1. INT J CURR SCI 2013, E 35-40
RESEARCH ARTICLE ISSN 2250-1770
Antibacterial properties of secondary metabolites from the endophytic
marine algal bacterial population against chicken meat microbial pathogen
Vinodhkumar Ta
*, Lubobi Ferdinand Shamalaa
, SS Maithilia
, G. Ramanathanb
and S. Sudhakara
a
Department of Microbiology, AVS College of Arts and Science, Salem-636 106, India
c
Department of Microbiology, V.H.N.S.N. College, Virudhunagar-626 001, India
*Corresponding author: sivamicrovinodh@gmail.com; Phone: +91- 9965464010
Abstract
The world’s oceans comprise the largest part of the biosphere and contain the most ancient and diverse forms
of life. The seaweed genus, Amphiroa is a group of marine algae belonging to the class Florideophyceae has come to
attention for more pharmacological products and have received comparatively less bioassay attention. From this
study endophytic bacteria were isolated from seaweeds along the East Coast of India, Palk Strait to find out the
potential of antibacterial activity of Total Heterotrophic bacterial(THB). Based on the morphological characters, 20
different strains were isolated. The effect of bioactive compound from chosen heterotrophic bacteria strains were
assayed for antibacterial effect through Minimum inhibitory concentration and Minimum Bactericidal concentration
and tested for the antimicrobial sensitivity against Vibrio, Streptococci sp and Yersinia sp by cross streak assay. Of
them, 8 strains were showed sensitivity against pathogenic bacteria. The isolated endosymbiotic strains which shown
sensitivity against three pathogenic bacteria were subjected for the Minimum Inhibitory Concentration (MIC) assay
by following standard methodology. It shows that the strain no SW4, SW20 has MIC value of 125 µg against Vibrio
sp the SW16 SW20 and SW4 showed MIC value of 250 µg against Vibrio sp, Streptococci sp and Yersinia pestis,
the SW16 showed MIC value of 250 µg against Vibrio sp, and Streptococci sp and Yersinia pestis but the MIC value
was found lower by 125 µg against Streptococci sp. At a MIC value of 125 µg/ml, the strain SW4 and SW20 showed
inhibitory activity against Vibrio sp. At a MIC value of 250 µg/ml, the strain SW4 and SW16 showed inhibitory
activity against Yersinia pestis.
Keywords: seaweeds; THB strains; antimicrobial activity; Amphiroa anceps
Received: 08th
January; Revised: 16th
February; Accepted: 14th
March; © IJCS New Liberty Group 2013
Introduction
Life originated in the sea and has sustained itself
to the present day. The world’s oceans comprise the
largest part of the biosphere and contain the most
ancient and diverse forms of life. The marine biotopes
contain an unmatched metabolic and organism diversity
(ESF Marine Board Feasibility Study Group Report,
2001). The marine environment is a rich source of both
biological and chemical diversity and has been the
source of unique chemical compounds with potential
for industrial development as pharmaceuticals,

2. Vinodhkumar et al., 2013
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cosmetics, nutritional supplements, molecular probes,
fine chemicals and agrochemicals. In recent years, a
significant number of novel metabolites with potent
pharmacological properties have been discovered from
marine organisms. Although there are only a few
marine-derived products currently on the market,
several robust new compounds derived from marine
natural products are now in the clinical pipeline, with
more clinical development.
Seaweeds refer to any large marine benthic algae
that are multicellular, macrothallic, and thus
differentiated from most algae that are of microscopic
size. They are rich and varied source of bioactive
natural products and have been studied as potential
biocidal and pharmaceutical agents. These plants form
an important renewable resource in the marine
environment and have been a part of human civilization
from time immemorial. In addition, there are a number
of seaweeds with economic potential. It will be of great
significance if these species could be the major role
players in drug development. They grow in the
intertidal, shallow and deep sea areas up to 180 meter
depth and also in estuaries and black waters on the
solid substrate such as rocks, dead corals, pebbles,
shells and other plant materials. They form one of the
important living resources grouped under three
divisions namely, Chlorophyceae (green algae),
Phaeophyceae (brown algae) and Rhodophyceae (red
algae). They are abundant on hard substrates and
commonly extending to depths of 30-40 m. About 624
species have been reported in India with a potential of
77,000 tons (wet weight) per annum. The red seaweeds
contribute 27.0, brown 0.2 and others 72.8%. About
206 species of algae have been reported from the
mangrove environment. Presence of endophytic
microorganisms in the plant host is in the most cases
beneficial for plant. Secondary metabolites produced by
endophytes provide a variety of fitness enhancements
such as increased resistance to herbivore, parasitism,
drought as well as growth enhancements. Endophytes
usually produce the enzymes necessary for the
colonization of plant tissues. It was demonstrated that
most endophytes are able to utilize, at least in-vitro,
most plant cell components. Most of investigated
endophytes utilize xylan and pectin, show lipolytic
activity and produce non-specific peroxidase and
laccases (Leuchtmann et al., 1992), chitinase (Li et al.,
2004) and gluconase (Moy et al., 2002). Endophytes
represent a huge diversity of microbial adaptations that
have developed in special and sequestered
environments, and their diversity and specialized
habituation make them an exciting field of study in the
search new medicines. The hunt of new drug is
particularly important in view of the fact that so many
diseases are developing immunity to some of the
current treatment. Contamination of poultry meat with
food borne pathogens remains an important public
health issue, because it can lead to illness if there is
malpractice in handling, cooking or post cooking
storage of the product. In developed countries, food
borne illness causes human suffering and loss of
productivity, and adds significantly to the cost of food
production and healthcare. It is also a possible cause of
mortality, which is even more of a problem in
developing regions, where the health status of many
individuals is already compromise. Numerically, the
most important agents are Salmonalle, Staphylococcus,
Vibrio and Campylobacter sp.

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The discovery, development and clinical use of
antibiotics during the 20th
century decreased
substantially the morbidity and mortality from bacterial
infections. However, since the 1980’s the introduction
of new agents for clinical use has declined reflecting
both the challenge of identifying new drug classes to
antibacterial drug discovery by the pharmaceutical
industry .Marine bacteria being a heterotrophy with
simple cell multiplication process, which can be
cultivated in large amounts inexpensively. This has
prompted the present study, to assess the possible
utilization of associated bacteria as resources, to meet
the sufficient supply of desired metabolites. The
present investigation was initiated to screen the
antibacterial efficacy of marine algae associated
bacterial population against the meat pathogens
Materials and Methods
Description of the Study area
Thondi is situated in the Palk Strait region of
Tamil Nadu. The study area lies in the latitude of
99°44”N and 79 10’ 45” E. Amphiroa anceps species
of seaweeds were collected during month of (January
2012) for the isolation of endophytic THB strains
Isolation of Endophytic Organisms
The 1 gm of fresh sea weeds species were
aseptically weighed, washed thrice with sterilized
distilled water and were plated with the Zobell marine
agar medium. The bacterial species enumerated on the
Zobell marine 2216e (Hi-media) agar medium were
counted and the total number of bacterial counts was
expressed as Colony Forming Unit (CFU).
Isolation of chicken meat associated pathogens
The fresh chicken meat samples were collected in
a sterile container. The chicken meat associated
pathogens were isolated by serial dilution method. The
chosen isolates were identified using standard staining
and biochemical test. (Bergey’s manual-Holt et al.,
1994)
Antibacterial sensitivity assay
The antagonistic activity was tested by following
cross streak assay method (Sivakumar et al., 2005)
using 20 different THB strains against chicken meat
associated pathogen viz, Vibrio, Streptococcus, and
Yersinia pestis. Single streak of the isolated strains
was done on sterile Muller Hinton Agar plates
followed by overnight culture of chicken meat
associated pathogens were streaked at
perpendicular to the original streak of isolates and
incubated at 37±2°C. Bacterial strains showed
maximum inhibitory effect against tested pathogens
were subjected for mass cultivation in broth and was
filtered by using Millipore filter. Filtrate was mixed
with equal volume of ethyl acetate (v/v) in separating
funnel and shaken well and then allowed to stand
without any disturbance for 15 minutes. After that, the
lower aqueous phase was discarded and the upper
solvent phase was concentrated in a vacuum
evaporator at room temperature for 24 hrs to obtain
powder form of crude extract and stored in a
refrigerator for further analysis (Kang et al., 2007).
For MIC determination 0.5 ml of various
concentration of extracts (31, 62, 125, 250, 500, 1000,
1500 and 2000 µg) was prepared with Dimethyl
sulphoxide (DMSO) and mixed with 0.5 ml of nutrient
broth. 50 µl of bacterial inoculums serves as positive
control. Nutrient Broth alone served as negative
control. Whole setups in duplicates were incubated at
37°C for 48 hrs. The MIC was the lowest

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concentration of the extract that did not permit any
visible growth after 24 hrs of incubation and it was
examined on the basis of turbidity. To avoid the
possibility of misinterpretations due to the turbidity of
insoluble compounds, the minimum bactericidal
concentration (MBC) was determined by sub culturing
the MIC dilutions on to the sterile agar plates. The
lowest concentration of the extracts which inhibits the
growth of tested bacteria are observed and tabulated.
Results
About 20 bacterial strains were isolated as
endosymbiont from the marine algae Amphiroa anceps
and all of them have been tested for the antimicrobial
sensitivity against Vibrio, Streptococci and Yersinia sp
by cross streak assay. Of them, eight strains were
shown sensitivity against three pathogenic bacteria.
The isolated endosymbiotic strains which shown
sensitivity against three pathogenic bacteria were
subjected for the Minimum Inhibitory Concentration
(MIC) assay by following standard methodology. It
shows that the strain no SW4, SW20 has MIC value of
125 µg against Vibrio sp the SW16 SW20 and SW4
showed MIC value of 250 µg against Vibrio sp,
Streptococci sp and Yersinia pestis, the SW16 showed
MIC value of 250 µg against Vibrio sp, and
Streptococci sp and Yersinia pestis.
The MIC value was found lower by 125 µg
against Streptococci sp. Surprisingly, the strain no
SW4 showed MBC value of 500 µg to 2 of the
pathogenic bacteria viz., Vibrio sp and Yersinia pestis.
However, the other strains did not showed any MIC
and MBC values as mentioned in the tables. The
presence and absence of bacterial turbidity and the
effective concentration of sensitivity (MIC and MBC)
by the isolated bacterial strains against Yersinia pestis
particularly the isolated strains of SW4 and SW16
against Vibrio sp and Streptococci sp.
Fig. 1. Marine algae Amphiroa anceps
Discussion
Life originated in the sea and has sustained itself
to the present day. The Marine ecosystem is a rich
source biological diversity. This has been exploding in
the discovery of unique chemicals having
potential for industrial development as
pharmaceuticals. In recent years, a significant number
of novel secondary metabolites with potential
pharmacological properties have been discovered from
marine organisms. The world’s oceans comprise the
largest part of the biosphere and contain the most
ancient and diverse forms of life. The endo symbiotic
heterotrophic bacteria have been isolated from sea
weed species. Several studies have indicated that
consumption of poultry meat has been associated with
incidence of outbreaks of food borne illnesses
(Lunden et al., 2003; Prakash et al., 2005). Reduction
of initial bacterial load in meat is of prime importance
in an attempt to improve the shelf-life of the product
(Lillard et al., 1984).

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Table 1. Antibacterial efficacy of marine algal
associated bacteria against chicken meat associated
pathogens through cross streak assay. SW – Seaweed;
+ - Presence of Activity; - - Absence of Activity
Endophytic
Bacterial Strain
Chicken meat bacterial pathogen
Strain no
Vibrio
sp
Streptococci
sp
Yersinia
sp
Sw13 - + +
Sw14 + - +
Sw4 + + -
Sw5 - + -
Sw6 - + -
Sw16 + + +
Sw18 - + +
Sw20 - + -
Hence, the present study was undertaken to produce
valuable secondary metabolites from the marine algae
against chicken meat associated pathogens. Based on
the morphological characters, 20 strains were isolated
and all of them have been tested for the antimicrobial
sensitivity against Vibrio, Streptococci sp and Yersinia
sp by cross streak assay. Generally, the endophytic
bacteria isolated from seaweeds showed maximum
sensitivity against several human bacterial pathogens.
Moreover, the bioactive compounds from
endophytic bacteria showed maximum sensitivity with
minimum concentration than the bioactive compounds
from epiphytic bacteria of other biological origin. The
earliest marine bio active compounds were isolated
from endosymbionts of sponges. The bioactive
secondary metabolites isolated from endosymbionts of
sponges are produced by functional enzyme clusters,
originated from the sponges associated
microorganisms. A wide range of chemical and
functional diversity has been observed among
bioactive compounds. Of the various classes of
compounds, polyketides, alkaloids, fatty acids,
peptides and terpenes are the most abundant ones.
Table 2. Minimum Inhibitory Concentration (MIC)
and Minimum Bactericidal Concentration (MBC) (µg
ml-1) of the extracts from endophytic bacteria against
chicken meat associated pathogen. SW – Seaweed
Strain
no.
Vibrio sp Streptococci sp Yersinia sp
MIC MBC MIC MBC MIC MBC
SW4 125 250 500 500 125 500
SW5 - - - - - -
SW6 - - - - - -
SW13 - - - - - -
SW14 - - - - - -
SW16 250 500 125 250 250 250
SW18 - - - - - -
SW20 125 250 250 500 500 -
Majority of them show antimicrobial, antitumor and
anticancer properties. Tritetracontane, Nonadecane,
Docazane, Indolirine, Trifluroaceticacid, n-octadecyl
ester are highly abundant and have been shown to be
multifunctional. Triterpenes attract attention because
of their biological activities of endosymbionts. Hence,
steps have been undertaken to find out the reason for
the maximum activity of endophytic bacteria from
seaweeds and complete structural elucidation of the
potential metabolites will also been focused in future.
Acknowledgment
The authors are thankful to the authorities of
AVS College of Arts And Science, Salem and

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V.H.N.S.N. College, Virudhunagar, Tamil Nadu, India
for providing required facilities to complete this work.
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