Antibacterial Activity of Aspergillus oryzae against some Human Pathogens.

¹Senthilkumar G., ¹Josphine A., ²Madhanraj P.* and ¹Panneerselvam A.

¹Dept. of Botany and Microbiology, A.V.V.M Sri Pushpam College (Autonomous), Poondi, Thanjavur (Dt) – 613 503, Tamil Nadu, India.

²Dept. of Microbiology, Thanthai Hans Roever College of Arts and Science, Elambalur, Perambalur - 621 212, Tamil Nadu, India.

ABSTRACT:

The marine fungi occurring on the wood samples were studied. Diversity of marine fungi colonizing the lignicolous wood samples was assessed and their distribution pattern was determined. The antibacterial activity of Aspergillus oryzae was tested against five human pathogens by agar well diffusion method. Based on the results, the compounds were separated by Thin Layer Chromatography (TLC) and these compounds were tested against three human bacterial pathogens. The separated compounds were further analyzed by Fourier Transform Infrared Spectroscopy (FTIR) to identify the functional groups, for the antibacterial activity.

KEY-WORDS: Aspergillus oryzae, FTIR, TLC, Sterol.

INTRODUCTION:

The marine biosphere is one of the richest of the earth’s innumerable habitats, yet one of the least studied and characterized for microbial flora. Currently, marine microorganisms are considered as untapped sources of metabolites and products, which may possess important properties. They have a diverse range of enzymatic activity and are capable of catalyzing various biochemical reactions with novel enzymes. Thus there is enormous scope for exploring the probabilities of deriving new products of economic importance from the potential marine microorganisms.

Mangroves a kind of special host plants, are a resource of abundant endophytic fungi, marine derived fungi have proven to be a rich source of structurally unique and biologically active secondary metabolites (Yanagihara et al., 2005).

Ecologically mangroves are colonizers, creating land forms out of sea. Mangroves perform a number of vital ecological functions like nutrient, recycling, maintanance of hydrological regime, coastal protection, Fish-fauna production, protecting coastal zones from erosion, storms and flood, and also in supplying food and shelter for large number of fishes.

Muthupet meaning ‘Land of Pearls’. Muthupet is the largest mangrove forest in Tamil Nadu. It is located along the palk strait in Thiruvarur and Thanjavur district and has total area of 119sq.km.

Driftwoods are one of the important and interesting objects for the study of fungal colonization as their origin is unknown and can be drifted to any part of the coast by wind in the breakdown of driftwood and thus in the nutrient cycles of marine habitat. They get exposed to sunlight and atmosphere at a frequent interval due to tidal variations.

Aspergillus oryzae was growing rapidly, pale greenish-yellow, olive-yellow or with different shades of green, Typically with dull brown shades with age. Colonies on medium attaining a diameter of 4-5cm within 7 days, consisting of a felt of long conidiophores often intermixed with aerial mycelium. Conidial heads radiate, pale greenish yellow, later becoming light to dull brown. Conidiophores hyaline, upto4-5mm in length, mostly rough-walled.

The need for diversity and development of new classes of antimicrobial compounds are increasing, due to trends in antibiotic resistance among different strains of bacteria, fungi and other microorganisms.

Inventory of biologically active compounds has gained importance in recent years. This involves the process such as extraction, separation, purification and characterization. The compounds resulted in the process are proved to interesting in their structure and have effective activity against various pathogens. Moreover the compounds (both extra and intracellular) are considered as a key factor to identify the organisms.

Nowadays, the chemical, biological and ecological diversity of the marine ecosystem has contributed immensely potent antitumour compounds (Singh et al., 2008).

MATERIALS AND METHODS:

About 5 human pathogenic microorganisms were selected for the present investigation. The bacterial strains selected were Streptococcus sp, Staphylococcus aureus, Enterobacter aerogenes, Bacillus subtilis and Klebsiella pneumoniae. These bacterial strains were originally obtained from Microbial culture collection unit (MCCU), Sri Gowri Biotech Research Academy (SGBRA), Thanjavur, Tamil Nadu, India.

Screening for Antibacterial activity:

Agar well Diffusion (Perez et al.,1990):

The antibacterial activity of the methanolic extract of the fungus were tested against the selected bacterial strains. The sterilized nutrient agar medium was poured into each sterile Petri plates and allowed to solidify. Using a sterile cotton swab, fresh bacterial culture was spreaded over the plate by spread plate technique. The well- cut was prepared, the methanolic extract of fungus was added into the well. All the plates were incubated at 37^oC for 24-48 hours. After the incubation period, the results were observed and the zone of inhibition around the each microorganism was also measured.

Separation of compounds using TLC:

The general principle was similar to that of column chromatography i.e. adsorption chromatography. Five different compounds are get separated by spraying suitable spraying agents. Qualitative phytochemical screening was done by Thin Layer Chromatography(Wagner,1996).

FTIR (Young et al., 2003):

One bioactive compound was separated and purified using Fourier transform infrared spectroscopy (FTIR) analysis to identify the functional groups present in it . The sterol was selected for the FTIR.

RESULTS AND DISCUSSION:

The methanolic extract of A.oryzae showed maximum zone of inhibition against K. pneumoniae (12mm) followed by Bacillus subtilis (11.5mm) and streptococcus sp. (11mm). There is no zone formation around the E. aerogenes and S. aureus (Table-1;Plate-1).

Table.1- Antibacterial activity of the Aspergillus oryzae

S. No	Organism	Standard (mm) Streptomycin	Zone of inhibition (mm)	Negative control
1.	Klebsiella pneumoniae	10	12	-
2.	Bacillus subtilis	8	11.5	-
3.	Streptococcus	7	11	-
4.	Staphylococcus aureus	10	-	-
5.	Enterobacter aerogenes	8	-	-

The present study was also carried out for the presence of bioactive compounds in A. oryzae. Bioactive compounds were separated by Thin Layer Chromatography (TLC) method which mainly concentrates on the presence of alkaloids, saponins, phenols, sterols and flavanoids (Table-2; Plate-2).

Table.2-Bioactive compounds analysis of Aspergillus oryzae

S. No	Compounds	Colour
1.	Sterol	Blue
2.	Alkaloid	Brown
3.	Flavanoid	Yellow
4.	Phenol	Blue
5.	Saponin	Yellow

This has been made possible by thin layer chromatography (TLC) with suitable solvent systems and only one initial spray reagent was used to detect the compounds. (Korzybski et al.,1967).

Crude extract and purified fractions of Aspergillus spp. showed a wide spectrum of anti-microbial activity. Similarly, several metabolites of the marine isolate, Aspergillus niger showed anti-bacterial and anti-fungal potential (Bugni et al., 2004).

The above compounds can serve as antibacterial agents. TLC results confirmed the presence of sterols, alkaloids, flavanoids, phenols and saponins. These compounds were screened for antibacterial activity against Klebsiella pneumoniae, Streptococcus sp. and Bacillus subtilis . In this, sterol have the maximum activity when compared with other compounds against Klebsiella pneumoniae and followed by B.subtilis and Streptococcus sp respectively(Table-3 ;Plate-3).

Table.3-Antibacterial activities for the screened bioactive compounds

S. No	Compounds	Zone of inhibition in mm
S. No	Compounds	*K. pneumoniae*	*B. subtilis*	*Streptococcus* sp
1.	Sterol	20	18	17
2.	Alkaloid	15	13	11
3.	Flavanoid	10	11	12
4.	Phenol	11	8	4
5.	Saponin	7	9	3

The sterols was selected and it was further analyzed by Fourier Transform Infrared Spectroscopy (FTIR).The presence of sterols in the developed chromatogram was detected by spraying the Folin-ciocalteu’s reagent. After the plates was heated at 100°C for 6 mins, a positive reaction was observed by the formation of blue color spot. It was evidenced, sterols in A.oryzae and had the exact Rf value.

FTIR was used to discriminate important wood destroying fungi. Mycelia of 26 strains belonging to 24 different species were grown on agar plates and subjected to FTIR (Annette,2009).

The fingerprint of IR spectra showed functional groups of purified sterols as strong bands at 3432.97, 2355.06, 2093.19, 1640.52, 1568.67, 1451.46, 1232.41, 1091.52 were showed the different functional groups of purified sterols as Amine; Secondary free; One band, Alkene, Unsaturated nitrogen compounds; Isocyanides, α, β-Unsaturated Compounds; Primary amines, Alkene; Disubstituted, -N3 stretching vibrations; azides, C-N Vibration; aromatic tertiary, respectively (Table-4).

Table.4- Detection of various Functional group by FT-IR from Aspergillus oryzae – Sterol

S. No	Group of frequency cm^-1 of the sample	Functional group assignment
1.	3432.99	Amine, Secondary free; one band
2.	2355.06	Alkenes
3.	2093.19	Unsaturated nitrogen compounds, isocyanides
4.	1640.52	Alkyl compounds; α,β-unsaturated compounds
5.	1568.67	Primary amines
6.	1415.46	Alkene, disubstituted
7.	1232.41	-N₃stretching vibrations, azides
8.	1091.52	C-N vibration, aromatic tertiary

Fungi has proven themselves invaluable sources of natural products for agriculture as well as biomedical development for over a half century. As fungi thrive in competitive environments, it is hypothesized that their metabolic compatibility has been strongly influenced by natural selection. Bioactive product discovery depends on the knowledge of habitats where fungi are abundant and the strength of culture collection. Studies on mangrove endophytic fungi were initiated fungal resource for novel metabolites and enzymes and their application are major goals of current research to accomplish environment-friendly technological development.

REFERENCES:

1. Annette, N. J., 2009. A novel procedure for strain classification of fungal mycelium By cluster and artificial neural network analysis of Fourier Transform infrared(FTIR) spectra. Analyst.134: 1215-1223.

2. Bugni. T.S., Ireland, C.M., 2004. Marine derived fungi a chemically and biologically diverse group of microorganisms. Nat. Prod. Rep. 21: 143-163.

3. Korzybski,T.,Kowszyk – Gindifer, and Kurylowicz., 1967. Antibiotics, Vol.2. Pergamon press, New York.

4. Perez, C., Paul, M., Bazerque, P., 1990. Antibiotic assay by agar well diffusion method. Acta. Biol. Med. Exp., 15: 113-115.

5. Singh, R., Sharma, M., Joshi, P., Rawat, D.S., 2008. Clinical status of anti-cancer agents derived from marine sources. Anticancer agents Med Chem. 8: 603-617.

6. Wagner, R. and Bladt, S.D.,1996. Plant Drug Diagnosis, A thin layer chromatography,pp:384.

7. Yanagihara, N., Sasaki-Takahashi, M., Sugahara, T., Yamamoto, S., Shinomi, M., Yamashita, E., Hayashida, M., Andoh, T., 2005. Leptosins isolated from marine fungus Leptosphaeraia species inhibit DNA topoisomerases I and/ or II and induce apoptosis by inactivation of AKT / Protein Kinase B. Cancer. Sci. 96: 816-824.

8. Young, X., Jun, L., An-guol., and Jing-Xia, C., 2003.Structure identification for the compound I separated and purified from taxoids produced endophytic fungi (Alternaria alternate var. taxi 1011) J.For.Res.14(3) : 217-233.

Received on 10.04.2011

Accepted on 10.06.2011

Research J. Pharmacology and Pharmacodynamics. 3(5): Sept –Oct. 2011, 250-252