The relationship between antibacterial activity and levels of 24 metals in 19 selected southern African seaweeds was examined. The antibacterial activity of ethanolic extracts of these seaweeds was assessed by agar diffusion against selected Gram positive and Gram negative test bacteria. Metal levels associated with seaweeds were determined using X-ray fluorescence (XRF) analysis. Extracts of the Phaeophyta exhibited the highest level of antibacterial activity, followed by the Rhodophyta and then the Chlorophyta, in decreasing order. Metal levels, however, were highest in the examples studied from the Rhodophyta and then the Phaeophyta and Chlorophyta, in decreasing order. There was no relationship between the observed antibacterial activity of the crude extracts tested and the levels of metals occurring in their tissues. Thus the antimicrobial activity of extracts from the seaweeds tested should also be generally recognised as safe (GRAS)

Critchley, T. Alan

Holy, Alex von

Vlachos, Vaso

Kochi University Repository

Bzall. Mar. Sci. Fish., Kochi Univ.
No. 21, pp. 7-12, 2001
On the GRAS status of seaweeds. I. Observations on the association
 between antibacterial activity of ethanolic extracts and meta1 levels
                      present in selected seaweeds.
Vaso VIAcHosi, Alan T. CRITcHLEy2" and Alex von HoLyi
i School ofMolecular and Cell Biology, University ofthe VVitwatersrand, fohannesburg, Private Bag 3,
 VVits, 2050, South Africa.
2Degussa, Texturant Systems Franee SAS, Research Centre, F-50500, BauPte, France,
Abstract: The relationship between antibacterial activity and levels of 24 metals in 19 selected southern African
seaweeds was examined. The antibacterial activity of ethanolic extracts of these seaweeds was assessed by agar
diffusion against selected Gram positive and Gram negative test bacteria. Metal levels associated with seaweeds
were determined using X-ray fluorescence Q(RF) analysis. Extracts of the Phaeophyta exhibited the highest level of
antibacterial activity, followed by the Rhodophyta and then the Chlorophyta, in decreasing order. Metal levels,
however, were highest in the examples studied from the Rhodophyta and then the Phaeophyta and Chlorophyta, in
decreasing order. There was no relationship between the observed antibacterial activity of the crude extracts tested
and the levels of metals occurring in their tissues, Thus the antimicrobial activity of extracts from the seaweeds
tested should also be generally recognised as safe (GRAS).
Key words: antibacterial activity, southern African seaweeds, macroalgae, metals.
Introduction
   Natural products, which are not harmful to humans, have been used in food preservation.
These natural products have been termed "GRAS" (generally recognised as safe ; Holzapfel et al.,
1995) . For centuries, seaweeds have been a source of food and medicine for the people of the Far
East (South and Whittick, 1987). These seaweeds have not posed any known health risks to
humans and can thus also be regarded as "GRAS". Secondary metabolites responsible for antibac-
terial activity in seaweeds are, by definition, also likely to be "GRAS" and therefore crude extracts
have potential use in food preservation and medicinal applicabions.
   Notwithstanding the above, it is well known that certain marine macroalgae accumulate heavy
metals to a level higher than those in the surrounding seawater (Seeliger and Edwards, 1977;
Chung and Lee, 1989) . Metals may occur naturally in the marine environment by mineral erosion
or weathering, but with a rapid increase in industrialisation in the form of mining, smelting and
fossil fuel combustion, levels of certain metals fparticularly heavy metals) in the marine envi-
ronment have escalated (Chung and Lee, 1989). Metals accumulated in seaweeds can be trans-
ferred to higher trophic levels through food chains. High levels of some of these metals can
become toxic to humans, thus if the antibacterial properties of seaweed extracts are to be used in
the food and pharmaceutical industries, one must.speculate as to whether the antibacterial activity
of seaweed extracts is due solely to antibacterial compounds (secondary metabolites) produced
by the algae, or whether accumulated metal concentrations associated with macroalgal tissues
also contribute to the antibacterial activity of their crude extracts.
7
8 VASOVIACHOSET AL.
   Many compounds in nature have the ability to inhibit the growth of microorganisms. However,
microorganisms have developed resistance to these natural compounds and microbiologists are
forced to continue their search for naturally occurring antimicrobial compounds. The marine envi-
ronment provides a unique and sti11 largely unexploited source of novel bioactive compounds in
the form of secondary metabolites (Tariq, 1991; K6nig et al., 1994). Surprisingly, it was only rela-
tively irecentiy that the potential of seaweeds as sources of antimicrobial compounds was recog-
nised (Pesando and Caram, 1984; Pesando, 1990) .
   The presence of antibacterial activity has been documented from seaweeds collected world-
wide, including southern Africa (Hornsey and Hide, 1974; Campos-Takaki et al., 1988; Niang and
Hung, 1984; Moreau et al., 1988; Caccamese et al., 1985; Rao, 1991; Vlachos et al., 1997).
Compounds reportedly responsible for these antimicrobial properties range from acrylic acid and
diterpenoids in the green seaweeds (Glombitza, 1970) and halogenated terpenoids in the red
seaweeds, to metabolites of mixed terpenoid-aromatic origin in the brown seaweeds (Pesando,
1990).
   This study investigates the association between antibacterial activity of crude seaweed extracts
and levels of selected metals in southern Mican seaweeds, known to exhibit antibacterial activity
(Vlachos et al., 1997) and which also contain elevated levels of selected metals (Vlachos et al.
1998).
Materials and Methods
Seaweed samPle collection
   Seasonally and locally abundant seaweeds were collected from the intertidal zone of
Swakopmund (Namibia), Palm Beach, Rocky Bay, Isipingo Beach and Mission Rocks (South
Africa) on the southern Mican coast.
SwaleoPmundsamPles (IVamibia):
   Phaeophyta- Bifurcariopsis capensis, Laminaria schinzii
   Rhodophyta- Mazzaella capensis, Plocamium rigidum.
Palm Beaeh samPles (Sozath Africa):
   Phaeophyta- Anthophycus longifolius, Sargassum incisifolium, Stypopodium zonale, Zonaria
              tournefonii'
                       '
   Rhodophyta- Amphiroa ephedraea, Beckerella pinnatifida, Gelidium abbottiorum,
              Osmundaria serrata'
                              '
   Chlorophyta- Halimeda cuneata.
Rocley Bay samPles (Sozath Africa?:
   Rhodophyta- Arthrocardia carinata.
Isipingo Beach samPle (South A,frica);
   Rhodophyta- Cheilosporum sagittatum.
Mission Rocks samPles (South Africa) .-
   Phaeophyta- Dictyopteris longifolia, Ec klonia radiata , Zonaria subarticulata;
The antibacterial activity of echanolic extracts and metal levels in seaweeds. 9
Chlorophyta- Codium duthieae.
   Several individual algae, comprising 200g wet weight, were collected in the field and pooled to
provide a single sample. Three such replicate samples were collected, air-dried and cleaned of
sand, crustaceans and epiphytes as described by Vlachos et al. (1996) .
Determination ofmetal levels
   Metal levels in the southern African seaweeds listed above were determined using X-ray fluo-
rescence Q(RF) analysis. Samples were prepared according to standard methods described in
Vlachos et al. 1998. Three replicate pellets of each sample were prepared and subjected to XRF
analysis (Philips PW 1400, Holland). Metal levels were then scored according to a key based on
concentration ranges (Table 1).
Table 1. Inhibiti,Qn z/Qne diameters and metal concentrations in selected southern African seaweeds
          INHIBITION ZONE DIAMETER
                 ge
                      ds
                 g
                      $. tsSEAWEED s g k-
          li;- SB: :/s, i/'g As
MIirlrALCONCENTRATIONS
Co Cr Cu Mo Pb Sr N203
Zonaria
subarticulata
Zonaria
tournefortii
++++ +++++ ++++ +++
+++ +++ +++ ++
+
+
+
+
+
+
+
+
+
+
+
+
+++ ++
+++++ +++
Plocamium
rigidunt
Gelidium
abbottiorum
Osmundaria
serrata
Codium
duthieae
llalimeda
cuneata
++++ +++ ++
++++ +++ ++
++ ++ ++
+++++ ++++ ++
+++++ ++ ++
+
+
+
+
+
+++
+
+
+
++++ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
++
++
+
++
++++
+++++
++++++
++
++++++ ++
Determination ofantibacterial activity
PreParation of Crzade extracts and selection oftest bacteria
   Milled seaweed material was extracted in 80e/o ethanol (in water) according to the method
described by Machos et al. (1996) .
   The 4 test bacteria used in the study were isolates of common food-borne pathogens or
spoilage organisms, viz. Bacillzas subtilis, StaPhylococcus aurezas, Acinetobacter lwoffii and
Escherichia coli.
Antibacterial activity assay
   Crude extracts were tested for antibacterial activity by agar diffusion (6.lmm wells in overlay
agar ; Vlachos et al. 1996) . A control of 800/o ethanol (in water) was used. Plates were incubated for
24 hours at 25Åé and inhibition zone diameters were measured and means calculated. For each
              ,plate, the inhibition zone diameters around the control well were subtracted from those around
the well of the seaweed extracts. Inhibition zone diameters were then scored according to a key
based on size (Table 1).
10 VASO VIACHOS ET AL.
Analysis ofresults '
   A multi-factorial analysis of variance of the means, at the 950/o confidence level, was used to
compare metal levels between seaweeds, as well as to compare the inhibition zone diameters
produced by ethanol extracts from seaweeds.
Results and Discussion
   Table 1 shows the inhibition zone diameters and the metal levels of the seaweeds showing the
highest level of antibacterial activity in each of the divisions Phaeophyta and Rhodophyta (Zonaria
szabantculata and Plocamizam rigidzam, respectively), as well as the seaweed(s) showing the
highest level of metals in each division (the phaeophyte Zonaria tournefortii and the rhodophytes
Osmundaria serrata and Gelidium abbottiorzam). The metals levels and inhibition zone diameters
of the 2 Chlorophyta used in this study are also shown in Table 1. According to Trevors et al.
(1985) of all the metals tested for in this study, only As, Co, Cr, Cu, Mo, Pb and Sr, and the metal
oxides A1203 and Fe203 are toxic to microorganisms when present in high concentrations and are
thus reported in Table 1.
Antibacterial activity ofselected southern African seaweed crzade extracts
   Extracts of the Phaeophyta showed antibacterial activity against all the test bacteria. Extracts
of the brown alga Zonaria subarticulata showed the broadest spectrum of antibacterial activity of
all the seaweeds used in this study (Table 1).
   Generally, extracts of the Rhodophyta were inhibitory only to the Gram positive bacteria and
Acinetobacter lwoLfi7i of the Gram negative bacteria. Plocamium rigidum showed the highest level
of antibacterial activity of all the rhodophyte extracts (Table 1).
   The extracts of both the Chlorophyta, Codizam duthieae and Halimeda cuneata, showed high
levels of antibacterial activity against StaPhylococcus aureus and Bacillus subtilis, respectively.
However, the level of antibacterial activity of these seaweed extracts was low against the
remaining Gram positive and all the Gram negative bacteria (Table 1).
Metal levels in selected southern African seaweed crude extracts
   Generally the highest levels of metals were found in the Rhodophyta, followed by the
Phaeophyta and then the Chlorophyta in decreasing order. The rhodophyte, Osmundaria serrata
contained the highest levels of As, Pb, A1203 and K20, and Gelidium abbottiorum, also a
rhodophyte, contained the highest levels of V, Fe203, Si02 and Ti02. Generally, Zonaria tourne-
fortii and Halimeda cuneata contained the highest metal levels of the Phaeophyta and
Chlorophyta, respectively.
RelationshiP between antibacterial activity and metal levels
   The decreasing order of antibacterial activity of extracts of the Phaeophyta, Rhodophyta and
Chlorophyta did not correspond to the observed order of metal levels in these algal groups. For
example, extracts ofthe rhodophyte, Plocamizam rigidzam, showed significantly greater (P< O.05)
antibacterial activity than extracts of Osmzandaria serrata against Bacillus subtilis and
StaPhylococcus azareus. By contrast, Osmundaria serrata showed higher levels (P< O.05) ofAs and
A1203 than Plocamium rigidum (Table 1). The levels of As, Co, Cr, Cu, Mo, Pb and Sr measured in
these seaweeds was probably too low to inhibit the growth of the test microorganisms.
The antibacterial activity of echanolic extracts and metal levels in seaweeds. 11
   The antibacterial activity of extracts of the phaeophyte, Zonaria subarticzalata, was greater than
that of Zonaria tourn efortii (P < O.05) against the 4 test bacteria, yet the levels of Sr and N203 were
significantly higher (P < O.05) in Zonaria tournefortii (Table 1) . As with the Rhodophyta, the levels
of Co, Cr, Cu, Mo, Pb and Sr levels measured here are probably too low to inhibit the growth of
the test microorganisms. Codizam duthieae extracts showed greater antibacterial activity than
Halimeda czaneata against StaPhylococcus aureus (P<O.05). Halimeda cuneata, however, showed
significantly greater (P< O.05) levels of Sr than Codizam duthieae.
   The apparently high As level associated with Osmundaria serrata does not call for concern
because As is accumulated in the non-toxic organic tpentavalent) form by seaweeds; it is the inor-
ganic (trivalent) form which is toxic (Kesava Rao et al., 1991) .
   The high metal levels in the Rhodophyta are not likely to have an effect on the antibacterial
activity of the seaweed crude extracts as the highest level of metals in the Rhodophyta were in
Osmundaria serrata and Gelidium abbottiorum, and the greatest antibacterial activity was reported
for Plocamium rigidzam. Furthermore, the antibacterial activity of the Rhodophyta is reportedly
due to halogenated terpenoids (Pesando, 1990), yet metals bind to alginic acid (Ramachandran et
al., 1994) and other polysaccharides (eg. agar and carrageenan) in seaweeds (Chung and Lee,
1989). Gelidium abbottiorum produces large quantities of agar (lpolysaccharide) (Aingworth and
Critchley, 1997) which may bind to metals and thus be an explanation for the high levels of metals
reported in this seaweed.
   From the results obtained in this study, the levels of metals associated with seaweeds from
southern Africa did not have a measurable effect on the antibacterial activity of ethanol crude
extracts. It is suggested that the antibacterial activity exhibited by these southern African seaweed
extracts can be attributed to naturally occurring secondary metabolites which are free of metals.
These secondary metabolites, once purified, may have a commercial application in pharmaceu-
ticals and/or food biopreservation, thus maintaining the concept that seaweeds are recognised as
"GRAS".
Conclusion
   It can be concluded that there is no relationship between the meta1 levels associated with
seaweed tissues and the corresponding antibacterial activity of their ethanol crude extracts
against the test bacteria used in this study. This finding implies that the antibacterial activity
displayed by the seaweed extracts tested in this study are due to secondary metabolites which are
free of metals. Ethanolic extracts, with antibacterial activity, made from the selected seaweeds
tested here, should be considered as "GRAS" and would be suitable candidates for further testing
in commercial applications.
Acknowledgements
   The authors gratefully acknowledge receipt of funding from the Foundation for Research
Development, University of the Witwatersrand and University of Namibia for various phases of
the work resulting in the final write-up. We thank Professor M. Ohno for interest shown in this
work.
12 VASOVLACHOSETAL.
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On the GRAS status of seaweeds. I. Observations on the association between antibacterial activity of ethanolic extracts and metal levels present in selected seaweeds.

On the GRAS status of seaweeds. I. Observations on the association between antibacterial activity of ethanolic extracts and metal levels present in selected seaweeds.

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