyl acetate fractions but absent capillary non-polar column with dimensions of 30 mm × 0.25 mm ID × 0.25 μm in hexane and methanol fractions. Also, the hexane and dichloromethane film. The carrier gas used is Helium with at flow of 1.0 ml/min. The injector was fractions contained the coumarins, which is absent in ethyl acetate and methanol. operated at 250 °C and the oven temperature was programmed as follows: 50 °C Steroids were present in all the solvents except the methanol fraction. Glycosides for 5 min, then gradually increased to 250 °C at 100C/min, and finally to 3000C at were found present only in the methanolic fraction. The results of the 7 0C/min for 10 min. The identification of components was determined by phytochemicals is in line with previous studies made by Bartolome et al., 2013 comparison with the NIST library data while the percentage composition was and Yang, 2014. computed from GC peak areas. Table 2 Quantitative Phytochemical Analysis of Bidens pilosa leaf extract RESULTS AND DISCUSSION Total Total Total Total Total Fractions Alkaloid Flavonoid Phenol Tannin Steroids The results of phytochemical screening of the B. pilosa leaf extract indicated the (%) (%) (%) (%) (%) appearance of the following secondary metabolites: carbohydrates, alkaloids, Hexane 0.80 0.23 - - 0.14 flavonoids, phenolic compounds, anthocyanins, quinones, terpenoids, DCM 0.92 - 0.26 0.78 - triterpenoids, steroids and cardiac glycosides (Table 1). Particularly, hexane, Ethyl - - 0.33 0.70 1.31 dichloromethane, ethyl acetate and methanol extracts of B. pilosa were good acetate sources of different classes of compounds. Alkaloids, cardiac glycosides and Methanol 1.12 0.35 - 0.61 - terpenoids were present in all the solvent extracts. This could be a useful insight in the possible compounds that could be isolated from the plant material. It is also The quantity of the most common phytochemicals present in the leaf of B. pilosa observed that in all the solvent fractions, saponin was absent. Tannin was present is presented in Table 2. It was observed that steroids were more abundant in the in all the solvent fractions except in hexane fraction. Steroids and Triterpenoids ethyl acetate fraction while the alkaloids were present in all the fractions except are present in all the solvents except in methanolic fraction. Carbohydrates, in ethyl acetate. Tannins were moderately present in all fractions except for flavonoids and quinones were present in the hexane and methanolic fractions but Hexane fraction. Phenols were present in small amounts in dichloromethane and were absent in dichloromethane and ethyl acetate fractions. Anthocyanin and ethyl acetate. Flavonoids were present in small amounts in hexane and methanol. betacyanins, quinones were found only in the hexane solvent.
Table 3 Zones of inhibition (in mm) of leaf fractions of Bidens pilosa against selected microorganisms Zone of inhibition in millimetres for the BP leaf Organism Hexane Dichloromethane Ethyl acetate Methanol Control fraction Fraction Fraction Fraction (gentamycin) Bacillus subtilis 10 - 15 11 15 Escherichia coli - - - 15 - Klebsiella pneumoniae - - - 10 16 Pseudomonas aeruginosa - - 14 16 10 Staphylococcus aureus - - - - 8 Candida albicans 10 20 - 40 - Rhizopus sp. - - 11 10 -
Table 4 Minimum inhibitory concentration of Bidens pilosa leaf fractions against against Bacillus subtilis and Candida albicans; while the dichloromethane selected microorganisms (mg/ml) fraction showed inhibition at 20 mm against Candida albicans only. It can be Dichloromethane deduced that methanolic fraction of the BP leaf is more active against the test Organism Methanol Fraction Fraction organisms and hence can inhibit their growth. In view of the result obtained from the zones of inhibition further processes to situate the minimum inhibitory Candida concentration was carried out and the result is shown in Table 4. The MIC was - - - 6.25 - - - - - 3.125 albicans found at 3.125 against Candida albicans, while the methanolic fraction also had Rhizopus - - - - 3.125 - - - - - 3.125 concentration against Rhizopus sp. This implies that the BP leaf has both anti-bacterial and anti-fungi properties. For the antimicrobial analysis, Table 3 showed the zones of inhibition against The modern practice being used presently in identifying the active components various test organisms. The methanolic fraction showing the highest number of present in crude extracts is bioassay-guided fractionation. activity against test organisms which were 11 mm, 40 mm, 15 mm, 10 mm, 16 The result in Table 5 revealed that the hexane/methanol fraction exhibited good mm, and 10 mm against Bacillus subtilis, Candida albicans, Escherichia coli, anti-TB activity, (MIC 6.25mg/ml), while other fractions were resistant. This Klebsiella pneumoniae, Pseudomonas aeruginosa, and Rhizopus sp. respectively. result gives a very useful insight to the fraction that could be used in extracting This was followed by the ethyl acetate fraction showing zones of inhibition at 11 active ingredients from the leaf of Bidens pilosa in the treatment of tuberculosis. mm, 14 mm and 15 mm against Rhizopus sp., Pseudomonas aeruginosa and Bacillus subtilis respectively. At 10 mm, the hexane fraction showed inhibition
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Table 5 Presence of resistant and sensitive strains from BP leaf extract against Mycobacterium tuberculosis isolate during seven weeks DS-MTB DS-MTB DS-MTB DS-MTB DS-MTB DR-MTB DR-MTB H37Rv BP leaf extract 1 2 3 4 5 1 2
Crude extract R R R R R R R R Aqueous Fraction R R R R R R R R Interface of Hexane/Methanol S S S S S S S S Chloroform fraction R R R R R R R R Methanolic fraction R R R R R R R R Hexane fraction R R S R R R R S Control drugs: susceptible – Rifampicin (40 mg); resistant- Levofloxacin (200 mg) Keys: DS-MTB – Drug susceptible Mycobacterium tuberculosis; DR-MTB – Drug resistant Mycobacterium tuberculosis; H37Rv = Mycobacterium tuberculosis strain; S = Sensitive; R = Resistant.
The identified compounds using GC-MS analysis with their retention time (RT), antioxidant, hypocholesterolemic, nematicide, pesticide, antiandrogenic, flavour, molecular formula, molecular weight and abundance are presented in Table 6. hemolytic and 5-Alpha reductase inhibitor properties (Mujeeb et al., 2014). The results of the GC-MS analyses of the crude extracts are shown in Figure 1. 9,12,15-Octadecatrienoic acid methyl ester(Z,Z,Z)-, has hepatoprotective, Identification of these compounds was confirmed by comparing the mass spectra nematicide, insectifuge antihistaminic, antiarthritic, anticoronary, antieczemic obtained with literature mass spectra where available. The result indicated the antiacne, 5-alpha reductase inhibitor antiandrogenic properties (Bartolome et al., presence of the volatile components in the methanolic extract of Bidens pilosa 2013). It was also observed that Tetradecanal, is an aldehyde with antifungal, leaf: cis-9-Hexadecenal, 9-Oxabicyclo[6.1.0]nonane, Hexadecanoic acid methyl antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricative ester, Dodecanoic acid 10-methyl-methyl ester, 10,13-Octadecadienoic acid properties (Mujeeb et al., 2014) while 13-Octadecenal (Z), is an aldehyde and methyl ester, 9,12,15-Octadecatrienoic acid methyl ester (Z,Z,Z)-, Tetradecanal, Oleic acid, both exhibit antibacterial properties (Duke, 1992; Bartolome et al., 2-Methyl-Z,Z-3,13-octadecadienol, 13-Octadecenal (Z)- and Oleic acid. It is of 2013 ). The bioactive compounds identified in the leaf part of the plant justifies interest to note that the compounds identified from the leaf extract have some its ethnobotanical claims. form of biological activity. For instance, cis-9-Hexadecenal, is an aldehyde and has antimicrobial property while Hexadecanoic acid methyl ester, has
Table 6 GC-MS analysis of methanolic extract of Bidens pilosa leaf Molecular weight S.No. Ret.Time Compound Molecular formula Abundance (%) (g/mol)
1 1.196 cis-9-Hexadecenal 238.415 C16H30O 95
2 1.196 9-Oxabicyclo[6.1.0] nonane 126.199 C8 H14 O 92
3 24.261 Hexadecanoic acid, methyl ester 270.457 C17H34O2 96
4 24.261 Dodecanoic acid 201.314 C12H24O2 90
5 26.160 10,13-Octadecadienoic acid, methyl ester 294.47 C19H34O2 99
6 26.252 9,12,15-Octadecatrienoic acid, methyl ester(Z,Z,Z)- 292.4562 C19H32O2 99
7 33.462 Tetradecanal 212.377 C14H28O 93
8 34.612 2-Methyl-Z,Z-3,13-octadecadienol 280.496 C19 H36 O 95
9 37.456 13-Octadecenal, (Z)- 266.469 C18H34O 90
10 43.063 Oleic Acid 282.47 C18H34O2 91
Figure 1 Gas Chromatogram of methanolic extract of Bidens pilosa leaf
CONCLUSION terpenoids, cardiac glycosides and steroids. This confirms previous studies carried out on B. pilosa by Silva et al, 2011. The alkaloids present in the leaf is The potentiality of Bidens pilosa leaf as enhancement formulation in the an indication that the leaf could be used as an analgesics and anti-malaria. treatment of tuberculosis has been explored in this study. The results obtained Tannins was established to be present in the BP leaf which makes the leaf potent from the phytochemical screening revealed the abundancy of alkaloids, tannins, as antioxidants, anti-fungal and anti-virus properties. Terpenoids, such as
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steroids, lupeol, oleanolic acid are known to possess sedative and anti-depressive SANCHEZ, J.G.B., KOUZNETSOV, V.V. 2010. Antimycobacterial effects, and this was found to be present in the leaf. The presence cardiac susceptibility testing methods for natural products research. Braz. J. Microbiol. glycosides is an indication of its usefulness in the treatment of heart failure. The 41(2): 270–277 doi: 10.1590/S1517-83822010000200001. GC-MS studies carried out on the methanolic extract of Bidens pilosa showed the SHAKERI, A., HAZERI, N., VLIZADEH, J., GHASEMI, A., TAVALLAEI, presence of bioactive compounds responsible for the potent activity of the plant. F.Z. 2012. Photochemical screening, antimicrobial and antioxidant activity of The plant serves as a potential source for drug formulation as a result of its Anabasis aphylla L. Extracts. Kragujevac Journal of Science, 34, 71-78. antimicrobial and anti-tubercular properties. SILVA, F.L., FISCHER, D.C., TAVARES, J.F., SILVA, M.S., DE-ATHAYDE- FILHO, P.F., BARBOSA-FILHO, J.M. 2011. Compilation of secondary Acknowledgements: The authors would like to gratefully acknowledge metabolites from Bidens pilosa L. Molecules, 16(2), 1070–1102. Covenant University for the grant support given for this study and also to the http://dx.doi.org/10.3390/molecules.16021070 staff of Multi-user laboratory, Ahmadu Bello University, Zaria for the use of SOFOWORA, A. 1996. Research in medicinal plants and traditional medicine in laboratory facilities. Africa. The Journal of Alternative and Complementary Medicine, 2(3), 365-372. https://doi.org/10.1089/acm.1996.2.365 REFERENCES WHO (World Health Organization). 2002. Traditional medicine strategy. 2002– 2005. Geneva: World Health Organization; 2002. ADEBAYO, A., BALADE, A., YAKUBU, O. 2017. Gas chromatography-mass YANG, W. 2014. Botanical, pharmacological, phytochemical, and toxicological spectrometry analysis of viburnum opulus (L) extract and its toxicity studies in aspects of the antidiabetic plant Bidens pilosa L. Evidence-Based Complementary rats. Asian Journal of pharmaceutical and clinical research, 10(6), 383-388. and Alternative Medicine Volume 2014, Article ID 698617, 14 pages. http://dx.doi.org10.22159/ajpcr.2017.v10i6.17350 http://dx.doi.org/10.1155/2014/698617 AIBINU, I., ADENIPEKUN, T., ADELOWOTAN, T., OGUNSANYA, T., ODUGBEMI, T. 2007. Evaluation of the Antimicrobial Properties of Different Parts of Citrus Aurantifolia (Lime Fruit) as Used Locally. African journal of traditional, complementary, and alternative medicines, 4(2), 185–190. BARTOLOME, A., VILLASENOR, M., YANG, W. 2013. Bidens pilosa L. (Asteraceae): Botanical Properties, Traditional Uses, Phytochemistry, and Pharmacology. Evidence-Based Complementary and Alternative Medicine, 2013, 1-51. http://dx.doi.org/10.1155/2013/340215 SATHISHKUMAR, T., BASKAR, S. 2014. Screening and quantification of phytochemicals in leaves and flowers of Tabernaemontana heyneana. Indian Journal of Natural Products and Resources, 237-243. CONNELLY, P. 2009. Horrible weed or miracle herb? A review of Bidens pilosa. Journal of the Australian Traditional-Medicines Society, 15(2), 77–79. COOPER, R. 2015. Natural product chemistry, sources, separations and structures. 6000 broken sound parkway NW, Suite 300 Boca raton: CRC press. DEWANTO, V., WU, X., ADOM, K.K., LIU R.H. 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem., 50(10), 3010-3014. DEBA, F., XUAN, T.D., YASUDA, M., TAWATA, S. 2008. Chemical composition and antioxidant, antibacterial and antifungal activities of the essential oils from Bidens pilosa Linn. var. Radiata. Food Control, 19(4): 346– 352, 2008. https://doi.org/10.1016/j.foodcont.2007.04.011 DUKE, J. 1992. Handbook of Biologically Active Phytochemicals and Thir Activities, CRC Press, 1992. FEDERAL MINISTRY OF HEALTH 2009. Acid fast direct smear microscopy, National Tuberculosis and Leprosy control programme, 9-15. FUJIKI, A. 2001. Culture examination for M. tuberculosis. In, TB Bacteriology Examination to stop TB. (ed). Pp 14-19. HAMILTON, A.C. 2004. Medicinal plants, conservation and livelihoods. Biodiversity and Conservation. 13(8), 1477-1517. https://doi.org/10.1023/B:BIOC.0000021333.23413.42 IJEH, I.I., NJOKU, O.U., EKENZE, E.C. 2004. Medicinal evaluation of Xylopia aethiopica and Ocimum gratissimum. J. Med. Aromatic Sci. 26(1), 44-47. KUMAR, V., ABUL, K., FAUSTO, N., MITCHELL, R. 2007. Robbins Basic Pathology (8th ed.). Saunders Elsevier. pp. 516-522. LADAN, Z., AMUPITAN, J.O., OYEWALE, O.A., AYO, R.G., TEMPLE, E., LADAN, E.O. 2014. Phytochemical screening of the leaf extracts of Hyptis spicigera plant. African journal of pure and applied chemistry, 8(5), 83-88. MAHESH, B., SATISH, S. 2008. Antimicrobial activity of some important medicinal plant against plant and human pathogens. World J. Agric. Sci., 4: 839- 843. MUJEEB, F., BAJPAI, P., PATHAK, N. 2014. Phytochemical evaluation, antimicrobial activity, and determination of bioactive components from leaves of Aegle marmelos. BioMed. Research International, vol. 2014, Article ID 497606, 11 pages, 2014. http://dx.doi.org/10.1155/2014/497606 NKAFAMIYA, I.I., MANJI, A.J. 2006. A study of cyanogenetic glucoside content of ome edible nuts and seeds. J.Chem.Soc.Niger, 31(1&2), 12-14. NWEZE, E.I., OKAFOR, J.I., NJOKU, O. 2004. Antimicrobial activities of methanolic extracts of Trema guinensis (Schumm and Thorn) and Morinda Lucida Benth used in Nigeria. Bio-Research 2(1), 39-46. OLUGBUYIRO, J.A.O., MOODY, J.O., HAMANN, M.T. 2009. Anti-Mtb activity of triterpenoid-rich fractions from Spondiasmombin L.. Afri J Biotechnol, 8(9), 1807-1809. KALITA, P., BARMAN, T.K., PAL, T.K., KALITA, R. 2013. Estimation of total flavonoids content (TFC) and anti-oxidant activities of methanolic whole plant extract of Biophytum sensitivum linn. Journal of Drug Delivery & Therapeutics, 3(4), 33-37. KUMAR, R.R., JADEJA, V.J. 2016. Endophytic Actinomycetes: A Novel Antibiotic Source. Int. J. Curr. Microbiol. App. Sci. 5(8), 164-175. http://dx.doi.org/10.20546/ijcmas.2016.508.018
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SEROLOGICAL AND MOLECULAR DETECTION OF VIRUSES INFECTING FIG TO IDENTIFY THE VIRUS-FREE PLANTS
Hemaid Ibrahim Ahemaidan Soliman
Address(es): Plant Genetic Resources Department, Desert Research Center, El-Matariya 11753, Cairo, Egypt.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.1.726-731
ARTICLE INFO ABSTRACT
Received 9. 3. 2018 Fig (Ficus carica L.) is one of the old and the most common fruit tree grown in all the Mediterranean countries, which is among those Revised 10. 6. 2018 exposed to diseases and pathogens. A recent investigation indicated that fig mosaic disease was the main pathogenic agent. In this Accepted 9. 7. 2018 study, field surveys were conducted in different areas of Egypt. A total of 300 leaf samples were collected randomly from symptomatic Published 1. 8. 2018 and symptomless fig trees of six cultivars, and tested by RT-PCR for the presence of Fig mosaic virus (FMV), Fig leaf mottle- associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FMMaV-2), Fig mild mottle-associated virus (FMMaV) and Fig latent virus 1 (FLV-1), using specific sets of primers. About 84% of the samples tested were found to be infected by at least one virus. Regular article FLMaV-1 was the prevailing virus with a (78.3%) incidence followed by FMV (73.7%), FLMaV-2 (50%), FLV-1(40.3%) and FMMaV (35%). The highest infection rate was observed in Sultani, Asuani and Adasi-Ahmer (100%), followed by Kahramani (80%), Kadota (64%) and Black Mission (60%).Ultra thin section for infected fig plants showed a large double-membrane particles (DMPs) 100-150 nm in diameter were located in the cytoplasm of parenchyma cells. Fig mosaic virus-specific antiserum raised in rabbits, proved useful for its detection in fig tissues. The plantlets from meristem tip tissue culture were transplanted successfully. FMV could be detected through DAS-ELISA in the virus-free fig plantlets.
Keywords: Ficus carica, fig mosaic disease, virus detection, RT-PCR, DAS-ELISA, electron microscopy
INTRODUCTION negative-sense, single-stranded RNA (Elbeaino et al., 2009a). The genome has long been thought to have four segments, but recently, discovered that FMV The fig (Ficus carcia L.) is one of the earliest cultivated fruit trees and its genome contains six RNA genome segments (Elbeaino et al., 2009b; Walia et cultivation has spread to many parts of the world, especially in the al., 2009; Ishikawa et al., 2012) and dsRNA isolated from FMD-affected fig Mediterranean, Middle East and naturalized in many places. Nowadays it is an trees had revealed a changing number of molecules of up to 7 kb in length important crop worldwide. Fig is a desert plant cultivated in Egypt especially (Acikgöz and Döken, 2003). Each segment has one open reading frame (ORF) north coast of the western desert which extends from the Alexandria to the Marsa (Ishikawa et al., 2013). The first segment, FMV vcRNA 1 (7093 nt), is common Matrouh. Lately, the plants showed a mosaic symptom associated with a great to all viruses of genus Emaravirus and codes for the virus’s 264 kDa RNA loss in the yield. The causal agent of the disease is associated with the high dependent RNA polymerase (RdRp). The second segment, vcRNA2, (2252 nt) population of mites and was identified as fig mosaic virus (Hemaid et al., 2010). encodes a 73 kDa putative glycoprotein. FMV vcRNA3 (1490 nt) encodes a 35 Fig mosaic has a wide distribution and has been reported as occurring in the kDa nucleocapsid protein. FMV vcRNA4 (1472 nt) encodes a 40.5 kDa protein following countries: United States, Turkey, England, Algeria, Tunisia, Syria, with function still unknown (Walia and Falk, 2012). The two most recently Spain, Italy, Jordan, New Zealand, Puerto Rico, Greece, Israel, China, and discovered segments, RNA5 (1752 nt) encodes a 59 kDa and RNA6 (1212 nt) Australia (Blodgett and Belgin, 1967). Fig mosaic disease (FMD) was first encodes a 22 kDa these proteins are the unknown function (Elbeaino et al., reported in California and symptoms include bright mosaic, spotting and 2012). All RNA segments possess untranslated regions containing at the 50 and distortion of leaves and mottling, reduction in the size of some fruits and 30 termini a13-nt complementary sequence (Ishikawa et al., 2012). occasional leaf malformation. Symptoms often appear clearly when the tree Ultrastructural studies have shown FM affected plants to have a characteristic becomes under heat or water-stresses (Condit and Horne, 1933). Fig mosaic is cytopathology. The cells of the symptomatic plants contain quasi-spherical, transmitted in nature by the eriophyid Aceria ficus which is transmitted from double-membrane-bound particles (DMPs) of 100 to 200 nm in diameter present plant to plant through wind (Flock and Wallace, 1955) and the virus may be in the cytoplasm (Martelli et al., 1993; Appiano et al., 1995). Infection with transmitted by vegetative propagation of infected cuttings from diseased trees FMV results in distinct double-membrane bodies or particles, called DMBs or and by budding or grafting of infected propagative buds or scions to healthy DMPs, 90-200 nm in diameter in the cytosol of infected parenchyma cells (Walia stocks (Blodgett and Belgin, 1967). Recent studies have reported FMD et al., 2009; Caglayan et al., 2009). Immunological assays such as enzyme- observation, in leaf tissues fig symptomatic of viral particles of different linked immunosorbent assay (ELISA) have been successfully applied for morphology (Serrano et al., 2004). Several viruses infecting fig trees have been detection of viruses in different organisms such as plants and insects Clark & reported Fig mosaic virus (Walia et al., 2009; Elbeaino et al., 2009a), Fig leaf Adams (1977). Two principle methods “direct” and “indirect” depends on mottle-associated virus 1 and 2 (Elbeaino et al., 2006; Elbeaino et al., 2007), antigen–antibody for ELISA test has been used in several virology studies. This Fig mild mottle-associated virus (Elbeaino et al., 2010), Fig latent virus 1 assay is visualized by means of a suitable detection system such as an enzyme- (Gattoni et al., 2009), Fig badnavirus-1 (Tzanetakis et al., 2010), Arkansas fig labeled antibody which reported by Van Regenmortel and Dubs 1993. The closteroviruses 1 and 2 (Elbeaino et al., 2011a), Fig fleck-associated virus most advantage for ELISA test is to test a high number of samples in short time (FFkaV) and Fig cryptic virus (Elbeaino et al., 2011b) but fig mosaic virus (Clark and Bar-Joseph 1984; Cooper and Edwards, 1986). (FMV) is the agent that occurs in symptomatic plants more often than any of the In this study we report a preliminary investigation carried out in some fig other fig-infecting RNA viruses, and is the major incitant of mosaic. Fig mosaic growing areas of Egypt to assess the presence of FMV, FLMaV-1, FLMaV-2, virus (FMV) is a member of the recently established genus Emaravirus (Adams FMMaV and FLV-1 using molecular tools (RT-PCR), to isolate and identified and Carstens, 2012). The FMV genome consists of segmented (multipartite)
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the FMV-infected plant tissues and used meristem culture to regenerate virus- Extraction of nucleic acids and RT-PCR free plants. Total RNAs were extracted from leaves of infected fig trees and healthy controls MATERIALS AND METHODS from in vitro plantlets using the RNeasy kit (Qiagen, Germany) following the manufacturer’s instructions. RNA aliquots of 50 ng were used as a template for Field survey and plant material RT- PCR. The detection of FMV, FLMaV-1, FLMaV-2, FMMaV and FLV-1 in RT-PCR was conducted using one set of specific primers (Table 1), whose To study the distribution of the disease, three hundred samples were collected nucleotides sequences and use conditions were previously described (Elbeaino et from naturally infected fig cultivars Asuani, Kadota, Black Mission, Kahramani, al., 2006; 2007; 2009; 2010; Gattoni et al., 2009). One-step RT-PCR protocol Sultani and Adasi-Ahmer. The chosen fifty samples from each cultivar of fig was used in a 25 µl reaction volume containing template, 2.5 µl of 10X PCR trees are located in Aswan, Kaliobeya, Monoufia, Giza, Sinai Governorates and buffer, 1.1 µl of 25 mM MgCl , 1.25 µl of 0.1 M DTT (dithiothreitol), 0.5 µl of the Northern West Coast, Egypt, which were selected randomly. Symptoms 10 mM dNTPs, 1.25 µl of specific primer set (10 µM), 0.25 µl of Taq varied from chlorotic blotches, vein clearing, vein banding, chlorosis, mosaic and polymerase(5 U/µl; Fermentas, Canada), and 0.035 µl of reverse transcriptase chlorotic ringspot, all suggestive of virus-like diseases. All of the samples were (200 units/µl; Fermentas). Reactions were performed at one cycle of 42°C for 30 assayed by RT-PCR to assess the presence of FMV, FLMaV-1, FLMaV-2, min; 35 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 1 min; and a final FMMaV, and FLV-1 in Egypt. 72°C extension for 10 min. PCR products were analyzed by electrophoresis in 1.5% agarose gels in TrisAcetate-EDTA buffer, stained with ethidium bromide and visualized under UV light.
Table 1 RT-PCR specific primers of five fig-infecting viruses (FMV, FLMaV-1, FLMaV-2,FMMaV and FLV-1) Amplicon Genus Virus (bp) Gene Primer sequence 5’-3’ Reference
FMV-s 5′-CGGTAGCAAATGGAATGAAA-3′ FMV Emaravirus 302 RdRp Elbeaino et al., 2009 FMV-a5′-AACACTGTTTTTGCGATTGG-3′ FLMaV1-s5′-CGTGGCTGATGCAAAGTTTA-3′ FLMaV-1 Closterovirus 352 HSP70 Elbeaino et al., 2006 FLMaV1-a 5′-GTTAACGCATGCTTCCATGA-3′ FLMaV2-s 5′-GAACAGTGCCTATCAGTTTGATTTG-3′ FLMaV-2 Ampelovirus 360 HSP70 Elbeaino et al., 2007 FLMaV2-a 5′-TCCCACCTCCTGCGAAGCTAGAGAA-3′ FMMaV-s 5′-AAGGGGAATCTACAAGGGTCG-3′ FMMaV Closterovirus 311 HSP70 Elbeaino et al., 2010 FMMaV-a 5′-TATTACGCGCTTGAGGATTGC-3′ CPtr1-s 5′-CCATCTTCACCACACAAATGTC-3′ FLV-1 Trichovirus 389 RdRp Gattoni et al., 2009 CPtr-a 5′-CAATCTTCTTGGCCTCCATAAG-3′ RdRp (RNA-dependent RNA polymerase) , HSP70 (Heat shock protein)
Cloning and sequencing followed by staining with reynolds lead citrate for 20 min. The grids were then washed several times with d.H2O and dried on a filter paper. The dried grids were Selected PCR products were ligated to pGEM-T Easy Vector (50 ng/μL) then examined under the electron microscopy (JEOL-CX100) in Electron following the manufacturer’s instructions (Promega), and then subcloned into Microscope Unit of Faculty of Science, Ain Shams University, Egypt. Escherichia coli DH5α cells. Plasmids were extracted from bacterial cells by the boiling method and further purified using polyethylene glycol as described in Negative staining Sambrook et al. (1989). Plasmids containing PCR amplicons of expected sizes were subjected to automated bidirectional sequencing using virus-specific sense Negative staining technique as described by Milne and Lesemann (1984). 15l and antisense primers (PRIMM). Nucleotide and protein sequence homologies of purified suspensions of infected fig leaves were adsorbed on gold coated grids were analyzed using the online BLASTn analysis software. for 2 min followed by staining with 2% uranyl acetate (w/v), and washed with d.H2O to remove the excess stain. The grids were left for 5 min to dry and then Electron microscopy examined by electron microscope.
Virus particle purification Production of virus free- fig plants
The method described by Jensen et al. (1996) with some modification was used Establishment of meristem tip culture for virus purification. 250 grams of leaves from mosaic-diseased fig trees were homogenized in 0.1M phosphate buffer pH 7.0 containing 0.5% 2- The shoot tip explants were collected from mature infected trees of fig (Ficus mercaptoethanol, 450 g bentonite per milliliter and 0.01 M EDTA (3-4 ml of carica L) cvs. Asuani, Kadota, Black Mission, Kahramani, Sultani and Adasi- buffer for each gm of material). The juice was stirred overnight with gentle Ahmer in Aswan Governorate, Giza Governorate, Kaliobeya Governorate, agitation, squeezed through two layers of cheesecloth, then clarified with 1/3 Monoufia Governorate, Northern West Coast and Sinai Governorate, Egypt, volume cold chloroform, and centrifuged 10 minutes at 8000 rpm in Sorvall GSA respectively. The shoot tips were sterilized 2.5% sodium hypochlorite for 20 rotor. The virus suspension was stirred for a minimum of 1 hour, centrifuged for minutes for surface sterilization and washed 4-5 times with sterile water. The 10 minutes at 1000 rpm in a Sorvall SA 600 rotor. The supernatant was apical meristems with two or three leaf primordial were obtained under laminar resuspended in 2 ml pad of 20% sucrose (w/v) in 1 x SSC buffer (0.15 M NaCl, air flow cabinet using a dissecting microscope. The explants were cultured on 0.015M Na citrate, pH 7.0) and ultracentrifuged for 3 hours at 50, 000 rpm in a MS medium supplemented with 0.05 mg L-1 NAA, 0.5 mg L-1 BAP and 0.5 mg beckman Ti 70.1 rotor and pellets were resuspended in 1 x SSC buffer pH 7.0 -1 was added to 1% Triton X-100 (v/v) with was stirred on ice for 30 minutes, L 2ip. After two months, numbers of shoots per plant were recorded, and the followed by a low-speed centrifugation at 8000 rpm for 10 minutes in an shoots were transferred on multiplication and rooting medium (Hemaid 2000; Eppendorf centrifuge 5415C. Sucrose gradients (10-40%, 2 ml/fraction) were Hemaid et al., 2010). All cultures were incubated in the growth chamber at prepared in the 1 x SSC buffer and one ml of the homogenized pellet was layered 26°C, under 3000 lux light and 8 hours dark and 16 h light photoperiod over the sucrose columns and centrifugation at 35,000 rpm for 12 hours in a conditions. The experiment was designed in a completely randomized Beckman SW 41 rotor. The columns were fractionated manually in Eppendorf experimental design with three replications and fifty explants included per tubes (1 mL/tube) and subjected to spectrophotometry at 260 and 280 nm. The replication. resulted suspension was examined by electron microscopy. Immunization and polyclonal antiserum production Leaf ultrastructure Antiserum was prepared by injection a new zealant white rabbit with purified Samples were excised from infected fig leaves. Samples were rinsed several preparation according to Lister et al. (1983). Purified virus preparations times in 4 % glutaraldehyde in 0.05M phosphate buffer (pH 7.2), and kept containing nucleoprotein mixed 1:1 with Freund’s incomplete adjuvant was o injected subcutaneously to a New Zealand white rabbit, followed by four overnight at 4 C. The specimens were post-fixed with 1% OsO in 0.05 M intramuscular injections over a period of four weeks. The FMV antiserum was phosphate buffer for 2 hrs at 4°C and then washed two times with distilled water obtained by bleeding the rabbit after 10 days of the last injection. The blood was each for 15 min. Samples were then dehydrated in a gradient acetone series, and incubated at 37C for 1 hr. followed by centrifugation at 14,000 rpm in GSA embedded in Spurr (1969) medium. The selected sections were stained with a mixture of 2% uranyl acetate and acetone (v/v) for 20 min. at room temperature,
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rotor at room temperature. The rough titre was determined by decorating virus Shahmirzaie et al., 2012; El Air et al., 2015; Amal et al., 2015). Also, the particles (Milne, 1993; Seifers et al., 1997) with progressive antiserum dilutions. incidence of FLMaV-2 was high (50%) than in other countries such as in Tunisia (4.3%), Italy (45%) and Lebanon (36.3%) (Elbeaino et al., 2007; Amal et al., IgG purification 2015). FMMaV was looked for in all the surveyed areas, its incidence was relatively lower (35%) than in other countries such as in Tunisia (60%) in cvs. The immunoglobulin G (IgG) fraction for the obtained antisera in this study was Takelsa and Sfax (El Air et al., 2015) and high than that of Turkey (2%) (Caglar separated according to the technique described by (Mckinney and Parkinson, et al., 2011). Finally, the incidence of FLV-1 was high (40.3%) than that reported 1987; Temponi et al., 1989; Perosa et al., 1990). The IgG fraction was purified in Syria (40%), Albania (17.5%) and other Mediterranean countries (29.4%) from rabbit sera with caprylic acid to precipitate albumin and other non-IgG (Elbeaino et al., 2009b) and in Tunisia (4.3%) (El Air et al., 2015). proteins. Then, the IgG fraction is precipitated with ammonium sulfate. Equal volume of 120 mM sodium acetate buffer (pH 4.0) was added to the rabbit serum placed on the magnetic stirrer. Caprylic acid was slowly added with continuous stirring for 30 min at room temperature. The tubes were centrifuged at 5000g for 10 min and the supernatant was saved. The supernatant was transferred to dialysis tubing and dialyzed versus PBS. The obtained IgG was further concentrated and purified by ammonium sulfate precipitation.
Direct double antibody sandwich (DAS-ELISA)
Direct double antibody sandwich (DAS-ELISA) demonstrated by Clark and Adams (1977) was used for detection of fig mosaic virus in plant tissue. Polyethylene microtitre plates were coated with the specific immunoglobin G (IgG) with 200 l/well diluted in coating buffer pH 9.6. Plates were washed three times, loaded with 200 µl per well of extracted samples and incubated overnight at 4°C. The wells were rewashed 3 times with PBS-tween 20, and 200 l of alkaline phosphatase conjugated antibodies were pipetted in each well and incubated at 37C for 4 hours, then finally washed and revealed with p- nitrophenylphosphate substrate. Absorbance values were measured at 405 nm by the Bio-rad ELISA reader model 3550 at 15 min. intervals. Figure 1 Agarose gel electrophoresis (1.5%) of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from infected fig plants. (FMV) Statistical analysis lanes 1-4 are PCR-positive results from infected plants, Lane 5 is the PCR-
negative from in vitro regenerated plants; (FLV-1 ) lanes 1-6 from infected plants The experiment was carried out based on complete randomized design. Each of and lane 7 from in vitro plant; (FLMaV-2) lanes 1-4 are PCR-positive results the experiments, excluding field performance study, was executed in five from infected plants, Lane 5 is the PCR-negative from in vitro regenerated plants replicates with 20 samples per replication. Analysis of variance (ANOVA) was lanes 1-5 are PCR-positive results from infected plants, Lane 6 is the PCR- used to statistical analysis of experimental data using MSTAT Software (2009). negative from in vitro regenerated plants and (FLMaV-2 and FMMaV) lanes 1- Differences between individual means were estimated according to Snedecor 6 are PCR-positive results from infected plants, Lanes 7-8 are the PCR-negative and Cochran (1982). All values are reported as means ± standard deviation. from in vitro regenerated plants. Lane M: DNA marker (1kb plus DNA ladder).
RESULTS AND DISCUSSION
Field survey and RT-PCR detection
RT-PCR assays of samples yielded five DNA amplicons of sizes, 302 bp, 352 bp, 360 bp, 311 bp and 389 bp, as results for FMV, FLMaV-1, FLMaV-2, FMMaV and FLV-1 infections, respectively (Figure 1). From a total of 300 samples, 252 (84%) were infected by at least one virus. FLMaV-1 was the prevailing virus with an infection rate of 78.3% (Table 2). The incidence of this virus was particularly high on cvs. Sultani, Asuani and Adasi-Ahmer (100%) grown in Northern West Coast, Aswan and Sinai province. While, the second virus for importance was FMV with an infection rate of 73.7%, which occurred mainly on cv. Sultani (100%) grown in Northern West Coast, on cv. Adasi-Ahmer (98%) grown in Sinai province and on cv. Asuani (96%) grown in Aswan province. FLMaV-2 ranked third as incidence (50%) and was substantially equally distributed in all cultivars and regions (24–90%) also the fourth virus for importance was FLV-1 (40.3%), which occurred mainly on cvs. Sultani (94%), Adasi-Ahmer (76%) and Kadota (28%), but was absent on cvs Black Mission, Kahramani and Asuani. While, FMMaV identical infection rates (35%) and was consistently found in cvs. Sultani (80%), Adasi-Ahmer (60%), Asuani (24%), Kadota (18%), Kahramani (14%) and Black Mission (12%). A single FM tree was found infected with at least five viruses: FMV, FLMaV-1, FLMaV-2, FMMaV and FLV-1. The diversity of the viruses and their vectors reveal the complexity of the disease and the symptomatology observed in FM trees. The results show that the greatest level of infection in the assayed samples was attributed to FLMaV-1 not to FMV and that most fig trees are infected with two viruses in the North Coast region was (100%) of Sultani cultivar. This result is not in agreement with other reports on the occurrence of fig virus infections where FMV has always been found to be the most widespread in mosaic diseased fig orchards (Hemaid, 2000; Castellano et al., 2007; Caglar et al., 2011; Shahmirzaie et al., 2012; El Air et al., 2015). These surveys showed that all five Figure 2 Electron micrographs of an ultra thin-section of infected fig leaves. (A viruses characterized in this study were present in the main Egypt fig-growing and C): showed groups of Double-Membrane Particles (DMPs) cytoplasm of areas, with levels of infections that were substantially in line with those reported mesophyll cells, (B): in the chloroplast showing complete bold stain, and (D): from other Mediterranean countries with a few exceptions. In particular, the showed a typical fig mosaic virus consisting of unique quasi-spherical from presence of FMV in fig trees was high (73.7%) especially in the North Coast purified viral preparation. Bar: 300 nm. region was (100%) of Sultani cultivar if compared to previous reports in Turkey (8.6%) (Caglar et al., 2011), in Tunisian (37%) (El Air et al., 2015) and Iran PCR amplification resulted in amplicons of the expected sizes (302 bp) of FMV, (7.6%) (Shahmirzaie et al., 2012). While, The incidence of FLMaV-1 was (352 bp) of FLMaV-1, (360 bp) of FLMaV-2, (311 bp) of FMMaV and (389 bp) particularly high (78.3%), when compared with that reported from fig orchards of of FLV-1 whose sequence identity with the nucleotide sequence. BLASTn many surrounding countries, including Iran (11%), Syria (4%), Tunisia (33.3%), analyses showed that sequences of FMV and FMMaV were 100% identical to the Lebanon (15%), Saudi Arabia (55%) and Egypt (68.3%) (Elbeaino et al., 2012; Italian (Genbank accession number AM941711and FJ611959), whereas
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similarities of FLMaV-1 (95–99%), FLMaV-2 (97– 100%) were found with the FN377573. The nucleotide sequence of these viruses was reported by (Elbeaino Italian (Genbank accession numbers AM113547, KC534878) and FLV-1 et al., 2007; Gattoni et al., 2009; Elbeaino et al., 2010; Ishikawa et al., 2012). obtained sequence was deposited in the EMBL database under the accession No.
Table 2 Incidence of FMV, FLMaV-1, FLMaV-2, FMMaV and FLV-1 infections in fig cvs. Asuani, Kadota, Black Mission, Kahramani, Sultani and Adasi-Ahmer growing provinces of Egypt as determined by RT-PCR assays Cultivar (Province) Tested treesInfected trees FMV FLMaV-1 FLMaV-2 FMMaV FLV-1 No. No. % No. % No. % No. % No. % No. % Asuani (Aswan) 50 50 100 48 96 50 100 18 36 12 24 0 0 Kadota (Giza) 50 32 64 19 38 15 30 12 24 9 18 14 28 Black Mission (Kaliobeya) 50 30 60 15 30 22 44 20 40 6 12 22 0 Kahramani (Monoufia) 50 40 80 40 80 48 96 19 38 7 14 0 0 Sultani (Northern West Coast) 50 50 100 50 100 50 100 45 90 40 80 47 94 Adasi-Ahmer (Sinai) 50 50 100 49 98 50 100 36 72 30 60 38 76 Total 300 252 84 221 235 150 104 121 Mean infection rate 84 73.7 78.3 50 35 40.3
Electron microscopy and cell ultrastructure incubated with multiplication medium supplemented with 2 mg L-1 BAP and 0.5 -1 Electron micrographs of sectioned cells prepared from FMD-infected F. carica L. mg L 2iP at 4 weeks (Figure 3B). The shoots transferred on rooting medium leaves represented in (Figure 2) showed the presence groups of double- contained on 2 mg L-1 IBA and 0.5 mg L-1 NAA of all cultivars were developed membrane particles (DMPs), considered to be FMV particles in parenchyma and to plantlets that represent the six cultivars as shown in Figures 4A and B. The subepidermical cells, always presented in rounded to ovoid 100-150 nm in size developed plantlets were studied for fig (Ficus carica L.) cvs. Asuani, Kadota, and elongated, straight to slightly flexuous up to or exceeding 1µm in length Black Mission, Kahramani, Sultani and Adasi-Ahmer as shown in Table (3). The (Figures 2A and C). Moreover, long elongated and flexuous virus-like particles highest response percentage of meristem explant forming shoots was in Sultani surrounding the chloroplast, in parenchyma cell also presented in Figure (2B). cultivar (100%), Adasi-Ahmer cultivar, Asuani cultivar (99%) and Kahramani Also, large quasi-spherical double membrane-bound particles DMPs of 100~150 cultivar (96%). Also, the highest response of mean shoot length was in Asuani nm in diameter were showed in purified extracted from tissues having yellow cultivar (1.54 cm), Adasi-Ahmer cultivar (1.45cm) and Kadota cultivar (1.35cm) mosaic and malformation (Figure 2D). This result is supported by (Appiano et compared with other cultivars. When shoots was transferred to multiplication al., 1995; Elbeaino et al., 2009c; Alhudaib, 2012), they observed similar results medium the results showed that the highest response of mean number of shoots for the infection with FMV results in distinct double-membrane bodies or was in Kahramani (3.62) cultivar, Asuani (3.45) and Sultani (3.54) cultivars particles, called DMBs or DMPs, 90-200 nm in diameter in the cytosol of respectively. While, the Adasi-Ahmer cultivar showed the highest mean number infected parenchyma cells. Comparable large quasi-spherical DMBs of 100-150 of roots (3.25) followed by Sultani (2.95) and Black Mission (2.65) and the nm in diameter were located in the cytoplasm of parenchyma cells. Electron highest response of mean roots length was in Asuani (2.64) cultivar when dense median core particles were observed as well (Martelli et al., 1993). transferred on root medium compared with other cultivars. Plants produced from the rooting stage were transfer to a greenhouse and they were repotted into sterile Meristem tip culturing for the elimination fig viruses soil consists equal parts of peat and sand (v/v) (Figures 4C and 4D). In vitro propagation through meristem culture is the best possible means of virus Thirty meristems out of 180 were developed from the six cultivars that cultured elimination and produces a large number of plants in a short span of time. It is a on MS medium supplemented with 0.05 mg L-1 NAA, 0.5 mg L-1 BAP and 0.5 powerful tool for large-scale propagation of horticultural crops including fig -1 plants. Fig (Ficus carica L.) plantlets have been produced from shoot tips and mg L 2ip (Figure 3A). The meristems commenced their initial growth by meristem tips (Murithi et al., 1982; Demiralay et al., 1998; Gella et al., 1998; increasing in size and gradually changed to light green color; within 3-4 weeks Hemaid 2000; Hepaksoy and Aksoy, 2006). small leaves appeared. After two months of incubation, the in vitro shoots that Table 3 In vitro developed fig (Ficus carica L.) cvs. Asuani, Kadota, Black Mission, Kahramani, Sultani and Adasi-Ahmer produced from meristem tips after eight weeks % of survived % of meristems Mean shoots Mean number Mean number Mean roots Cultivar meristems forming shoots length (cm) of shoots of roots length (cm) Asuani 99 99 1.54±0.252a 3.45±0.097c 2.24±0.206f 2.64±0.102a Kadota 95 93 1.35±0.195c 3.00±0.124d 2.56±0.125e 1.75±0.119b Black Mission 90 87 1.04±0.095f 2.95±0.078e 2.78±0.115c 1.54±0.145c Kahramani 96 96 1.25±0.087d 3.62±0.102a 2.65±0.108d 1.28±0.097f Sultani 100 100 1.15±0.072e 3.54±0.147b 2.95±0.104b 1.48±0.128e Adasi-Ahmer 99 99 1.45±0.091b 2.55±0.095f 3.25±0.215a 1.50±0.109d Values are presented by mean ± SE Same letters represent no significant differences between means at P ≤ 0.05 level
Kahramani, Sultani and Adasi-Ahmer. The extraction of healthy fig plant tissues from in vitro plantlets used as negative control and the purified virus preparation were used as positive control. FMV-antiserum was produced from virus particles. The virus specific titer for the polyclonal rabbit antibodies raised against purified FMV was at least 1/1000. DAS-ELISA on leaves extracts from samples already tested in RT-PCR assay, using specific immunoglobin G (IgG) from bleeds diluted 1:1000 gave the optimal binding specificity. The extraction of the purified virus was used as positive control. The results indicated that most infected trees gave positive results with FMV antiserum diluted 1:1000 while healthy tissues from in vitro plantlets gave negative results. Our results indicated that samples Figure 3 Fig regenerated plants from meristem tip culture on MS medium taken from the in vitro regenerated cultivars showed not any color, whereas, all supplemented with 0.05 mg L-1 NAA, 0.5 mg L-1 BAP and 0.5 mg L-1 2ip the infected field samples (symptoms showing) developed yellow color after after two months (A); Shoot multiplication of Ficus carica L. cultivars on MS DAS-ELISA test. This indicated that the antiserum reacted strongly with all infected plants, but the in vitro regenerated plantlets did not react. This may be medium supplemented with 2 m g L-1 BAP and 0.5 mg L-1 2iP (B). due to the regenerated plantlets becoming 100% free from fig mosaic virus
through meristem culture. This result is supported by Hemaid (2000), in which Detection of FMV by DAS-ELISA similar results were observed for the detection of fig mosaic virus through DAS-
ELISA test. Plantlets that are derived from tissue culture can be tested by DAS-ELISA. The fig samples were prepared (0.2 g of fresh leaves in 2 mL) and 200 l/well of each sample from fig (Ficus carica L) cvs. Asuani, Kadota, Black Mission,
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Caglayan, K., Medina, V., Gazel, M., Serce., C., Serrano, L. (2009). Putative agents of fig mosaic disease in Turkey. Turkish Journal of Agriculture & Forestry, 33: 469–476. https://doi.org/10.3906/tar-0807-20. Castellano, M.A., Gattoni, G., Minafra, A., Conti, M., Martelli, G.P. (2007). Fig mosaic in Mexico and South Africa. Journal of Plant Pathology, 89, 441–443. http://www.sipav.org/main/jpp/volumes/0307/030720.pdf. Clark , M.F., Adams A. N. ( 1977). Characteristics of the microplate method of enzyme – linked immunosorbent assay for the detection of plant viruses. Journal of General Virology, 34: 475 – 483. http://doi: 10.1099/0022-1317-34-3-475. Clark, M.F., M. Bar-Joseph. (1984). Enzyme immunosorbent assays in plant virology. Pages 51–85 in Methods in virology, edited by K. Maramorosch and H. Koprowski. Academic Press, New York, USA. https://doi.org/10.1016/B978-0- 12-470207-3.50009-7. Condit, I. J., Home, W. T. (1933). A mosaic of the fig in California. Phytopathology, 23:887-896. Cooper, J.I., Edwards, M.L. (1986). “Variations and limitations of enzyme- amplified immunoassays”, Developments and applications in virus testing, edited by R.A.C. Jones and L. Torrance. Association of Applied Biologists, Wellesbourne, UK, pp.139-154.(16) Polyclonal and Monoclonal Antibody-Based Methods for Detection of Plant Viruses. Available from: https://www.researchgate.net/publication/283344435 Polyclonal_and_Monoclonal_Antibody- Figure 4 Rooting and ex vitro acclimatization of Ficus carica L. (A): Rooting Based_Methods_for_Detection_of_Plant_Viruses. of Ficus carica L. on MS medium supplemented with 2 mg L-1 IBA and 0.5 mg Demiralay, A., Yalçin-Mendi, Y., AkaKaçar,Y., Cetiner, S. (1998). In vitro -1 propagation of Ficus carica L. var. Bursa Siyahi through meristem culture. Acta L NAA , (B): healthy plantlets with normal roots, (C): Acclimatization of Horticulturae, 480: 165-167. http://doi: 10.17660/ActaHortic.1998.480.28. Ficus carica L. plants after three weeks and (D): fig seedlings after two months. ElAir, M., Mahfoudhi, N., Digiaro, M., Dhouibi, M.H., Elbeaino, T. (2015). Incidence and distribution of viruses in Tunisian fig orchards. Journal of Plant CONCLUSION Pathology, 97:327–331. http://doi: 10.4454/JPP.V97I2.030. Elbeaino, T., Digiaro, M., De Stradis, A., Martelli, G.P. (2006). Partial Fig (Ficus carica L.) is one of the old and the most common fruit tree grown in characterization of a closterovirus associated with a chlorotic mottling of fig. all the Mediterranean countries, particularly in Egypt for which no information Journal of Plant Pathology, 88, 187–192. was previously available. A recent investigation indicated that fig mosaic disease http://www.sipav.org/main/jpp/volumes/0206/020608.pdf. was the main pathogenic agent. In this study, field surveys were conducted in the Elbeaino T., Digiaro, M., De Stradis, A., Martelli, G.P. (2007). Identification of areas of Northern West Coast, Aswan, Kaliobeya, Monoufia, Giza and Sinai a second member of the family Closteroviridae in mosaic-diseased figs. Journal Governorate to detect any of FMV, FLMaV-1, FLMaV-2, FMMaV and FLV-1. of Plant Pathology, 89, 119–124. This is the first report of this virus occurring in the Egyptian fig orchards in most http://www.sipav.org/main/jpp/volumes/0107/010714.pdf. governorates of Egypt.In addition to that, a total 300 fig samples were collected Elbeaino, T., Digiaro, M., Alabdullah, A., De Stradis, A, Minafra, A., Mielke, N., from the surveyed areas. Molecular studies, by using RT-PCR were detected in Castellano, M.A., Martelli, G.P. (2009a). A multipartito single-stranded negative- test samples with overall incidence of (84%). The most spread virus was sense RNA virus is the putative agent of fig mosaic disease. Journal of General FLMaV-1 (78.3) and FMV (73.7%), flowed by FLMaV-2 (50%), FLV-1 (40.3%) Virology, 90:1281–1288. http://doi: 10.1099/vir.0.008649-0. and FMMaV (35%). Also, were detecting about 100% of mixed infection Elbeaino, T., Digiero, M., Martelli, G.P. (2009b). Complete nucleotide sequence between FLMaV-1 and FMV in some areas. Portions of the fig virus's genomes of four RNA segments of fig mosaic virus. Archives of Virology, 154:1719–1727. were sequenced and showed high similarity with published ones in GenBank (98- http://doi: 10.1007/s00705-009-0509-3. 100%). FMV-antiserum was produced from virus particles. Symptomatology Elbeaino T., Nahdi, S., Digiaro, M., Alabdullah, A., Martelli, G.P. (2009c). identification of FMV was recorded in the infected samples that judged by DAS- Detection of Fig leaf mottle-associated virus1and Fig leaf mottle-associated virus ELISA as mosaic symptoms observed in almost all growing areas of Egypt. The 2 in the Mediterranean region and study on sequence variation of the HSP70 results of DAS-ELISA on leaves extracts from fig samples already tested in RT- gene. Journal of Plant Pathology, 91, 425–431. PCR assay, using IgG purified from blood, showed that the IgG obtained from http://sipav.org/main/jpp/index.php/jpp/article/download/973/759. blood diluted 1:1000 gave the optimal binding specificity and confirmed the Elbeaino, T., Heinoun, K., Digiaro, M., Martelli, G.P. (2010). Fig mild mottle- molecular assays. Tissue cultured plantlets were established from these plants associated virus, a novel closterovirus infecting fig. Journal of Plant Pathology, and used to determine the effectiveness of various methods for detecting a virus 92,165–172. http://www.sipav.org/main/jpp/volumes/0110/011017.pdf. in in vitro cultured fig plants. Elbeaino T., Abou Kubaa, R., Digiaro, M., Minafra, A., Martelli, G.P. (2011a). 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