International Research Journal of Microbiology (IRJM) (ISSN: 2141-5463) Vol. 3(10) pp. 328-332, October 2012
Available online http://www.interesjournals.org/IRJM
Copyright © 2012 International Research Journals
Full Length Research Paper
Phytochemical screening, microbial load and
antimicrobial activity of underexploited seaweeds
Abirami R. G* and Kowsalya S.
Department of Food Science and Nutrition, Avinashilingam University for Women, Coimbatore- 64103,
Tamil Nadu- India
Accepted 16 April, 2012
Underexploited seaweeds of south India namely Acanthophora spicifera, Gracilaria edulis,
Padina gymnospora, Ulva fasciata and Enteromorpha flexuosa collected from Gulf of Mannar
Coast, Ramanathapuram district India, were screened for their antimicrobial potential against
Staphylococcus aureus, Streptococci, Escherichia coli, Klebsiella aerogenes, Proteus vulgaris,
Aspergillus niger and Candida albicans by disc diffusion method. The phytochemical analysis
had showed the presence of all phytochemicals except alkaloids which was absent in all the
selected seaweeds and quiniones were absent in brown seaweed like Padina gymnospora and
green seaweed like Ulva fasciata, Enteromorpha flexuosa. There were no detectable Salmonella
typhii and faecal streptococci in all selected seaweeds, other tested organisms were found to be
within the desirable limit. The methanolic extracts were found to inhibit the growth of all bacteria
and fungal organisms tested. The antimicrobial effects of extracts were comparable with the
standard antibacterial agent amikarin and fluconazole as standard antifungal agent and were
found to be active against all the organisms tested. Thus this study proved remarkable
antimicrobial potentials for the selected seaweeds.
Keywords: Seaweeds, antibacterial, antifungal, amikacin and fluconazole.
INTRODUCTION
Seaweeds are considered as such a source of bioactive
compounds as they are able to produce a great variety
of secondary metabolites characterized by a broad
spectrum of biological activities. Compounds with
cytostatic, antiviral, antihelmintic, antifungal, and
antibacterial activities have been detected in green,
brown and red algae (Lindequist and Schweder, 2001;
Newman et al., 2003). There are numerous reports
concerning the inhibiting activities from macroalgae
against human pathogens, fungi and yeasts. Marine
macroalgae, commonly known as seaweeds, are one of
the living renewable resources of the oceans with
potential food applications. Consumption of seaweeds
as sea vegetables in human diets has been the
common practice in several Asian countries (Nisizawa,
2002).
Antibiotic treatment of microbial diseases has been
applied for many years. The prevention and treatment
of these infectious diseases by applying products from
*Corresponding Author E-mail: abirami.rg@gmail.com
marine organisms appears as a possible alternative.
Hence, the interest in marine organisms as a potential
and a promising source of pharmaceutical agents has
increased during the last years (Lindequist and
Schweder, 2001; Mayer and Hamann, 2002; Newman
et al., 2003). Hence the present study was planned to
evaluate the phytochemical components, microbial load
and antibacterial activity of methanolic extract of
selected seaweeds.
MATERIALS AND METHODS
Selection of Seaweed material
Based on the results of pilot study on consumption
pattern of seaweeds: (Kowsalya and Abirami, 2011) in
the Mandapam area of Ramanathapuram district, Tamil
Nadu-India. We studied the nutritive contribution of Ulva
lactuca and Kappaphycus alvarezii. Now many edible
seaweeds are available in this area but are
underutilized by the masses. Hence it was thought of
interest to select red seaweeds like Gracilaria edulis,
Abirami and Kowsalya 329
and Acanthophora spicifera, brown seaweeds Padina
gymnospora and green seaweeds like Ulva fasciata and
Enteromorpha flexuosa are grown in abundance as
dominant community. The selected seaweeds were
authenticated in Central Salt and Marine Chemicals
Research Institute, Mandapam, TamilNadu. The
collected samples were washed immediately in
seawater and then washed thoroughly with fresh water,
transported to the laboratory in an iced condition.
Initially, the seaweeds were shade dried at room
temperature for 48-72 hours.
The shade dried
seaweeds were powdered and used for further
experiments.
Preparation of seaweed extract
Methanolic Extract
The dried seaweeds were coarsely powdered and 250g
of this seaweed powder was packed in soxhlet extractor
of one liter capacity. The solvent methanol was added
into the flask and heated. The temperature was
maintained at 600C to 700C throughout the extraction.
The soluble active constituents of the extract remained
in the flask and the process was repeated until the
compounds were completely extracted. The liquid
extract was then cooled and concentrated by using a
0
rotary evaporator at 30–45 C. The extract was stored in
labeled sterile screw-capped bottles at 40C until it was
used (Celikler et al., 2009).
Gracilaria edulis, Padina gymnospora, Ulva fasciata
and Enteromorpha flexuosa were evaluated against
bacterial strains like Gram positive Staphylococcus
aureus, Streptococci, Bacillus cereus and Gram
negative Klebsiella aerogenes, E.coli respectively.
Microbes were grown in Mueller Hinton Agar medium
by using agar well diffusion method. The bacterial
strains were inoculated on nutrient broth and incubated
for 24 hours at 30±10C. Adequate amounts of
autoclaved Mueller Hinton Agar medium were
dispensed into sterile plates and allowed to solidify
under aseptic conditions. Then bacterial strains were
placed over plates containing Mueller Hinton Agar
medium using cotton swabs well were made by using
well cutters, 40 and 60µg concentration of extracts were
used in the medium (39g/1000ml). Briefly, the selected
seaweed extracts were dissolved in DMSO. Then wells
were filled with extracts of different concentration under
sterilized condition in laminar airflow chamber, and then
petridishes were covered with thin films and kept the
0
plate under incubation overnight at 37 C. Also amikacin
was used as positive control against bacterial strains
(10 and 20µg/disk). After incubation, all the plates were
observed for zones of growth inhibition and the
diameters of these zones are measured. Inhibitory
activity of DMSO also tested. All tests were carried out
under sterile conditions in triplicate (NCCLS, 2001).
Antifungal activity
The selected five species of seaweeds were analyzed
for the microbial load for coliforms, Escherichia coli,
Salmonella typhii, Staphylococcus aureus, faecal
streptococci and standard plate count by standard
techniques (James et al., 2008).
Antifungal activity of methanolic extracts of
Acanthophora spicifera, Gracilaria edulis, Padina
gymnospora, Ulva fasciata and Enteromorpha flexuosa
were tested against Aspergillus niger and Candida
albicans using the diffusion plate method. In this, 0.1ml
of fungal spore suspension (growth for three days on
10ml of nutrient dextrose agar) was thoroughly mixed
with 25ml of melted potato dextrose agar and was
poured into sterilized petri plates. When the agar
solidified, 8mm diameter wells were made on each of
the seeded plates. These cups were filled with test
samples of various concentrations (40µg and 60µg) and
standard fluconazole (100µg/ml) was kept in control.
The petri plates were incubated at 280C for 2-4 days.
All the culture plates were examined from 24h onwards
till 48hours and the results were tabulated. The
inhibition zones produced by the test samples were
compared with the inhibition zone produced by
fluconazole used as the standard (Latha and Latha,
2011).
Antimicrobial activity of underexploited seaweeds
RESULTS AND DISCUSSION
Seaweeds are known to contain bioactive compounds
that display antibacterial, antiviral and antifungal
properties. The determination of antimicrobial activity of
the methanol extracts of Acanthophora spicifera,
Phytochemical content of underexploited seaweeds
Phytochemical screening
The methanolic extracts obtained from underexploited
seaweeds were used for phytochemical studies.
Phytochemicals
like
alkaloids,
glycosides,
carbohydrates, proteins and amino acids, flavonoid,
sterols, saponins, tannins, gums and mucilage,
terpenoids, phenols, starch and quinones were
analysed by qualitative chemical method.
Microbial analysis of underexploited seaweeds
The preliminary phytochemical analysis of the seaweed
used in the present study has been studied and
330 Int. Res. J. Microbiol.
Table 1. Phytochemical content of underexploited seaweeds
Phytochemicals
Alkaloids
Glycosides
Carbohydrates
Proteins
&
aminoacids
Flavonoids
Sterols
Saponins
Tannins
Gums/Mucilage
Terpenoids
Phenols
Starch
Quinones
A.spicifera
+
++
+
G.edulis
+
++
+
P.gymnospora
++
+
U.fasciata
+
++
+
E.flexuosa
+
++
+
+
+
++
+
+
+
++
+
+
+
+
++
+
+
+
++
+
+
+
+
+
+
+
+
++
+
-
+
+
+
+
+
+
++
+
-
+
+
+
+
+
+
++
+
-
+ Presence, - Absence
Table 2. Microbial load in selected seaweeds
Microbial species
Coliforms
E.coli
Salmonella tyhpii
Staphylococcus
aureus
Faecal streptococci
Standard plate count
A. spicifera
G. edulis
P. gymnospora
U. fasciata
E. flexuosa
<2/g
<4/g
<1/g
-
<1/g
<5/g
<1/g
-
<2/g
<3/g
<7/g
30 cfu/g
90cfu/g
20 cfu/g
25 cfu/g
43cfu/g
reported in the table 1. The phytochemical screening of
the all selected seaweeds showed that the seaweeds
contained carbohydrates, protein, gums and mucilage,
phenols, starch. Alkaloids were absent in all the
selected seaweeds, Phenols found to be plentiful as
reported by Wang et al., (2009). Quinones was absent
in the brown seaweed like P. gymnospora and green
seaweed such as U. fasciata, E. flexuosa.
Microbiological profile of selected seaweeds
It is important to examine the microbiological aspects
relating to seaweed as they could be consumed raw
and may originate from an environment which may
contain faecal and other human clinical pathogens. The
results of microbiological analysis of selected seaweeds
were presented in the Table 2.
The common food pathogens have been conducted;
the results indicated that there were no detectable
Salmonella typhii and faecal streptococci in all selected
seaweeds. Staphylococcus were found in red seaweed
A.spicifera, brown seaweed P.gymnospora and green
seaweed E.flexuosa, whereas E.coli was <3/g in
E.flexuosa and <1/g in Padina gymnospora which was
below the norm of the Superior Council of Public
Hygiene of France (Mabeau, 1991). The standard plate
count was found to be 30 cfu/g in Acanthophora
spicifera and 90 cfu/g in Gracilaria edulis which was
also within the aerobic and anaerobic counts prescribed
for seaweeds (<100 cfu/g) by Burtin (2003). E.coli and
Staphylococcus aureus count in Padina gymnospora
was <1/g and <5/g respectively.
The results of microbial analysis of the selected
seaweeds showed the presence of Coliforms and
E.coli. Both coliform and E.coli counts were found to be
very low (<1 to <3/g) which was within the permitted
level prescribed by Burtin (2003). Similar results were
also observed in studies of Burtin (2003). Thus the
selected species of seaweeds meet the safety limits in
terms of bacteriological criteria.
Antimicrobial activity of selected seaweeds
The antimicrobial activities of the selected seaweeds in
terms of the diameter of zone of inhibition are shown in
Table 3.
The methanolic extract of selected seaweeds showed
considerable antibacterial activity against the test
Abirami and Kowsalya 331
Table 3. Antibacterial activity by agar well diffusion method
10 µg
Staphylococcus
aureus
Streptococci
E.coil
Proteus vulgaris
Klebsiella
aerogenes
E. flexuosa
U. fasciata
P. gymnospora
G. edulis
A. spicifera
DMSO
amikarin
Microorganisms
Diameter of zone inhibition in (mm)
25 µg
50 µg
40 µg
60 µg
40 µg
60 µg
40 µg
60 µg
40 µg
60 µg
22
7
12
12
10
12
8
10
11
15
9
12
20
24
20
22
5
5
7
6
9
10
8
12
9
12
10
12
12
10
8
14
14
14
8
17
8
7
6
12
9
6
9
17
12
10
12
19
15
12
15
9
15
14
15
11
15
17
Inhibition zones 15 mm was declared as strong (bold), from 8 to 15 mm as moderate and from 1 to 8 mm as weak
activities.
Aspergillus niger
Candida albicans
10µg
22
20
7
9
40µg
14
12
60µg
16
12
40µg
9
10
60µg
9
12
40µg
7
11
E. flexuosa
U. fasciata
P. gymnospora
G. edulis
A. spicifera
DMSO
fluconazole
Microorganism
Table 4. Antifungal activity by agar well diffusion method
60µg
9
12
40µg
16
9
60µg
16
9
40µg
9
9
60µg
10
12
Inhibition zones 15 mm was declared as strong (bold), from 8 to 15 mm as moderate and from 1 to 8 mm as
weak activities.
organisms. The methanolic extracts of selected
seaweeds showed strong inhibitory effects against all
the microorganisms tested at different concentrations
(40 and 60µg/disc).
The highest zone was found in the methanolic extract
of Acanthophora spicifera and Ulva fasciata against
S.aureus at 60µg/disc concentration. The maximum
zone of inhibition was against Sterptococci for the
methanolic extract of E.flexuosa. Ulva fasciata showed
strong activity against E.coli (19mm) at 60µg/disc
concentration. All other seaweed extract showed
moderate activity against the tested organisms. Ulva
fasciata and Enteromorpha flexuosa showed strong
activity against Proteus vulgaris strong against at
similar concentration.
The red seaweed G.edulis and green seaweed
E.flexousa showed strong activity against Klebsiella
aerogenes
at
60µg/disc
concentration.
The
effectiveness of extraction methods highlight that
methanol extraction yields the highest antimicrobial
activity compare to the other solvents stated in our
previous experiment (Abirami and Kowsalya, 2011).
The effect of extracts on other microorganisms showed
variable antibacterial activity, these results were similar
to the results of Tuney et al., (2006). It is evident from
the clear zone of inhibition obtained in the present study
against all the organisms tested, that the selected
seaweed extracts are bactericidal in nature. In
conclusion, methanolic extracts of selected seaweeds
were potentially a good source of antibacterial
substances with a broad spectrum of activities in
preventing the growth of all the microorganisms tested.
Antifungal activity of selected seaweeds
The antifungal activity of the selected seaweeds in
terms of diameter of zone of inhibition are shown in
Table 4. The antifungal activity of seaweed depends on
type of seaweed species, solvent used for the
extraction and fungal strains. The crude extracts
exhibited good activity against C.albicans and A.niger
by the methanol extract of all selected seaweeds like
Acanthophora spicifera, Gracilaria edulis, Padina
332 Int. Res. J. Microbiol.
gymnospora, Ulva fasciata and Enteromorpha flexuosa.
The highest zone of inhibition were found in
A.spicifera and U.fasciata (16mm), whereas all other
seaweed spices showed moderate and weak activity
against Aspergillus niger. For activity against Candida
albicans only moderate activity was noticed in
A.spicifera, G.edulis, P.gymnospora and E.flexuosa,
whereas no activity was found in U.fasciata.
CONCLUSION
The phytochemical, microbial load and antimicrobial
activities were analyzed in the present study by using
methanol extract of different seaweeds. In this growth
inhibition of several bacteria and fungi strains of
selected seaweeds has been reported. Seaweeds are
great production of secondary metabolites, which are
not found in terrestrial environment. Thus marine algae
is the richest source of know novel bioactive
compounds.
From the above study, the methanolic extracts
possessing high antibacterial effects should be further
studied for their therapeutic use. This result could be
related to the presence of bioactive metabolites in the
selected seaweeds, which are soluble in methanol. The
structures of the bioactive metabolites of the species
will be examined in future. The present study suggests
that these seaweed extracts possessed antibacterial
activity against bacterial pathogens and antifungal
activity against fungal pathogens thus supporting their
folkloric usage, promising a future scope for the use of
these marine seaweeds against microbial populations.
This study suggests the possibility of using seaweed
extracts as natural antimicrobials in the food industry.
The microbial diversity of the seaweed demonstrated
that raw seaweeds are of good microbiological status.
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