Screening of Antimicrobial activity of bodily luid from three different local ish species around Madanapalle

The ish skin acts as a protective shield against environments that are high in infectious agents. A thin layer of mucus which acts as a defence shield against colonization by aquatic parasites, bacteria and fungi which was mediated by peptides and polypeptides, was covered on the external body surface of the ish. In the present study, we had shown the activity of epidermalmucus of tap water, mineral water and saltwater ish exhibit strong antibacterial activity. Here, we have isolated supernatant of a ish Pamphlet (Pomfret), Tilapia (Oreochromis niloticus) and Cat ish (Clariasgariepinus) mucus. This Bodily luid (Fish mucus) activity was correlated with a strong antibacterial activity (minimal inhibitory concentration for the three ishes) against both Gram-ve and Gram +vebacteria. In this study, the mucus isolated shows an inhibiting effect on the selected microorganisms. The antibacterial activity of ish mucus may be due to the presence of antibacterial glycoproteins and able to kill bacteria by forming large pores in the target membrane. Fishmucus is believed to play an important role in the prevention of colonization by parasites, bacteria and fungi and thus acts as a chemical defence barrier. Our results suggest that ish secrete antibacterial which are able to kill bacteria in the target membrane.


INTRODUCTION
Fish are mostly cold-blooded aquatic vertebrates usually having scales and breathing through gills with the Composition of minerals, enzymes, pigments/ lavors, proteins or are wealthy in items (Hellio et al., 2002). Utilization of bodily luid (Fish Mucus) for research on natural obstruction among ish and their watery circumstance contains of a bodily luid layer made out of biochemically assorted emissions from epidermal and epithelial cells (Ellis, 1999). High potential mucus production is necessary for the Protection from abrasion injury (Blackstock and Pickering, 1982) and to limit frication against the water, the bodily luid layer covers the surface of the exterior body (Bressler and Bressler, 1989). Its have an assortment of naturally dynamic components in the bodily luid, in reality, go about as humoral barrier factors, considering the fact that the ish invulnerability is less modern than that of greater creatures (Alexander and Ingram, 1992). During the past years, a ish bodily luid has additionally been assumes a function in the aversion of parasites, microscopic organisms, and fungi (Ebran et al., 2000).
The antimicrobial agent play an important role in  aquatic organisms which includes ishes, which are continually expressed to pathogenic microorganisms through the surrounding water. The antibacterial activity of ish bodily luid was once acknowledged for a lengthy time then again, previous deals with antibacterial assessments has been coordinated toward marine microbial strains. As per the studies conducted by Dalmo et al. (1997) an in lammatory response such as elevated production of antimicrobial substances is often encountered (Dalmo et al., 1997). It used to be accounted for that epithelial tissues produce antimicrobial which ills in as the mainline of hosts safeguard against microbial intrusion in an assortment of vertebrates along with humans (Ganz, 1999). In this existing study, a series of solvent extracts of mucus from three marine ishes have been display screen for their invitro recreation in opposition to terrestrial Gram negative positive, gram positive bacteria.

Collection of Fish
Fish Collection and Maintenance of Mucus sample used to be accumulated from a Pamphlet (Pomfret), Tilapia (Oreochromis niloticus) and Cat ish (Clariasgariepinus) (Bodyweight; 160g ± 2.324 g) are formed at kaikaluru, that have been received from a neighborhood ish market in Madanapalli, Chittoor, Andhra Pradesh. Then they are saved in three rectangular plastic tanks which has a capacity of above 500 Litres. The ish accustomed to lab conditions in three different types of water, i.e., tap water, mineral water and saltwater and maintained for seven days (Fast, 2002;Austin and Intosh, 1988) by feeding them once a day with industrial feed with feeding strategies of ad libitum (Lekang, 2015). The cleaning of all three tanks was done once a day by changing of 50% water. After Seven days of acclimatization, the ish were used for bodily luid(mucus) collection. Dead ish or ish with skin lesions are not considered for the collection of bodily luid. They are separated from the tanks. Only wholesome ishes had been chosen for mucus collection.

Bodily luid Collection
Bodily luid collected by using way of a modi ied technique of (Subramanian et al., 2008), as shown in Figure 2. Fish modi ied into starved for one day preceding to physical luid series. At the day of bodily luid collection, three ishes was once washed and transferred into a separate sterile polyethylene bag for 10-20 minutes and moved the front and lower back to slough off the bodily luid. Then, the individual ishes transferred again to restoration tanks.

Figure 5: Antimicrobial assay of Fish bodily luid
Physical luids received from three ish used to be then pooled and saved in the fridge at 2 • C till in similar use. The pooled bodily luid mucus sample was further divided one by one with crude, acidic, and aqueous solvents as three elements (Diamond et al., 1991).

Bacteria Culture Conditions
Antimicrobial activities of bodily luid mucus extracts have been examined towards a wide variety of human and ish pathogens along with every gram +ve(Lactobacillus vulgaris, Staphylococcus aureus) and gram -ve bacterium (Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella paratyphi, Salmonella typhi, and Vibrio cholera). All of the microbes have been grown at 38 • C in Luria-Bertani (LB) broth and maintained at equal temperature, without ish pathogen aeromonas hydrophila. The ish pathogen turns out to be grown in nutrient broth at 38 • C.

Bodily luid mucus Extraction and Protein Quanti ication
For crude extract, 20 ml of bodily luid was centrifuged at 4500 rpm for 20 minutes. The obtained supernatant are then saved at 4 • C (Jais et al., 1998). The aqueous extract of bodily luid changed into organized use of a technique as mentioned through (Hellio et al., 2002). 50 ml of bodily luid blended with 50 ml of distilled water and homogenized with the use of a homogenizer. The aggregate used to be then centrifuged at 25,000 for forty minutes at 4 • C (Remi, CPR-23 plus, RA-2313). The acquired Supernatant was collected and iltered with Whatman no.1 ilter paper. The obtained iltrate was collected and stored in the refrigerator at 4 o C. The acidic extract of bodily luid used to be geared up by using a modi ied approach of (Subramanian et al., 2008). The acidic bodily luid mucus mixture was prepared by 30ml of the bodily luid was mixed with 30ml of 3% acetic acid and placed in a boiling electrical water bath for nearly 8mins. Later acidic mixture was cooled with the help of ice to reduce temperature and homogenized by the use of homogenizer. The combination used was then centrifuged at 25,000 rpm accurately 45 minutes by maintaining 4 o C temperature. The supernatant amassed and iltered by using a syringe with 0.22 µm clear out. Elutes which are obtained are again stored at in the refrigerator at 4 • C. Protein quanti ication turns into determined based totally on Bradford protein assay (Bradford, 1976) via the utilization of bovine serum albumin as regularly occurring (Lowry et al., 1951).

Anti-microbial Assay
As mentioned by Rusell et al., antimicrobial activity was represented in diameter phrases by which area of inhibition is measured by using mm units The spectrum of antimicrobial activity grew to be described by means of way of (Russel and Fur, 1977) antimicrobial exercise become expressed in phrases of a diameter of area inhibition had been measured in mm via the usage of vernier callipers and recorded. The Inoculated micro-organism are incubated at 35 o C for 24Hr into a nutrient broth, fungal cultures have been incubated in potato dextrose broth at 25 o C for 48Hr. Those cultures had been spread-plate on Mueller Hinton agar antibiotic susceptibility checking out by means of the usage of sterile cotton and with the help of (1 cm), borer wells are made within the plates. The test bodily luid (0.1ml) was delivered into the well and the plates should be incubated.

FTIR studies
FT-IR spectroscopy samples of mucus from three marine ishes relied on a pattern (5 mg) combined with 50 mg of dried potassium bromide (Kbr) and compressed in addition to preparing as a pellet for studying spectrum as sown in Figure 3.

Protein level
The bodily luid become predicted for its protein stage and it effects 0.724mg/ml of protein content

Protein Quanti ication
The protein quanti ication was used to identify and monitor proteins during puri ication and to access the homogeneity by using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis. SDS-PAGE of bodily luid based on molecular size ranging >20KDa showed many protein bands shown in Figure 4.

Antimicrobial assay
To identify the antibacterial activity of bodily luid extracted from three ishes pamphlet, Tilapia and cat ish were used for inhibition assay on thin agar plates. The antibacterial activity of mucus of the pamphlet, Tilapia and cat ish are represented in the Graph. The bodily luid shows a strong inhibition in the growth of tested bacteria. Maximum zone of inhibition was observed against Vibrio cholera (26mm), Staphylococcus aureus (23mm)and Salmonellaparatyphi(12 mm), whereas the other bacteria show asigni icant inhibition in their growth Salmonella typhi (17mm), Lactobacillus Vulgaris(17 mm), Pseudomonas aeruginosa (20 mm), Proteus mirabilis (20mm), Klebsiella oxytoca(12mm), Escherichia coli (11mm) and Klebsiella pneumonia(13 mm). The comparative antibacterial effect of the mucus of the ishes pamphlet, Tilapia and cat ish with standard drug Chloramphenicol are shown in Figure 5 and Figure 6.

DISCUSSION
The antibacterial activity in mucus samples of three ishes pamphlet, Tilapia and cat ish were tested among different bacteria strains of E.coli, Lactobacillus anguillarum, Clastridiumglutamicum and Staphylococcus aureus, later it was compared against different samples of bacterial cultures. Based on growth curves of bacteria and water was considered as negative controls. The antibacterial activity of ish mucus may be due to the presence of antibacterial glycoproteins and able to kill bacteria by forming large pores in the target membrane (Ebran et al., 1999). In the serum and mucus, number of antibacterial factors were been founded in three ishes in which immunization acts against microbial diseases. (Rainger and Rowley, 1993) Based on the ish epidermis and epidermal mucus study author Hellio et al. (2002) had found antifungal by conducting different activities of antibacterial, antifungal and cytotoxic of body luid extracts from ish. Bodily luid from solid phase extraction of three ishes was tested against E.coli, Lactobacillus anguillarum, Clastridiumglutamicum and Staphylococcus aureus for antibacterial activity. Its resulted L.anguillarum and C.glutamicum were noticed most sensitive micro-organisms during the activity.

CONCLUSIONS
In this research, results suggest that ish mucus have bactericidal properties and thus play important role in the protection of ish and showed that mucus had high potential source of an antimicrobial activity on against the invasion of pathogens. Further study will surely open new window to formulate new drugs for the therapy of infectious diseases caused by pathogenic and opportunistic microorganisms. These properties of mucus suggest that it may be bene icial in aquaculture and human healthrelated applications.