Indian Journal of Experimental Biology Vol 42 , September 2004, pp. 928-932

Isolation, characterization and optimization of antifungal activity of an actinomycete of soil origin

S K Augustine, S P Bhavsar, M Baserisalehi & B P Kapadnis*

Department of Mi crobiology, University of Pune, Pune 411007, India

Received 20 Ocrober 2003: revised 20 May 2004

About 3 12 ac tinomycctcs were isolated from soi l samples on chitin agar. All these iso lates were purifi ed and screened for thei r antifungal acti vity against pathogenic fun gi. Out or these, 22% of the isolates exhibited acti vity agai nst fun gi. One promising iso late with strong antifungal ac ti vity against pathogenic fun gi was se lected for furth er studies. This iso late was from Punc. and was active against both yeasts and molds. Various fermentati on parameters were optimi zed. Based on mor­phological and biochemical parameters, the iso late was identilied as Srreplomyces. The correlation of antifungal acti vity with growth indicated growth dependent production of antimetabo lite. Maximum an tifunga l metabolite production (600 units/ml) was achieved in the late log phase, which remai ned constant during stationery phase, and it was extrace llular in nature.

Keywords : Antifungal ac ti vi ty. Screening, Srreprotnyces sp. PU 23.

Fungi are eukaryotic and have mac hinery for pro­tein and nucleic acid synthes is similar to that of hi gher animals. It is , therefore, difficult to find out compounds that selectively inhibit fun ga l metabolism and ex hibiting no toxicity to humans'. There is an ev idence that some fun ga l strains are resistant to cer­tain antimycotic drugs with res ulting therapeutic fail­ures". There is lack of effec tive and safe antifungal anti biotics3

. Therefore, there is a press ing need of non-tox ic and effec ti ve antifungal antibiotics. The pioneering work of Waksman showed that actinomy­cetes are capable of producing med ically useful anti­biotics". Actinomycetes are di verse group of hetero­trophic prokaryotes forming hyphae at some stage of their growth , hence referred as fil amentous prokaryo­tes5 They are a successful group of bacteria that occur in a multiplicity of natural and man-made environ­ments6 and a unique group having different morpho­logical , cultural , biochemical and physiological char­acters7. Approaches to search for and di scovery of new antibiotics are generally based on screening of naturally occurring actinomycetes8

. The main objec­tive of the present study was to isolate, screen and characterize naturally occurring antifungal actinomy­cetes and to optimize the fermentation parameters.

*Corresponden t author Tel 912025690643 ; Fax 912025690087 Emai l bpkapadnis@yahoo.com

Materials and Methods Collection of soil samples- Soil sa mples were

collected from three Indian states viz., Maharashtra , Karnataka and Kerala. The samples were collected in sterile containers and maintained at 4·~c until analysis.

Isolation and screening of antifirngal actinomy­cetes- Actinomycetes were isolated on chitin agar9

which were incubated at 37°C for 7 days. All these cultures were purified by streak pl ate technique and confirmed by colony morphology and sc reened for their antifunga l activity. One promi sing strain , PU 23, was selected and grown on differen t aga r med ia viz.., starch casein, glucose asparagine, glycerol asparagine, potato dextrose, Sabourauds dextrose and yeast ex­tract malt extract, to have medium that st imulates maximum antifunga l activity. After incubation for 7 days at 37°C, agar di scs of actinomycete growth were made with a sterile cork borer and placed on Sabou­raud dextrose agar plates (pH 5.6) seeded with the funga l culture. The plates were incubated at 28°C and observed for antibiosis after 24 hr in case of yeas ts and 96 hr in case of mold s. Similarly, the antifungal activity of the culture supernatant of the actinomycete in abovesaid liquid medium was tested by agar well diffusion method .

Characterization of selected actinomycete iso­late-Th e growth of the organism was studi ed at 22", 28°, 37" and 42°C temperatures. Morphological fea­tures of the actinomycete were studied under phase

AUGUSTINE et a!.: ANTIFUNGAL ACTIVITY OF AN ACTINOMYCETE OF SOIL ORIGIN 929

contrast microscope (Nikon, Japan). Same culture was subj ected to cu ltural, physiological and biochemical characterisation 10

. Cultural characterization was done on ISP media viz., yeast extract malt extract agar (ISP-2), oatmeal agar (ISP-3), inorganic salts starch agar (ISP-4), glycerol asparagine agar (ISP-5), pep­tone yeast extract iron agar (ISP-6), tyrosine agar (ISP-7) and glucose asparagine agar (ISP-5) at 37°C. The substrate and aerial mycelium colour including soluble pi gments were assigned using the colour har­mony manual (4111 edition; 1958; Container corpora­ti on of America; Chicago, Illinois). Utilization of dif­ferent carbon and nitrogen sources such as D-glucose, D-ga lactose, D-fructose, D-mannitol , D-xylose, L-arabinose, L-rhamnose, raffinose, sucrose, adonitol, D-L-alanine, L-cyste ine, L-histidine, L- leucine, L-phenylalanine and L-va line was studied. The cells were analysed for 2, 6- di aminopimelic acid 11 and

p whole cell sugars content -.

Optimiwtion of a11.t({ungal metabolite productiOJz­Antifungal metabolite production was carried out in 100 ml starch casein medium (starch, 1 %; casein, 0.1 %; KH2P04, 0.05 %; MgS04.7H20 , 0.05 %; pH 7) in 500ml Erlenmeyer flasks. The unit of antifungal activity was arbitrarily defined as the amount corre­sponding to 0.3 mm (di am.) of inhibition zone of As­perg illus niger under defined conditions.

Tell/perature- Five 500 ml Erlen meyer flasks, each with I 00 ml starch casein medium were inocu­lated with the ac tinomycete spores at the rate of lxl06

spores mr 1 of the medium. These flasks were incu­bated at different temperatures viz., 24°, 28°, 32°, 37° and 42°C on rotary shaker (Steelmet, India) for 7 days .

pH- The initi al pH of the starch casein fermenta­tion medium was adjusted to 4, 5, 6, 7, 8 and 9 sepa­rately with 0.1N NaOH/O.IN HCI. All flasks were inoculated as mentioned above and incubated at 37°C on rota ry shaker (250 rpm) for 7 days.

Agitatio11.- Five 500 ml Erlenmeyer flasks, each with 100 ml starch casei n broth were inocul ated as mentioned above and incubated on rotary shaker at I 00, 150, 200, 250 and 300 rpm for 7 days at 37°C.

0 /ycerol concentration- Effect of glycerol at va· ed concentrat ions viz. , 1.0, 1.1 , 1.2, 1. 3, 1.4, 1.5 and 1.6% v/v was studi ed on anti funga l metabolite production . The in oc ulum size and incubati on condi­tions were as mentioned above.

Ti l11 e course of antifungal merabolite production under optil11U l11 conditions- The 500 ml Erlenmeyer

fl as k with 100 ml starch casein broth was inocul ated with spores at the rate of 1x l06 spores mr 1of prod uc­tion ·medium. The flasks were incubated at 37°C on shaker at 250 rpm. After every 24 hr, the cu lture broth was analysed for antifungal metabolite content by well diffusion method and biomass in terms of OD5.t0,

for 12 days . Besides, the pH was monitored by dig ital pH meter.

Detection of antifungal metabolite in the cell mass and supernatant-To test if the antifunga l metabol ite production was intracellular or extracellular, the cu l­ture was centrifuged at 10,000 rpm for 20 min . The supernatant and ethanol extract of the dri ed biomass were tested for their antifungal activity with ethanol as control.

Results Isolation and screening-In all 312 strains of acti­

nomycetes were isolated and screened aga inst yeasts and molds. Amongst them, 22% showed antifunga l ac tivity against fungi. The anti funga l ac tivity of the strain PU 23 agai nst the pathogenic fungi was good when grown on starch casein med ium indicat ing that the starch casein aga r med ium was good for inducing antifunga l act ivity in the strain PU 23 (Fig. I). In the shake flask study, the cu lture supernatant of the ac­tinomycete also showed good anti funga l activity.

Characterization of actinomycete isolate-The se­lected isolate, PU 23, had an opti mum temperature for growth at 37°C. It could grow very we ll on all the JSP media and produce water soluble brown pigment on all the medi a except oatmea l agar and yeast ex tract malt extract agar, however, it had ye ll ow colour on reverse on the latter medi a. The aerial mycelium was grey on all media except tyros ine agar and yeast ex­tract malt extract agar on which it was white. The spore chains were spiral type and each had more than 15 spores per chain (Fig.2). The cell wall of the strain PU 23 contained L-diaminopimelic acid. The bio­chemical properties have been shown in Table 1. The taxonomic properties described above apparent ly sug­gested that the isolate PU 23 belongs to the genus Streptomyces and des ignated as Streptomyces sp. PU 23.

Optimization of temperature, pH, agitation and glycerol concentration for antifitngal metabolite pro­duction- The optimum conditions for antifunga l metabolite production were pH 7, temperature 37°C. ag itation 250 rpm and glycerol 1.5% (Fig.3) and the act ivity was eq ui valent to GOO unitslrnl.

930 INDIAN J EXP BIOL, SEPTEMBER 2004

Fig. !-Strong antifungal activity exhibited by starch casein grown Streptomyces sp. PU 23 against - Molds: (a) Aspergillus niger NCIM 586 and (b) Fusarium oxysporum NCIM 1072; and Yeasts: (c) Candida albicans NCIM 7102; and (d) Cryptococcus hwnicolus NRRL 12944.

Fig. 2 -Streptomyces sp. PU 23. a) Spore chains as seen under phase contrast microscope (x400), and b) Growth on starch casein medium showing grey spore mass.

AUGUSTINE et al.: ANTIFUNGAL ACTIVITY OF AN ACTINOMYCETE OF SOIL ORIGIN 931

Time course of antifungal metabolite production in Streptomyces sp. PU 23-The antifungal metabolite production was monitored over a period of 12 days. The rate of antifungal metabolite production corre­lated with growth rate of Streptomyces PU23 and was highest (600 units/ml) in the late log phase (Fig.4). The pH of the broth was between 6.8 and 7 through­out fermentation.

Testing for extra- and intra-cellular metabolite production-The ethanol extract of the biomass did not show antifunga l activity. However, the culture supernatant inhibited all fun gi tested (Table 2). There-

Table I - Biochemical characteri stics of St reptomyces sp. PU 23

Coagulati on of milk

Decomposition of cel lulose

Hydro lysis of starch

Liquefaction of gelatin

Reduction of nitrate

700

c 600 . ,-----0 ., " "0 500 .

~ " K i 400 .. c "' ~ 0 .0 2. 300 B ~ ,._ !'!

" .g 200 0. c: :X: E <( "- " 100 1-

0 .

A B

-

8 e-~ !::!-0

-~ ~

c Optimized parameters

+

+

+

,-----~ "' :::-c

· ~ ~ " c v

" ~ " ,., G

D

Fig. }--Condit ions required for optimum production of antimetabolite

700

u

~ :::> 15 500 Q.::::-. <l> E 400

~~ .g § 300 -v ~

E 200 ., c <t 100

2 3 4 5 6 7 8 9 10 11 12

Fetmertation time (days)

0.35

0.3

0.25

0.2

0.15

0. 1

0.05

0

E

~ Tii 0 0

Fi g. 4--Time course of antimetabolite production by

Streptomyces sp. PU 23 .[ +-Antimetabolite produced (units/ml); and • - 0. D. at 540 nmj

Table 2- MIC values* of the supernatant** from Streptomyces sp. PU 23 against fungi in comparison

with that of nystatin

Target organi sms MIC (units/ml) Supernatant Nystatin

Aspergillus niger NCIM 586 1.8 2. 18

Fusarium oxyspomn1 NCIM 1072 1. 8 2. 18

Candida albiccms NC IM 7 102 3.75 1.1

Cryptococcus humicolus 3.75 I. I NRRL 12944

*Determined by broth tube di lution procedure using two fold dilution in Sabaroud dextrose broth at 28°C.

** Inoculum ( IXI06 spores ml' 1) in 100 ml starch casein broth

in 500 ml Erlenmeyer flask, incubated on rotary shaker (250 rpm) for 7 days at 37°C, followed by centri fugation to obtain su pernatant.

fore, the antifungal metabolite was ex trace llu lar 111

nature.

Discussion There are reports regarding bi oactivity amongs t

chitinolytic actinomycetes 13. Further, we also propose

based on our findings that c hitinolytic actinomycetes exhibit promising ant ifungal ac ti vity. The antifunga l ac tivity has been observed both in solid as we ll as in culture broth unlike fumaradimyc in , which is inacti­vated in the fermentation broth 14 . Production of ant i­fungal metabolite has been known to be influenced by components of medium and cultural conditions, (such as aeration, agitation, pH, te mperature and g lycero l concentration), which often vary from organism to o rgani sm 15

• C ultural conditions such as pH, tempera­ture, aeration and agitation were found to affect anti­fungal metabolite production by Streptomyces sp. PU 23. The optimum pH for metabo lite production was found to be 7. It has been reported that the change in pH of the c ulture medium induces production of new products that ad verse I y affect anti biotic producti on 16

.

It has been reported that the production of he I voic acid and cerulenin by Cephalosporium caeruleus is affected by change in the pH 17

. In Streptonzyces sp. PU 23, the optimum temperature for metabolite pro­duction and growth is sa me i.e. 37°C. Deviation from this temperature affects the yie ld of the antifunga l metabolite 18

• Agitation affects aeration and mi xing of the nutrients in the fermentation medium. T he opti­mum rate of ag itation (250rpm) was found to facili­tate increased antifungal metabolite production in the

932 INDIAN J EXP BIOL, SEPTEMBER 2004

present study, which might be due to better transfer of oxygen and enhanced uptake of nutrients. It has been reported that the yield of cephalosporin C increases with increase in dissolved oxygen 19

•

Rate of production of the antifungal metabolite was directly proportional to growth rate. Maximum me­tabolite production (600 units/ml) was achieved at late log phase, which remained constant during stationary phase. However, there are many reports about the an­tibiotic production in the stationary phase as antibiotic is a secondary metabolite20

. The antifungal metabolite of the strain was extracellular in nature. In most of the cases, antibiotics are extracellular21

. Further studies on the extraction, purification and characterization of the antifungal metabolite are currently in progress.

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2 1

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