3. 3
•Bhattacharya et al., (2012) studied the antagonistic relation among different microbial
community in Vallapattanam and Pappinishery mangrove soils. Of the 28 bacteria
isolated majority of them were gram positive rods. 12 actinomycetes showed
antagonistic activity towards bacteria isolated from the same soil sample. While
antifungal activity was reported against native fungi namely, Aspergillus flavus,
Penicillium, Trichoderma, Candida albicans, C.parapsilosis, Trichophyton,
T.mentagrophytes and Cryptococcus.
•Ravikumar. S et al., (2011) observed diversity of actinomycetes in Manakkudi
mangrove ecosystem, Southwest coast of India. The existence of actinomycete is found
maximum in rhizosphere soil than the non-rhizosphere soil. Efforts have been undertaken
to isolate potentially useful actinomycetes.
4. 4
•Sateesh V.Naikpatil et al., (2011) opined that mangrove system still formed the unexplored source
of many microorganisms particularly actinomycetes. In this research fifty three rare actinomycetes
strain were isolated using selective approaches from mangrove forest of Karwar. Of the
selected isolated actinomycete belonging to the genus Micromonospora and Actinomodura
were selected for antibacterial activity.
•Md. Anwar - Ul Islam, et al., (2011), carried research on isolation of actinomycete colonies from
various soil samples having antibiotic principles collected from different places in and around
Rajshahi, Bangladesh. Out of the 30 isolates, 16 were found to have moderate to good activity
against four Gram positive and four Gram negative bacteria thus making the soil an interesting
source to explore novel antibiotics.
•Usha. R et al., (2010) carried work on actinomycetes isolated from Pichavaram
mangrove soils which possess specific substances demonstrating inhibitory activity
against prokaryotic and eukaryotic microorganisms. Of all the isolates Streptomyces sp. KUAP 106
was found to possess antimicrobial and antiangiogenic property. The organism was found to
be Streptomyces parvulus KUAP 106 and suggested to be an anticancer antibiotic.
5. 5
•Sahoo. K & Dhal N.K., (2009) studied potential microbial diversity in mangrove soils. Mangrove
forests are distributed in 112 countries and territories comprising of 181,000 sq.km comprising of
quarter of total coastline of the world. Analysis of microbial biodiversity will help in isolating and
identifying new and potential microorganisms having novel applications.
•Sunil Kumar. R, (2000) studied various microbial diversity dwelling in mangrove soil system.
Studies were carried out on mangroves found in West Bengal, Orissa, Andhra Pradesh, Tamil Nadu,
Karnataka, Kerala, Goa, Gujarat, Maharashtra and Andaman & Nicobar Islands. However
information is still needed primarily on species composition and diversity of
microorganisms in mangrove soils in Indian subcontinent.
7. • Soil sample from different directions were collected from Koringa
Mangrove Forest, near Kakinada region of Andhra Pradesh.
• Sample from North was selected and is designated as KMF-N
ISOLATION OF ACTINOMYCETES:
• One gram of soil sample is mixed in 50mL of sterile water and placed on Rotary
shaker at 120rpm for 30mins.
• After 30mins, dilutions were made upto 10-10 order.
• One ml of soil sample+ 1mL of Rifampicin (25μg/mL medium) + 1mL of
Cycloheximide(50μg/mL medium) added to 50mL of Starch Casein agar medium
PLATED.
7
8. ISOLATION AND MAINTENANCE OF CULTURES
Plates were incubated at 28oC for 7 days GROWTH OF ACTINOMYCETES
ACTINOMYCETES
Screening of actinomycetes on Growth of KMF-A1 on YEME medium.
Starch Casein Agar medium.
•Small portion of organism- removed- streaked on YEME medium- KMF-A1
•YEME medium was prepared by using 50% sea water and used to maintain
isolated cultures.
8
9. Determination of antimicrobial activity
CROSS STREAK METHOD
AGAR OVERLAY METHOD
CUP PLATE METHOD
CROSS STREAK METHOD:
Molten agar medium was poured into sterile petri plate After
solidification, our sample KMF-A1 was streaked as single straight line in the
centre Incubation for 7days.
After 7 days test organisms (Gram positive, Gram negative and fungi )
were streaked perpendicular to the isolate without touching Streptomycete
growth.
Incubation period: 37oC for 24 hours (Bacteria)
27oC for 48 hours (Fungi)
9
10. Cross streak method of KMF-A1 against bacteria
Cross streak method of KMF-A1 against fungi 10
11. AGAR OVERLAY METHOD:
• Isolate KMF-A1 showed positive result against fungi while antibacterial activity
was not observed with cross streak method.
• Molten YEME agar medium Plated KMF-A1 was stabbed centrally
• Plates were incubated at 28oC for 7 days colonies of few millimeters size.
• Test organisms Inoculated in molten Saboroud’s agar medium poured in
the petri plate without disturbing Streptomycete colony. Plates were incubated at
27oC for 48 hours.
• TEST ORGANISMS USED WERE:
Candida albicans
Aspergillus niger
Penicillium chrysogenum.
11
14. Initially production of the isolate KMF-A1 was carried out in different media
containing 50% sea water. For each medium, activity was determined by
performing cup plate method. Better zones of inhibition were obtained with
Production Medium (PM)-7 against Candida albicans.
Inhibition zone diameters of crude extract obtained in different production media.
SNo. Production
medium
(PM)
Inhibition zone diameters (mm)
1. PM-1 13
2. PM-2 14
3. PM-3 NS
4. PM-4 NS
5. PM-5 11
6. PM-6 14
7. PM-7 21
14
15. Composition of Production Medium (PM-7): (%w/v)
Soyabean meal 1.5%
Glucose 1.5%
Glycerol 0.25%
NaCl 0.5%
CaCO3 0.1%
Distilled water (q.s) 50mL
Sea water (q.s) 50mL
After production, the media was centrifuged and mycelia
were separated. The color of the clear supernatant solution
is Reddish brown. The antimicrobial activity of the crude
supernatant was carried out against C.albicans and
Pectinotricham llanense using Cup Plate Method. The
diameter of zones of inhibition was measured against
standard Ketoconazole.
Crude extract of KMF-A1
15
16. Determination of antifungal activity of crude supernatant with Candida albicans.
CRUDE SUPERNATANT
Determination of antifungal activity of crude supernatant with Pectinotricham llanense
16
17. 17
•The extraction of supernatant was carried out with various solvents like
Ethyl acetate, Methanol, Hexane in Rotary Evaporator at temperature
40οC and its antifungal activity was determined using Cup Plate method.
• The concentrated product after extraction was pale brown in color. The
sample was subjected to lyophilization and the activity of the freeze dried
sample was determined. The test organism is Candida albicans.
18. DETERMINATION OFACTIVITY
SUPERNATANT
FREEZE DRIED SAMPLE
SUPERNATANT
ETHYL ACETATE FRACTION
AQUEOUS FRACTION
ANTI FUNGAL
ACTIVITY OF FREEZE
DRIED SAMPLE
DETERMINATION OF
ANTIFUNGAL ACTIVITY
WITH FRACTIONS
EXTRACTED WITH
ETHYL ACETATE.
4
18
20. The proteolytic activity was studied with milk casein agar medium
by measuring hydrolyzed zone after incubation at 28oC for 7 days.
The ratio of the diameter of the hydrolyzed zone and that of the
growth on the milk-casein agar medium was measured 5.1.
Determination of Proteolytic acitivity of KMF-A1 using Skim Milk Agar medium.
20
21. Soil Samples from different directions were collected from Koringa Mada
Forest near Kakinada region, Andhra Pradesh.
Four cultures of actinomycetes were isolated from “North” sample and selected
for further study. Isolate KMF-A1 showed better antimicrobial activity
compared to all other isolates. This was selected for fermentative production
of the antibiotic.
Screening of actinomycete isolated from mangrove soil resulted in isolation
of biologically active compound that is effective against pathogenic fungi. In
the course of our screening, we found mangrove soils are good source of
actinomycetes possessing antimicrobial properties.
21
22. Extraction of active principle was carried out using various solvents like
Ethyl acetate, hexane and methanol. The activities of both organic and
aqueous fractions were determined each time. After many trials, it was found
that the active principle was retained in the aqueous fraction only.
The antibiotic activity in different production media was determined
against Candida albicans. Ketoconazole was used as the standard drug.
Microscopy and SEM studies showed thin, entangled spirals, mycelia of
various lengths with matured hyphae showing 10 to 50 spores per chain.
Thin, spiny spores were observed in 14day culture grown on YEME agar
medium.
Major macroscopic characteristics include white tuft colony gradually
turning pale purple and deep violet or red on reverse side.
22
23. •Alongi D M, Present state and future of the world's mangrove forests. Enivron. Cons., 29(3),
2002, 331-349.
•Bhattacharya Sourav, Chaitali Nag and Arijit Das., Evaluation of antagonistic activities of microbes
from Vallapattanam and Pappinishery mangrove ecosystems of Kannur district in Kerala, India,
International Journal of Pharmacy & Life Sciences., 3(5), 2012, 1650-1659.
•Cross and Mccarthy, A. J., Taxonomic study of thermomonospora and other monosporic
Actinomycetes, Journal of' General Microbiology,130, 1973, 5 - 25.
•Kim, C.J., K.H. Lee, O.S. Kwon, A. Shimazu, and I.D. Yoo. Selective isolation of actinomycetes by
physical pretreatment of soil sample. J. Appl. Microbiol. Biotechnol. 22, 1994, 222-225.
•Nolan, R.D. and T. Cross. 1988. Isolation and screening of actinomycetes, p. 2-8.
•Okami, Y., Okazaki, T., Studies on marine microorganisms isolation from the sea. J. Antibioti.,
192, 25, 1988, 456-460.
23
24. •Ozgur Ceylan., Gulten Okmen and Aysel Ugur., Isolation of soil Streptomyces as source antibiotics
active against antibiotic-resistant bacteria, EurAsia J Bio Sci, 2, 2008, 73-82.
•Poch G K & Gloor J B, Auranticins A and B: two new depsidones from a mangrove isolate of the
fungus Preussia aurantiaca. Jol.of Nat.Prod.Lloyd., 54(1), 1991, 213-217
•Rathna Kala R, Chandrika V (1993). Effect of different media for isolation, growth and maintenance
of actinomycetes from mangrove sediments. Indian J. Mar. Sci. 22: 297-299.
•Sahoo. K. and Dhal. N.K., Potential microbial diversity in mangrove ecosystems: A Review,
Indian Journal of Marine Sciences, 38(2), 2009, 249-256
•Salle, A J., Laboratory Manual on Fundamental Principles of Bacteriology (3rd ed.), 1948,
Mc Graw-Hill Book Co.
•Sateesh V.Naikpatil and Rathod J.L., Selective isolation and antimicrobial activity of rare
actinomycetes from mangrove sediment of Karwar, Journal of Ecobiotechnology, 3(10), 2011,
48-53.
24
25. •Usha.R., Ananthaselvi. P., Venil. C.K. and Palaniswamy. M., Antimicrobial and Antiangiogenesis
activity of Streptomyces parvulus KUAP106 from mangrove soil., European Journal of Biological
Sciences., 2(4), 2010, 77-83.
•Wael N. Hozzein, Walaa Rabie and Mohammed Ibrahim Ali, A., Screening the Egyptian
desert actinomycetes as candidates for new antimicrobial compounds and identification of a new
Desert Streptomyces strain, African Journal of Biotechnology, 10(12), 2011, 2295-2301.
•Williams, S. T. and Davies, F. L.J. Gen. Microbiol., 48, 1967, 171.
•Wulf Crueger and Anneliese Crueger., A textbook of industrial microbiology, Second edition,
Panima Publishing Coorporation, 2005, 229-233.
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