Enhanced candicidal compound production by a new soil isolate Penicillium verruculosum MKH7 under submerged fermentation

Genus Penicillium is a potential producer of a vast array of secondary metabolites like tremorgenic toxins, pigments, potentiators of nerve growth factor, inducers of osteoblast differentiation and most importantly antibiotics including macrocyclic polylactones [25–30]. In this report, characterization of the candicidal compound revealed the presence of lactone ring and carbonyl groups as prominent components. Breinholt et al. [27] had also reported the isolation of antifungal macrocyclic polylactones from P. verruculosum. Production of secondary metabolites is influenced by a number of cultivation parameters ranging from precursors to trace elements [31]. The combined effects of all the involved factors cannot be determined through traditional practices like the classical one-factor-at-a-time approach [32]. Interactions among factors and multifactor optimization cannot be evaluated using this method. These limitations can be overcome by statistical tools like RSM that enable the study of the effects of several factors simultaneously and determine the optimum values of the variables so as to maximize the response [33, 34]. PBD was applied to determine the main factors affecting metabolite production. In this investigation, from the PBD analysis it was found that among the variables tested, four factors viz. initial pH, temperature, peptone and MgSO₄.7H₂O, were found to have significant effect on the production of bioactive metabolite by the strain P.verruculosum MKH7. Medium pH, temperature [35] and peptone [36] have been reported to be some of the most important experimental parameters influencing bioactive secondary metabolite production by Penicillium species. Dextrose and peptone were found to be important also in the production of sclerotiorin from P. sclerotiorum [8]. However Brian et al. [30] reported that glucose was the best carbon source for wortmannin synthesis from P. wortmanni while glycerol was most favourable for mevastatin production by P. citrinum [37]. The reason for this dissimilarity might be the use of different strains and different culturing conditions, yet another reason might be the selection of different carbon sources in the original medium [24]. Moreover, the sources for growth and secondary metabolism may be different. For example, glucose may be beneficial for growth but the same may not be true for secondary metabolite formation [38]. Peptone played a crucial role in the biosynthesis of the metabolite sclerotiorin by P. sclerotiorum [39]. Several factors influence the effect of nitrogen sources on synthesis of secondary metabolite. The sources that are important for growth may negatively affect secondary metabolic pathways; there are reports on the negative effects of ammonium salts [38].

Under the RSM optimized conditions of initial pH7.4, temperature 27 °C, peptone 9.2 g/l and MgSO₄.7H₂O 0.39 g/l, a maximum metabolite production of 211.24 mg/l was predicted, simultaneously proved by triplicate experiments conducted under the same conditions. MgSO₄.7H₂O was found to be essential also for the synthesis of mevastatin from P. citrinum [37]. Trace elements like Zn, Mn, Fe, Cu are important for microbial growth because of their presence in metalloenzymes or as enzyme activators [24] while Mg and Ca are macronutrients in fungal nutrition [37]. Contrary to this, in our case the effect of Mg on metabolite yield was significant. Similar inference was also made on the production of sclerotiorin by P. sclerotiorum [8]. Temperature and pH affect the regulation of molecules like ATP which in turn influence the regulation of metabolic pathways, coupled reactions and functional yields at the membrane and cell wall level [39]. A change in the concentration of hydrogen ions may change the redox fluxes and oxidative state of energy molecules like ATP, thereby causing diverse metabolism and generating different products [40]. Brian, 1946 [41] reported that high initial pH (6) of media was best for development of fungistatic activity by Penicillium terlikowskii. Similar observation was also made for the production of antimicrobial agent by P. viridicatum [42]. Metabolic activity of fungus may be terminated by low temperature while high temperature kills the fungal cell [43]. The optimal temperature range for the production of the antibiotic and nephrotoxin, citrinin, by P. viridicatum was 25–30 °C [44]. Previous studies have shown that a temperature of 25 °C was found to be optimal in a number of cases [43].

The use of polyene antibiotics in clinical practice is restricted due to problems in their stability which is affected by extreme values of pH and temperature leading to total loss of drug potency. The stability of lactone antibiotics depends on the tetraene chromophore of the molecule and heating beyond 100 °C leads to cleavage of these four conjugated double bonds resulting in the degradation of the antibiotic [45]. According to Stark, 2000 [46], neutral aqueous suspensions of natamycin can remain stable at 50 °C for several days and a slight decrease in biological activity was observed after heating for 20 min at 110 °C. The polyene antifungal agent nystatin is more active at low temperature (30–25 °C) while amphotericinB is at 41 °C and their activity against C. albicans is stable at pH between 5 and 7 [47]. Similar observation was made by Raab, 1972 [45], on the stability of natamycin at different pH. Likewise, phoslactomycin B is most stable at a pH of 6.63 [48]. High pH results in saponification of the lactone and additional decomposition due to a series of retroaldol reactions while low pH might bring about the hydrolysis of the glycosidic bond [49].

So far as our knowledge goes, till date there are no reports available on the production of antibiotics by Penicillium verruculosum through media optimization using RSM. The enhanced yield of the antibiotic strongly suggests that the fungus P. verruculosum MKH7 can be efficiently used for antibiotic production on a large scale. Optimization not only led to a 7 fold increase in metabolite yield but the same was achieved at much lesser time (8–10 days compared to the earlier 12–15 days). In conclusion, statistical methods can be effectively utilized for arriving at optimal solutions and in analysing the interactive effects of the parameters thereby leading to improved metabolite production.