Research Paper: Iyyanuchamy and Periyanayagasamy., 2011: Pp.6-10 SCREENING THE BIOACTIVE POTENTIAL OF PROTEIN ISOLATED FROM CYPRINUS CARPIO Iyyanuchamy, K and A. Periyanayagasamy* Department of Microbiology, Sri Paramakalyani College, Alwarkurichi 627412. *Research and Development Centre, Bharathiar University, Coimbatore, India. ABSTRACT Screening the bioactive potential of fish protein was analysed against bacterial pathogens. Regarding fish, several compounds have been extracted and these are employed as remedies in the official medicine. Fish protein was isolated from a fresh water fish, Cyprinus carpio collected in three different regions at Tamirabarani, Veranam and Manimuthar, Tamilnadu, India and characterized with screening of antibacterial efficiency. The isolated protein samples have effective bioactive compound, it was confirmed by antimicrobial activity. Manimuthar protein sample effectively inhibit all tested bacterial pathogens compared to other samples. In conclusion, the finding in this study suggests that the fish protein possess antibacterial activity against pathogens. Keywords: Protein, pathogen, bioactive potential and medicine. INTRODUCTION Microbes play a complementary role in digesting foodstuffs, which cannot be tackled by the native enzymes of the host organisms (De Ridder et al., 1985) the human colonic micro biota plays an important role in human nutrition and health, by promoting nutrient supply, preventing pathogen colonization and shaping and monitoring mucosing immunity (Svenson et al., 1994). The symbiotic microbes are mostly common in herbivorous forms as the type of food is a broad spectrum of vegetable matter (Sornaraj et al., 2003). The carnivores on the other hand, do not face such a problems, since they take more or less uniform type of materials as food. They too lodge populations of some species of microbes, which are symbiotic and complement the native enzymes (Ranjitsingh et al, 2003). All these associations are beneficial and such relationships should have existed from early evolutionary times. Regarding fish, several compounds have been extracted and these are employed as remedies in the official medicine (Hamada and Nagai, 1995). Today, some of these compounds are important as tools for biochemical research or as new leads for the development of anticancer and antiviral drugs (Higa, 1996). Agosta (1996) says that ''one of the most promising discoveries of the 1990s is a new chemical compound derived from dogfish sharks (Squalus acanthias Linnaeus 1758) that kills parasites, fungi, and bacteria. Hence in the present paper is screening the antibacterial efficiency of fish protein against bacterial pathogens. MATERIALS AND METHODS Protein isolation, purification and characterization: In the present study, protein was isolated from a fresh water fish, Cyprinus carpio collected in three different regions at Tamirabarani, Veranam and Manimuthar, Tirunelveli District, Tamilnadu, India. The isolated protein purified with help of thin layer chromatography and ion exchange chromatography methods and characterized by western blot techniques as per standard Antimicrobial assay: Anti microbial activity of protein isolated from fishes was screened using filter paper disc agar diffusion method and compared with standard antibiotic, streptomycin. Twenty millilitres of the sterilized medium was taken in each petridish. 24 hour broth culture of sub-cultured organisms was inoculated evenly over the surface of the agar plates. Sterilized Whatman filter. 1 discs thoroughly moistened with different concentrations of samples were placed on the surface of the plate. Discs moistened with sterile water were used as control. The plates were incubated at 37 o C for 24 hour. After incubation the zone of inhibition was measured. In well plate method instead of disc, wells were prepared using cork puncture. All other protocols were same to disc diffusion MIC and MBC test: Minimum inhibition concentration (MIC) of the extracts was determined from the culture plates that had the lowest concentrations and prevented and growth of bacterial strain. Minimum bacterial concentration (MBC) was determined by using the method of Samy and Ignacimuthu (2001). The whole plant extracts were diluted to obtain concentration ranging from 10 µg -100 µg /ml. The test containing 3ml of Muller Hinton broth and 0.1 ml bacterial suspensions and 0.1 ml plant extract were incubated at 37 c for 24h. Bacterial turbidity was measured at 650 nm to determine bacterial inhibition. Streptomycin at 20 and 40µg /ml was used as a reference for determination of minimum bactericidal concentrations. The tubes containing only the growth medium and each of the organisms were used as control. The minimum bactericidal Rising Research Journal Publications 6
concentration that showed the reduction of the bacterial colony as measured from the turbidity of the culture assay optical density value. Total bacterial count of each bacterial species was estimated by counting the number of bacteria in each test tube incorporated with different concentrations of plant extracts and control. The average of three counting was taken as the total number of colony forming bacterial suspensions. All determinations were made in triplicate of extracts. RESULTS AND DISCUSSION Thin Layer Chromatography: The complex proteins were obtained in Manimuthar sample because the RF value was very less (0.9 cm) compared to other samples (Table 1). Western Blot analysis: Separated protein sample were screened with antiserum (raised against same protein) by western blot It shows three specific proteins obtained in Manimuthar sample. It helps to develop immunity and stress tolerant capability of test animals (Table 2). Antimicrobial assay: The isolated protein samples have effective bioactive compound, it was confirmed by antimicrobial activity. Manimuthar protein sample effectively inhibit all tested bacterial pathogens (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Kliebsiella pneumonia,, Serratia marcescens,, Staphylococcus aureus and Bacillus subtilis). The percentage of inhibition increased when the concentration of protein increased (Table 3-5). Minimum inhibitory Concentration: The least concentration of Manimuthar protein sample has potential inhibitory effect growth of bacteria compared to other samples (Table 6). Minimum Bactericidal Concentration: The least concentration of Manimuthar protein sample has potential killing effect of bacteria compared to other samples (Table 7-9). It is interesting to note that fish protein may have antibacterial activity due to the presence of various aminoacids and peptides. Therefore present investigation may demonstrate antibacterial activity of three different fish protein extracts. In conclusion, the finding in this study suggests that the fish protein possess antibacterial activity against pathogens. The present study is a preliminary attempt in evaluating the antibacterial activity of fish protein. Further pharmacological investigations are warranted in this direction for establishing its detailed mechanism of action and for substantiating its traditional and folk claims. REFERENCES De Ridder, C., Tangoux, C. and Devas, L. 1985. Description and significance of a particular intra digestive symbiosis between bacteria and adeposit feeding echinold. J. Exp. Mar. Biol. Ecol., 91: 65-76. Svenson, B.G., Hall berg, T., Nilson, A., Sehutz, A. and Hagmar, L. 1994. Parameters of immunological competence subjects with high consumption of fish contaminated with persistent organochlorine compound. Int. Arch. Occup. Environ. Health, 654: 351-358. Sornaraj, R. 2000. Impact of pesticides on the physiology and biochemistry of a fresh water fish. Ph.D. Thesis, Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India. Ranjitsingh, A.J.A., Sornaraj, R. and Dhasarathan, P. 2003. Bacteriological indicators in fish exposed to pesticides. Bull. Environ. Contam. Toxicol., 70: 85-89. Agosta W, 1996. Bombardier beetle and fever trees: A close up look at chemical warfare and signals in animals and plants. Addison-Wesley, Reading, MA. Higa, T. 1996. Effective Microorganisms -Their role in Kyusei Nature Farming and sustainable agriculture. In Proceedings of the Third International Conference on Kyusei Nature Farming. Ed. J.F. Parr et al., USDA, Washington, USA:20-24. Hamada, Minoru and Tadashi Nagai. 1995. Inorganic component s of bone s of f i sh and the i r advanc ed ut i l i z a t ion. Journa l of Shimonos eki University of Fisheries 43(4):185-194 Rising Research Journal Publications 7
Table 1: TLC analysis of fish protein Character Sample Tamirabarani Manimuthar Veranam 1. RF value 0.8 c.m 0.9 c.m 0.7 c.m c.m 2. of spots 2 1 1 3. Pigmentation Light brown Light green Table 2: Western Blot analysis of fish protein Character Sample Brown Green Tamirabarani Manimuthar Veranam 1. of bands 1 2 1 2. Mol weight range (KD) 60-90 70-80 70-100 Table 3: Antibacterial effect of protein (Tamirabarani) extracts against pathogenic bacteria in disc diffusion 0.7 0.8 1.5 1.2 2 Pseudomonas aeruginosa 1.1 1.5 1.8 2.0 3 Proteus vulgaris 0.5 1.3 1.5 1.7 4 Kliebsiella pneumoniae 0.4 1.2 1.5 1.8 5 0.4 1.1 1.5 1.6 6 Serratia marcescens 0.1 0.4 7 0.1 1.1 1.2 1.5 8 Staphylococcus aureus 0.2 1.2 1.1 1.3 9 Bacillus subtilis 0.5 0.7 1.1 1.2 1.4 Table 4: Antibacterial effect of protein (Manimuthar) extracts against pathogenic bacteria in disc diffusion 0.5 0.7 1.6 2.2 2.6 2 Pseudomonas aeruginosa 0.7 1.2 1.6 2.3 3 Proteus vulgaris 0.7 0.8 1.6 2.1 2.8 4 Kliebsiella pneumoniae 0.7 0.9 1.5 2.2 2.5 5 0.7 1.2 1.5 2.4 3.1 6 Serratia marcescens 0.9 1.3 1.4 2.3 2.4 7 0.8 1.2 1.3 2.2 2.1 8 Staphylococcus aureus 0.5 0.8 1.1 1.6 1.8 9 Bacillus subtilis 0.4 05 1.5 2.1 Rising Research Journal Publications 8
Table 5: Antibacterial effect of protein (Veranam) extracts against pathogenic bacteria in disc diffusion 0.8 1.1 1.3 2.5 3.0 2 Pseudomonas aeruginosa 0.8 1.2 2.9 3.0 3 Proteus vulgaris 0.7 1.3 2.5 2.6 3.0 4 Kliebsiella pneumoniae 1.8 2.5 2.7 2.8 5 0.8 1.6 1.8 2.3 2.7 6 Serratia marcescens 0.8 1.2 2.1 2.6 2.9 7 0.7 1.5 2.1 2.3 3.0 8 Staphylococcus aureus 0.9 2 2.2 2.2 2.9 9 Bacillus subtilis 0.2 1.3 2.1 2.6 Table 6. The minimum inhibitory effect of fish protein extracts against pathogenic bacteria in disc diffusion Extract K.pneumoniae marcescens aureus B.subtilis 1 Tamirabarani 20 30 40 20 40 50 60 60 40 2 Manimuthar 10 20 10 10 10 20 20 20 20 3 Veranam 20 30 40 30 50 50 60 40 50 Table 7. The minimum bactericidal concentration of fish protein (Tamirabarani) extracts against pathogenic bacteria in disc diffusion K. pneumonia marcescens Staphylococcus aureus Bacillus subtilis 1 rmal 1.8 1.7 1.9 2.0 2.0 2.0 1.9 1.8 1.9 2 20 1.1 1.4 1. 8 1.2 1.2 1.0 1.5 1.6 1.6 3 40 1.1 1.2 1.2 1.2 1.2 1.5 1.5 1.4 1.2 4 60 1.0 1.0 1.0 1.0 1.1 0.8 0.9 0.9 5 80 0.4 0.5 0.5 0.5 0.5 0.7 0.5 6 100 0.1 0.4 0.8 0.2 0.2 0.5 0.5 0.1 8 Streptomycin 100 0.1 0.2 0.1 0.4 0.5 0.2 0.1 Rising Research Journal Publications 9
Table 8. The minimum bactericidal concentration of fish protein (Manimuthar) extracts against pathogenic bacteria in disc diffusion N o K. pneumoniae marcescens aureus Bacillus subtilis 1 rmal 1.8 1.7 1.9 2.0 2.0 2.0 1.9 1.8 1.9 2 20 0.8 0.8 0.9 0.9 0.8 0.7 0.8 3 40 0.5 0.7 0.8 0.7 0.7 0.7 4 60 0.4 0.4 0.5 0.5 0.5 0.5 5 80 0.2 0.4 0.4 0.4 0.4 0.4 6 100 0.1 0.2 0.1 0.2 0.1 0.2 0.2 8 Streptomycin 100 0.1 0.2 0.1 0.4 0.5 0.2 0.1 Table 9. The minimum bactericidal concentration of fish protein (Veranam) extracts against pathogenic bacteria in disc diffusion K. pneumoniae marcescens Staphylococcus aureus B. subtilis 1 rmal 1.8 1.7 1.9 2.0 2.0 2.0 1.9 1.8 1.9 2 20 1.7 1.6 1.8 1.8 1.7 1.8 1.7 1.6 1.7 3 40 1.4 1.6 1.4 1.4 1.3 1.6 1.6 1.4 1.6 4 60 1.1 1.6 1.9 1.2 1.4 1.5 1.5 1.2 1.1 5 80 0.9 1.2 1.1 1.2 1.2 1.4 1.5 1.1 1.1 6 100 0.8 1.1 1.1 1.1 1.1 1.2 1.3 1.1 1.2 8 Streptomycin - 100 0.1 0.2 0.1 0.4 0.5 0.2 0.1 ******************* Rising Research Journal Publications 10