Screening and optimization of some inorganic salts for the production of ergot alkaloids from Penicillium species using surface culture fermentation process Memuna Ghafoor Shahid1*, Muhammad Nadeem2, Shahjehan Baig2, Tanzeem Akbar Cheema1, Saira Atta1 and Gul Zareen Ghafoor1* 1Department of Botany, GC University, Lahore, Pakistan 2 FBRC, PCSIR Laboratories Complex, Lahore, Pakistan Abstract: The present study deals with the production of ergot alkaloids from Penicillium commune and Penicillium citrinum, using surface culture fermentation process. Impact of various inorganic salts was tested on the production of ergot alkaloids during the optimization studies of fermentation medium such as impact of various concentration levels of succinic acid, ammonium chloride, MgSO4, FeSO4, ZnSO4, pH and the effect of various incubation time periods was also determined on the production of ergot alkaloids from Penicillium commune and Penicillium citrinum. Highest yield of ergot alkaloids was obtained when Penicillium commune and Penicillium citrinum that were grown on optimum levels of ingredients such as 2g succinic acid, 1.5 and 2g NH4Cl, 1.5g MgSO4, 1g FeSO4, 1 and 1.5g ZnSO4 after 21 days of incubation time period using pH 5 at 25°C incubation temperature in the fermentation medium. Ergot alkaloids were determined using Spectrophotometry and Thin Layer Chromatography (TLC) techniques. Keywords: Ergot alkaloids, Penicillium commune, Penicillium citrinum, culture conditions, spectrophotometry, TLC. INTRODUCTION also the potential candidates for the production of ergot alkaloids, including Penicillium sizovae, Penicillium Natural products synthesized by microorganisms have chermisinum, Penicillium roquefortii, Penicillium played a major role in the discovery and manufacturing of corylophilum, Penicillium regulosum (Moussa, 2003), drugs, which are in-use for the treatment of several human Aspergillus fumigatus, Aspergillus flavus and Aspergillus ailments. These natural products are commonly called as tamari, respectively (Flieger et al., 1997). The most secondary metabolites, and these constitute an important prominent ergot alkaloids produced by Claviceps and group of bioactive compounds that can be used in Penicillium species are ergometrine, ergotamine, ergosine, pharmaceutical, cosmetic and food industry (Devi and ergocristine, ergocriptine, ergocornine, festuclavine, Prabakaran, 2014). Alkaloids are the largest group of epicostaclavine, fumigaclavine and isofumigaclavine natural products synthesized as secondary metabolites in (Kozlovsky et al., 2013). plants, animals and in fungi (Polak and Rompala, 2007). Alkaloids have been reported as a group of organic The toxicity of ergot alkaloids make them substances, containing at least one nitrogen atom in the pharmacologically very useful. All of the types of ergot ring structure of their molecule. Alkaloids in fungi were alkaloids have been used in making different medicines to initially recognized in Claviceps purpurea (member of cure migraine, to reduce postpartum bleedings, for Clavicipitaceae family) the agent causing ergot of rye. inducing labour contractions, for the inhibition of Generally, the members of Clavicipitaceae family infest lactation, to terminate pregnancy and to inhibit mammary grass species, including cereal grains, and these members tumors (Floss et al., 1973; Masureker, 1992; Fleiger et al., are capable of producing a number of different important 1997; Moussa, 2003). Taking into consideration the ergot alkaloids (Danicke and Diers, 2013). These above-mentioned scenario and the day by day increasing compounds have proven to be important demand of ergot alkaloids as pharmacological and pharmacologically and agriculturally (Wallwey and Li, therapeutic agents, a strong need has been felt to develop 2011; Ryan et al., 2013). Penicillium is well known all a cost-effective process for the biosynthesis of ergot over the world, for producing secondary metabolites and alkaloids for commercial use in Pakistan using some commercially valued extracellular enzymes (Gulliamon et novel sources. Hence, the present study was designed and al., 1998; Tiwari et al., 2007, 2011). The secondary conducted on the optimization of culture conditions for metabolites produced by Penicillium include alkaloids, the enhanced production of ergot alkaloids using surface antibiotics, hormones and mycotoxins, etc. Therefore, culture fermentation technique. many species of genus Penicillium and Aspergillus are *Corresponding author: e-mail: [email protected] Pak. J. Pharm. Sci., Vol.29, No.2, March 2016, pp.407-414 407 Screening and optimization of some inorganic salts for the production of ergot alkaloids from Penicillium species MATERIALS AND METHODS and ergot alkaloids production by Penicillium commune and Penicillium citrinum. Procurement of fungal species Penicillium commune and Penicillium citrinum were Effect of concentration levels of MgSO4 obtained from Fungal Culture Bank, Institute of Effect of different concentration levels of MgSO4 i.e. 0.5, Agricultural Sciences, University of the Punjab, New 1.0, 1.5, 2.0, 2.5 and 3.0% were studied to find out the Campus, Lahore and Institute of Industrial Biotechnology, suitable MgSO4 concentration level for the growth of GC University, Lahore. mycelium and maximum yield of ergot alkaloids by Penicillium commune and Penicillium citrinum. Maintenance of fungal cultures The cultures were maintained on malt extract agar (MEA) Effect of concentration levels of FeSO4 medium slants (slants were prepared by taking 2g of malt Effect of different concentration levels of FeSO4 i.e. 0.5, extract and 2g of agar in 250ml Erlenmeyer flask and 1.0, 1.5, 2.0, 2.5 and 3.0% were evaluated to find out the dissolved in 100ml of distilled water). Spores from 7-10 suitable FeSO4 concentration level for the maximum days old culture slant were transferred to the freshly mycelial growth and yield of ergot alkaloids by prepared slants and placed in incubator at 25°C for 10 Penicillium commune and Penicillium citrinum. days for the growth of mycelium. The slants having proper growth after incubation were stored in refrigerator Effect of concentration levels of ZnSO4 at 4°C for further studies. Effect of different concentration levels of ZnSO4 i.e. 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % were evaluated to find out the Composition of growth medium and fermentation suitable ZnSO4 concentration level for maximum growth studies of mycelium and production of ergot alkaloids by Various self constructed fermentation media were Penicillium commune and Penicillium citrinum. screened for the production of ergot alkaloids and M5 fermentation medium was selected for the maximum Effect of pH production of ergot alkaloids. Surface culture The optimum pH level was evaluated for the growth of fermentation technique was used for the production of mycelium and production of ergot alkaloids by ergot alkaloids in M5 fermentation medium. The growth Penicillium commune and Penicillium citrinum by medium contained (g/100ml) ingredients such as sucrose adjusting the pH of the fermentation medium at different 5, yeast extract 0.5, succinic acid 0.5, tryptophan 0.5, pH levels i.e. 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. The pH of asparagine 0.5, NH4Cl 0.2, KH2PO4 0.5, MgSO4 0.03, each growth medium was adjusted before sterilization FeSO4 0.001 and ZnSO4 0.002. pH of the medium was with 0.1N HCl and ammonia solution. adjusted to 5.2 and after autoclaving 5ml of spore suspension (106-7 spores/ml) was transferred to the Effect of various incubation time periods respective flask containing self constructed fermentation Effect of various incubation times such as 7, 14, 21 and medium. After inoculation, the growth medium was 30 days was determined on growth of mycelium and incubated at 25°C for 21 days. production of ergot alkaloids by Penicillium commune and Penicillium citrinum by incubating the fermentation Optimization of fermentation condition media for above mentioned time range in incubator. The self-constructed fermentation medium was further optimized to enhance the production of ergot alkaloids. Determination of Ergot Alkaloids Effect of following inorganic salts and fermentation Each flask culture after 21 days of incubation was filtered condition was studied for the maximum yield of ergot under aseptic condition to separate the grown mycelial alkaloids: mass (intracellular) and culture liquid filtrate (extra cellular). The collected mycelial mass and liquid culture Effect of concentration levels of succinic acid medium was stored at 4°C for further analysis. The In order to get the maximum yield of ergot alkaloids, amount of ergot alkaloids present in extra cellular and various concentrations i.e. 0.5, 1, 1.5, 2, 2.5 and 3 g were intracellular filtrates was measured by drawing standard employed in the fermentation medium to find the suitable curve of reference salts such as ergotamine, bromocriptine concentration of succinic acid for mycelium growth and mesylate and dihydroergotamine methane sulfonate salts. ergot alkaloids production by Penicillium commune and Penicillium citrinum. Assay for extra cellular ergot alkaloids (culture liquid filtrate) Effect of concentration levels of ammonium chloride The extra cellular filtrates separated from mycelium were Different NH4Cl concentrations such as 0.5, 1.0, 1.5, 2.0, centrifuged at 5000rpm/min for 5min at 4°C. After 2.5 and 3.0% were used in the fermentation medium to centrifugation the supernatants were collected and named find out the suitable