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Indian Journal of Experimental Biology Vol. 41 , August 2003, pp. 837-845
Bacterial dynamics associated with algal antibacterial substances during post harvest desiccation process of Sargassum stolonifolium Phang et Yoshida
Charles Santhanaraju Vairappan
Borneo Marine Research Institute. Universiti Malaysia Sabah, Locked Bag 2073. Kota Kinabalu , 88999. Sabah, M alaysia
Recl' il 'ed 13 II lIg lIsl 2002: rel' ised 24 April 2003
Brown algae of genus SargasslIlII are known to produce relatively higher amount of alginic ac id. Optimal ex tracti on of thi s algalcolloid for local consumption requires in-depth studi es on post-harvest treatment of the alga l fronds. Present in vestigation endeavors to es tabli sh the dynamics and inter-relationship of moisture content and bacteria found on the surface of the alga and alginic ac id content during post-harves t desiccation of SargasswlI slOlollifoli lllll Phang el Yoshida. Harvested fronds were subjected to desiccation for 31 days and bacterial dynamics were monitored with rel ati on to moisture content and water ac ti vit y index (a".). There was 85% decrease in moisture content, however. a". showed a more gradual decrease . Total bacterial count increased during the first week and attained maximal va lue on day 7. Therea fter. a drasti c decrease was seen until day 14. fo llowed by a gradual decl ine. Six spec ies of bacteria were isolated and identi fied. i .e. A~OIIl (} lIaS P"IICIaW . AZOlllollas sp .. Escherichia coli , MicrococclIs sp., Pmlells vulgaris and Vibrio algill oly liclIs. Calcu lated ratios for increase in alginic acid content and dcc rease in moisture content were almost the same throughout the desiccation process, impl y ing that extracel lular alginase-producing bacte ria did not usc the algi nic aeid produced by the algae as its carbon source. It became apparent that drastic decrease in hacter i ~1I count after day 7 could not be attributed to sa linity. moisture content. a" or lack of carhon source for the bacteri a. The possible exposure of these bacteria to algal ce ll sap which is formed due to the rupture of al gal cell s was seen as the most li ke ly reason for the drop in bacterial populati on. Scanning electron microscope (SEM ) micrograph taken on day 10 of desiccati on showed the presence of cracks and localiti es where bac teria were exposed to algal ce ll sap. I II vilro antibacterial tes ts we re ca rried out to verify the effect of alga l ex tracts. Separati on and purifica ti on of crude alga l extracts via bioassay guided separation methodology revea led the identity of acti ve compounds ( i.e. gy lcolipids and free fally ac ids) invo lved in thi s inherentl y ava ilable antibacterial defense mechanism during algal desiccation.
Keywords : A lgal co lloid. Anti -bacteri al defence mechanism. Bacterial dynamics. SorgasslIlI l
Seaweeds have been utili zed for human consumpti on since time immemori al. It has been used as sa lad, animal fodd er, source of biogas. and extraction of commerci all y importan t chemicals t
• Main products of seaweed are gelling and viscous substances such as agar, carrageenan and algi ni c acid. Members of the brown algae family Sargassaceae, besides being a prolific source of alginic acid, are also know n to contain several other bioacti ve substances2
. Perhaps the most intriguing of its potenti al is its an timicrob ial acti vi ti es3-7.
In Malays ia, SargassulI/ grows in abundance along the coastal waters of Peninsula Malaysia, Sabah and Sarawak . Allhough Sargassum beds are known as good shelters and nursery grounds for fishes , it is generally nOI appreciated by the coasta l co mmunities and is· often regarded as weeds. In an effort to optimize the utili zation of marine resources, efforts
* rur correspondence: Phone: +6088-:>20000 Ex!. 2587: E- mai l : csv@lu ms.edu .my
are now being channeled to use SargasslIIll slolonifoliul1l Phang et Yoshida, as a source of alginic ac id for loca l needs. Hence, post harves t handling of collected alga l fronds has been identi fied as an area that requires in-depth study . Algal frond s are usuall y harvested in large quantities for process ing and in the absence of immediate processing; these fronds starr to emit foul smell due to aerobic and anaerob ic fermentation which is triggered by the presence of moisture, heat and bacteria . Such fermentation processes often occur due to the presence of fermentative bacteria . Some of these bacteria produce ex tracellular alginase enzymcs, hence information on the surface bacteria 's ability to utili ze alginic acid as their carbon source is important to safe-guard the quality of alginic ac id. Hitherto, no data is available on the dynamics of algal surface bacteri a in relation to alginic ac id content during po t-harvest desiccati on process of the alga. In an effort to understand thi s aspect, the present study was undertaken on the dynamics of surface bacteri a and its interaction with
838 INDIAN J EXP BIOL. AUGUST 2003
the alga l ce ll sap during des iccati on. Thi s phenomenon was elucidated by subject ing the alga to post-harvest des icca!ion process fo r 3 1 days, during \vhich dynamics of total bacteri a and bacterial species counts were monitored in relati on to the decrease in algal weight and water ac ti vity index (011")' Total alginic acid content was monitored to verify its utili zati on by surface bacteria as their carbon source. Subsequently , SEM observat ion was carried out to understa nd the interac ti on between surface bac teria and alga l cell sap. Crude alga l ex tracts th at were obtained via so lid/liquid phase ex trac ti on with benzene, di ethyl et her, chloroform:methanol:water (2: I: 1.8) and bu ller so lution. were subj ected to antibacteri al bioassay aga inst its surface bacteria. Further, bi oassay gui ded separati on of these ex trac ts yie lded ac ti ve co mpounds that were subsequentl y identified using IR , IH-NMR. I:lC_N MR and Gc.
Materials and Methods Salllple p,.eparatiol1 - Sa"gosslIIlI StOIVlli/olilllll
was harvested duri ng low tides at the roc k y shores of Batu Feringghi , Penang Island , Malays ia. The fronds were cleaned of ba rn ac les, gastropods and other contaminants at the site. These were immedi ately transported to the lab in sterile pol yeth ylene containers under coo led condition. The fronds were drained of excess seawater and remaining water was blotted using steril e Whatman o. I filter papers until neg ligibl e weight flu ctuations were observed. Bacteri al enumerati on of the bloLled and unbl olled fronds was carri ed out to veri fy any signifi cant loss of surface bacteria due to blotting. Alga l fronds were then di vided into fo ur lots consisting of nine sa mpl es respec ti ve ly, each sample weighing about 20-25 g. These samples were subjected to de icca tion in a sterile chamber within a clean room at 28°C under 12 hr li ght (approximately 30-40 ~lmo l photon m·2s·1
illumination). Relati ve humidity of the des icca ti on chamber was
about 85-88% and there was free air exchange between the chamber and clean room. Padding of des iccati on chamber was made of glass and could not bind any moisture from the ex udates and thus affect the humidity measurements. Clean room was prevented from contamination through a one-way fi Iter sys tem and routine bac ter ial monitoring was carr ied ou t as warranted in any clean room facility. Desiccati on of these algal frond s was monitored for 3 1 days . The first lot of sample was used to determine algal weight. The second lot of sample was used to
monitor water ac tivity index (a,,), while the third lot was used to monitor total bacteri a and bacteri al spec ies counts. The fourth lot of samp le was used to monitor the content of tOlai algini c ac id during desiccation .
Sample weight -Algal weight was monitored peri odicall y throughout the de 'icca ti on process, on day 0, 4, 7, 14, 2 1, 28 and 3 1, under aseptic conditions. After measurements, fronds we re immedi ately returned to desiccate in steril e chamber. Each data represents an average of 27 readings (n=27).
Wate,. ocri l1ity illdex (0".)- Water ac ti vity index (a".) was determined by using a cani ster-type Durotherm hyrgometer (hair hygrometer) according to Bone et a l , ~ . Since the sensiti vity of thi s in strument is about +/-0.003 0"., a total of 27 readings were taken fo r 9 samples to represent one data point (n = 27). The hygrometer cani ster was UV-steriled before use. Thus, water ac ti vity index (a".) was measured indirec tly, as Eq uilibrium Rela tive Humidity Measurements were taken peri od ically throughout the desicca tion process on day O. 7, J 4, 2 I, 28 and 3 I.
Bacterial enumeratiol/ - The alga l surface ( I cm·1)
was sc raped using sterile sca lpel. Precauti ons were taken so as not to injure the surface layer ce lls and ex posing the surface bacteri a to the alga l internal fluids. Scrap ings were then diluted into 10 ml of sterile-filtered seawater. Dec imal di lut ions were made of the alga l scrapings in the range of I x 10 1- 1 X 10K
and 0. 1 ml from each diluti on was spread on Spread Plate Agar (3 % NaCl , Himedia) . Thi s scraping method was used for thi s study after a preliminary bacteri al enumerati on was done using two other methods, homogenization and cotton bud swab method. The homogeni zation and the colton bud swab method showed a drop of30 and 15%, respecti vely, in t01a1 bac teria l count as compared to the sc raping method.
The Spread Pl ate Agar was chosen for thi s study after a preliminary bac teri al enumerar ion using-I ) Nutrient agar in seawater; 2) Nutri ent agar in aged seawater; 3) Nutrient agar+ 3% aCl: 4) Spread Pl ate Agar+ 3% aC I; and 5) Difco marine aga r. The plates were incubated at 28 °C for 2 days. Bac terial coloni es with different morphological characters were coun ted and isolated. Purifi ed cultures were maintained on Nutri ent agar (3% NaCI , Himedia) slants. Quantitative enumeration of total bacteria and bacteri al spec ies counts were recorded. Isolated pure cultures were identi fied using conventional methods
VA IRAPPA N : BACTERIAL DYNAMICS DURING POST-HARVEST DESSICATIO PROCESS 839
described by Shewan et al. 9, Chan and Mc Manus lo,
Baumann et al. II, Baumann et al. 12 and Lee el al. 13.
Electron microscopy-Alga l thalli were cut (ca. 0.5 x 0.5 cm pieces) and fixed for 24 hr in glutaraldehyde (4%) in O.IM cacody late buffer (p H 7.2). Specimens were then rin sed in O.IM cacody late buffer before post-fixat ion in 1% OS04 at 4°C for 2 hr, foll owed by dehydration with graded acetone se ries and finally subj ected to critical point drying. Dehydrated spec imens were mounted on stubs and coated with 10-30 nm layer of gold before observati on with Leica Cambridge S360 electron microscope.
Alginic acid quantification - Alginic ac id content was estimated on day 0, 4, 7, 14. 2 1, 28 and 3 1. Samples were oven dried ground to powder and treated with CaCl2 (I %), approximately 3 times the sample volume, fo r about 4 hr to eliminate mannitol and other simple sugar. This was repeated 3 times before the residues were washed with distilled water. Then , 5% of HCI was added and pred igested for 4 hr. Digested sample was washed 4 times with di stilled water, mi xed with Na]CO) (3 %) for 2 hr at 45°-50°C and stirred at roo m temperature for 8- 12 hr at 200 rpm. This mixture was then diluted with an eq ual amount of distilled water and 2 ml of HeOl was added as a bleacher before it was cen trifuged at 4000 rpm. fo r 20 mill . The supern atant obtained was further concentrated to 113 of its original volume. Equal amount of absolute ethanol was added in order to prec ipitate the sodium alginate, centri fuged and freeze dried before its weight was taken .
Calculatioll. oj rat ios Ratio of moisture decrement = Wl=o 1 WI=30 (i) Rati o of algini c ac id increment = ACI=30 1 ACI=o (ii )
Whereas, Wl=o= Alga l weight (g) at the beginning of desiccati on process; WI=30 = Algal weight (g) at the end of des iccati on process; ACI=o = Alginic ac id content (%) at the beginn ing of desiccation process; and AC=3o = Alginic ac id content (%) at the end of desiccation process .
Antibacterial activity bioassay Extraction - Alga l fronds were divided into two
lots-( I) lot was partially dried (minimal weight f1uacuation) under shade; and (2) lot was used as fresh algae for phosphate buffe r (P H 7.8) and chloroform/ methanol/water (2 : I: 1. 8) ex tract ions. First batch of fresh algae ( 100 g) was homogeni zed fo r ca. 60-90 sec. in 400 ml of sterile 0.2 M phosphate buffer (P H
7.8) and was kept at 4°_6°C for 24 hr under continuous ag itation. Ex trac t was filt ered and ce ntrifuged at 1000 g for 20 min, and the resulting clear supernatant was lyophili zed to yield about 2g of freeze-dried powder. Second batch of fresh algae ( l Oa g) was homogen ized in Waring blender fo r 2 min with a mi xtu re of 100 ml chloroform and 200 ml methanol. To the mixture is then added 100 ml of chl oroform and after blend ing for 30 sec, 100 ml of di sti lied water is added and blending continued fo r another 30 sec. The homogenate was filtered and res idues recovered. Thc above-described ex traction was according to the method as suggested earlier '4 .
Partially dried alga ( 100 g) was extracted with diethyl ether and benzene, respecti vely. The resulting solutions were concentrated in vacuum and partiti oned between Et20 and di sti li ed H20. The Et]O solution was washed with water, dri ed over anhydrous Na2S04, and evaporated to leave dark green oil. These steps gave the non-polar crude diethyl ether and benzene ex tracts.
Bioassay-The ex tracts and isolated compounds were tested against pure bacterial isolates obtained from the algal surface. One loopful of each organi sm was precultured in 10 ml of peptone water (3 % aCl ) overni ght. The turbidity of precultured solution was adjusted to optical density (00 ) of McFarland 0.5 (ref. 15). Then, 0. 1 ml of each precultured suspe nsion was spread on nutrient agar (3 % NaCl ) plates. Paper di scs (Whatman, 6 mm) impregnated with 8.0 mg/disc of the respective extracts were placed on the agar and incubated at 28°C fo r 24 hr.
Separation, pllri/ication and isolatioll oj acti ve compounds- Most ac ti ve crude extract was dissolved in CHCl3 fractioned using Si03 gel colu mn chromatography with CHCI 3/MeOH/H20 gradient eluent system. The fraction s were subjected to
antimicrobial bioassay. Acti ve frac ti ons were checked with thin layer chromatography (TLC) and submitted to PTLC Preparati ve Thick Layer Chromatography (PTLC) to obtain pure compounds.
Characterization oj active compounds-During iso lation and purification , compounds were developed on silica gel 60 F254 TLC plates in va rious so lvent systems, visuali zed with molybdo( VI ) phosphori c ac id and orcinol-H2S04 reagent l6 . 'H-NMR (400 MH z) and 13C_N MR (100 MH z) spectra were measured in CDCI) (fatty ac ids) and DMSO-d6:D20 (98 :2) (glyco li pids) with trimethy lsil ane (TMS) as the internal standard by Ll sing a JEOL-J NN-EX-400 spectrometer.
840 INDIAN J EXP SIOL. AUGUST 2003
CC Analysis of FOlly Acid Methyl Esters-Active fa tty acids were determined by gas chro matography of methy l es ter de ri vati ves using Shimadzu GC-6A B w ith Il ame ioni zation de tec tor. Fatty ac id methy l este rs were obtained by reac tion of isolated active fatty ac id with me thanolic hydrogen chlo ride' 7 fo llowed by si I ica gel chromatography with hexanediethyl ethe r (9 : I , v/v) as e lue nt. The conditi ons for GC measurements '8 were as follows: column ,
RASCOT Si lar 5CP (0.25 mm i.d . x 50m, Nihon C hromato Works Ltd .); inj ecti o n and detector
temperature. 230°C ; column temperat ure, 180°C; ca rri e r gas, H2; fl ow rate, I ml/min.
Resu lts Posl-harvest desiccotion - As shown in Fig. I ,
tota l algal weight showed dec reas ing tendency during post harves t desiccation process. A drasti c 64% decrease in alga l weight was observed in the firs t 7 days, followcd by gradual decrease to a constant weight at the end of the des iccati o n process. The a lga l fronds lost about 85 % mo isture during cnt ire process.
However, water acti vity index (a ,,) showed a more grad ua l decreas ing tendency. It decreased from 0.99 0 ... , at the beginning of the des iccation process, to about 0 .89 0 ... , on day 3 1. The corre lati o n observed between water ac ti vity index (a ... ) and a lga l weig ht decrement agrees with the sugges ti on by Troller and C hrist ian ,~ on water content and water ac ti vity index (a ... ). Hence, thi s findin g showed that water content in seaweed th allu s was no t in direc t equilibrium with wate r vapor in its surroundings.
11.7 16 .' 1.10 -- A LGA L WEICIIT Me --- " Ana ACTI YrrV 1 . ... ""3
II.' ~ TOTAL &ACTl.aJAL "-' .96 COUNT
u MO -IS .5 11 .• .,?!. 'i'
~ 3 co ' .91
... '" :> ~
..c 11 .4 t .' 0 .9 "" Co
~ as :~ 7 .2 .... u t . ) as ... ... .. '" ~
CAl - .. u ~
' .1 4 .' .. < ..c • . 14
2 .4 .. ) . 1 -0
l- •••• 14 11 21 11
Des i cc a I i o n p e r i 0 d (d ays)
Fig. I - Currcla tion of algal we ight in rela tion to watcr ac ti vity index and tolal bacteri al count 01' Sargassl/lII SIIi/Ollifo/i1/1II
(Phaeophy ta) . during desiccat ion at normal room tempcratu re
Pre liminary data on to ta l bacterial co unt showed about 6% drop in the to ta l bac te rial cou nts fo r the blo tted fronds as compared to the unbl otted frond s. Bl otting was vital to avo id possible bac teri a l contamination due to excess seawate r on the alga l fronds. Fig. I c learly shows unique patte rn in the total bacte ria l count. Initi a l total surface bacterial count of 792 CFU/cm2, inc reased unti l 7th day of desiccation , reaching a max imum at 1490 CFU/c m2. However, thi s did not persist and was fo ll owed by a d rop to 864 CFU/c n/ on 14th day . After thi s point, the decrease in to tal bac terial count was more g rad ua l for the next 2 weeks with a final count of 360 CFU/cm2
Fig. 2 shows the respecti ve bacte ri al counts of the s ix bac terial spec ies be long ing to four fa mili es of bacte ri a (Vib rionaceae, k:,ofObacteraceae, Enlerobacleriaceae and Micrococcaceae) iso iated th ro ughout this process. Iso lated bacteria were identi fied using mo rpho log ica l, bi oche mi cal and spec ific sugar test, with reference to Be rgey's Manu a l2o. These bacteria were-Aerolllollas pl/nCfa la , A-:,olllona' sp ., Escherichia coli , Micrococcus sp., Proleus I' ldgaris and Vibrio alginolyticus. The presence o f ex tracellul a r alg inase was detected in Aeromonas plll1Cfata, Micrococcus sr . and Vibrio alg inolylicus, hence suggesting the ir abi lity to d iges t a lg ini c aci d.
Initi all y . s ix species o f bacteria were iso lated from the a lga l surface with these re lati ve abundance-
7.0 16.'
~u • A "0 ..... 1 ,"Itel. ,.
0 A l ••••• ' ., . 14 .• u .-. --. • Me .:: IIKIo.rld,. <.11 u S., u
'0 III M lcr.ee cc ... II' . 11. ' -= ... .... IiJ rr.'~.' ."'~.ru
u ~ MO .. , • v" rt. ."I •• 'ylllc ., 11 .1
.... ~
c
= --<>- T OTAL aACTllklAL 0 • . 1 COVI<T t .' C
u = 0
'" u ... l .S ' .0 u as ... Co 1 . ' , .. ... ... -; 1 . 1 •••
u ~
"'" "-... 1.. l .1 '" -u 0
'" !-= ' .7 1. '
• • • 7 1. 1 1 11 II
D e s ic c a t i 0 D p er i 0 d (days)
Fig. 2 - Total bactcria l and bac tcrial ~ pecics coun t vJriati on in SarglisslIlIl SIO/Ollij(J/i1ll1l (Phaeophyw), during desiccation at normal room tempcrature
::-
,A ,
VA IR A PPAN: BACT ERI AL DYNAMI CS D URIN G POST-HA RVEST DESS ICATION PROCESS 84 1
Escherichia coli> Proteus vulgaris> Vibrio alginolyliclls > AlOJllonas sp. > Micrococcus sp. > Aerolllollas punctata. Similar pattern of abundance was also observed at 71h
day of des iccation. However, on 141h day, there was a drastic change in the bacteri al di versity as the total bacteri al count dropped. At thi s point, there were still six species of bac teria but the tendencies were as -Escherichia coli > Azomollas sp. > AerOIllOllas pllnctata > Micrococcus sp. > Protell s vulgaris> Vibrio algilloIyticll s. There was an obvious drop in Proteus vlllga ris and Vibrio alg illolyticus and a relati ve increase in Aerolllonas punctata and Az.oll lOllas sp. Then, number of bacterial decreased to fi ve with the absence of Vibrio alg inolyticus coupled with a gradual decrease in the bac terial spec ies counts, comparable to the decrease in the total bacterial count. Finally, on 31Si day, there were five spec ies of bacteria with these following tendency; Escherichia coli > AlOl11onas sp. > Aeromollas punctata > MicroCOCCllS sp. > Proteus vulgaris.
Subsequently, data obtained from the monitoring of algini c ac id quantity in thi s seaweed also gave some valu able insights. As show n in Fig . 3, algal alginic ac id conten t showed a increasing tendency throughout the desiccation process due to decrease in the total moisture content. Alginic ac id content showed an increase from 3.72%, at the ~ginning of des iccati on process, to 26.97%, at the end of desiccati on. Ca lculated rati os of alginic ac id increment (7.25) and
1 1.. __ ALGAL wucn 11 .'
---At- ... a c &PtTA.CI: O. ALGINIC ACID COKTtPn'
~ 11.' 14.' ~
... 15 .'
.,., 1' .' .-..
§ ~
- 11. ' Il.' • ... .,., -.-... .. ~ , .. 11 .' ...
os 0 .,., : 7.15 .. A C ID l"c aaWUfT
-< ••• I .' .... ... .. .. H 4.'
u
3.' .. .. p.,
• • 7 14 11 11 11
Des ic cati on p el'i o d (d ays)
Fig . 3 - Va ri at ion in a lgi ni c ac id contc nt as comparcd to a lga l moisturc dcc rcment in Sargassl l lIl .I'lOlolli/oli/l1l/ (Phacophyta) , duri ng des icca tion at no rm al room tempe ratu re
algal weight decrement (7.36) implies no apparent loss in the inherent ly available alginic acid.
Electroll microscopy-General ultrastructure of the thallus has been shown in Fig. 4. The thallus is undergoing dehydration where its surface ce ll layers show areas th at are under stress (S R). Besides these, there are also fea tures th at show cracks on its thallus (CR). Hence, at these vicinities, the surface bacteri a are exposed to the internal fluid of the seaweed, thus resu lting in the decrease in number of bacteri a. The gap between the cracked layers of thi thallus holds numerous dead bacteria (DB) which could be easil y differentiated with the healthy ones (VB ). This micrograph clearly suggested the poss ible presence of antibacterial substance in the internal fluid of this alga.
Antibacterial acti vity- Active anti -mi crobial principles in S. stolonijoliLIIII were extrac ted using the four different so lvent systems, phosphate bu ffe r (pH 7.8) , C/M/W (2:2: \. 8), di ethyl ether and benzene. et weight of the va rious crude ex tracts was 9, 7, 2 and 3% of the algal weight used during ex traction, respecti vely.
Results for the antibacteri al tests are given in Table 1. The benzene and diethyl ether crude ex tracts showed ant ibacterial ac tivity against 50% of the tested bacteria with strong inhibition aga inst Aerolllonas punctata and Vibrio algillolyticus. Minimum inhibitory concentrati on (MIC) fo r these ex tracts va ri ed between 10 and 80 Ilg di sc· l
. Lowest MIC value for benzene and diethyl ether ex tract was I 0 ~tg disc· l
. Both the extracts showed best
Fig. 4 - Sca nning Elec tron Mi crograph of Sorgas.I' lIIl1 510101li
/oli /lll/ taken during d rasti c decrease in total bac terial COUllt ( la'" day) . [S R - regions show ing stressed tha llus ce ll layers and in the ve rge of ruptu re: IR - regions with cracks: DB - deformed bac teri al cells and VB - viab le bac te ri a l cell s l
842 I DIAN J EXP BIOl. AUGUST 2003
antibacterial inhibition with low MJ C values against Vibrio alg inolyticus . On the other hand , chloroform: methanol: water (C: M: W; 2:1:1.8) crude ex trac t inhibited 100% of the bacteri a strains. Potent antibacterial acti vity was seen against Aeroll1ollas punctata and Vibrio alginolyticus with a MIC value of 10 pg di sc· l
. Phosphate buffer extract onl y inhibited about 33 % of the tested bac teri a with good inhibition aga inst Aerolllonos pUl1ctata and Vibrio alg illolyticus with a M IC va lue of 1001lg di sc· l
.
Antibacterial colI/poullds - Bioassay guided separation methodology indicated two groups of compounds as the ac ti ve principles . in separable mi xture of fatty ac ids and M G DGs I ~-ga l acto
pyranosy l ( I '-3 )]- 1 ,2 -di ~ cy l g l ycero l s (Compound 1). The fa tty ac id mi xture consisted of saturated fa tty ac ids and mono-unsaturated fatty acids. Saturated fatty ac ids were palmiti c ac id (67%) and stearic ac id ( 12%). while mono-un saturated fatty ac ids were palmitoleic ac id (C 16: I: 5%), oleic ac id (C 18: I; 7O/C ) and gadoleic ac id (C20: I; 9%). MGDGs showed charac teri stic dark red spots due to sugar moieties when vi. uali zed with orcinol-sul furi c ac id reagent, suggestin g that the e compounds are gy\Colipids. I H-
MR spectrum of co mpound 1 ex hibited signals of two termin al methyl groups at 80.85 and 0.92 (br t, .1 =7.3 Hz), broad methylene groups at 8 1.24, two methylene groups at '62.27 (t, 1=7.3 Hz) flanked by a carbonyl function, indicating the presence of two acy l groups. The I H-I H COSY spectrum showed a 5-spin ABXA' B' system at 8 3.6 1 ( I H, dd , 1 = 11.2, 5.9 Hz)J 3.80 ( IH , dd , 1= 11.2, 5.4 Hz) , 4. 14 ( IH. dd, .I= 11 .7, 6.8 Hz)J 4.32 ( IH, dd , .I= 11.7, 2.4 Hz) and 5.10 ( IH ,
Table I - Anti bactcrial act ivi ty and M IC or crude ex tract of Sargassu/II srololli(oli lllll aga inst it s su rface bacteria
Bacteria tested
AerOIll() lIaS plillcrara A:olllollas sp. Escherichia coli Micrococcus sp. Proteus vlligaris Vibr io aiginolyriclis
Bacteria tes ted
AerOIllOllas pli l/ crara A:OIllOIl(lS sp. E.l cherichia coli Micrococclis sp. Prorells 1'lIlgaris VilHiD alg il/olyricus
Bu rfer
+
++
Anti bacterial act ivity C/M/W Etf)
+++ ++ ++ + +
++ ++ +++ ++
Benzene
+
+ +
Crude ex trac t MIC* values (I-\g di sc- I) Burrer C/M/W EtlO Benzene
100
100
10 40 20 60 40 10
60
80 10
60
80 10
m), which are characteri stic of signals due to a 1,2-di acy lglycerol moiety. Detailed analys is of the remaining protons in tH_I H COSY coupled with I.1C NMR and HSQC spectra showed th at sugar moiety of compound 1 was ~-ga l actos ide hav ing an anomeri c proton at () 4. 1 I ( I H, d, 1 = 6.8 Hz). Therefore, structure of co mpound 1 was identifi ed as m-galac topyranosy l ( I ' ,3)] - 1 ,2-d iacy lglycero ls (MG DGs)21.22 .
Since compound 1 consisted of a mixture of molecu lar spec ies, the compos ition and positional distributi on of fatty ac ids in co mpound 1 (MGDGs) were determ ined by enzy mati c hydrolys is fo ll owed by al kal ine hydrolys is. Li pase-catalyzed regiose lecti ve dcacy lat ion at I-positi on of 1 using li pase type XI from Rhiw PllS delell/ar in Triton X- IOOJboric-borax buffer (pH 7.7), produced exc lusively la and mi xture of fatty ac ids. The resulting fatty acids were converted to their corresponding methyl ester by treat in g with trimethylsil yldiazometh ane in methan I-hexane. GC analys is of meth yl ester confirmed that fatty ac ids at I-positi on of 1 were 16: 0 (58%), 16: 1(9%), 18: 0 ( 13%), 18: 1(10%), 18: 4 (8%) and 20: 5 (2%). Alkal ine trea tment (NaOMe-MeOH) of la gave a mixture of methyl esters, GC analys is showed that fatty ac ids located at 2-position of compound 1 were 16:0(60%), 16: 1 ( 14%), 18: 1 (9%), 18;4 (7 °lt ), 20: I (6%) and 20: 5 (4%) .
Ac ti ve compounds, fatty ac ids and MGDGs (1 ), were distributed in va ri ous extracts in different di stinct quanti ti es. Active fa tty ac ids constituted 2% of benzene extract, 3% of diethyl ether ex tract, 6% of CJMJW extract and 2% of the buffer extrac t. On the other hand , MGDGs were onl y present in signi ficant
H
H
H
H
la
OR2
.
O~ORI RI & Rz : acyt gr oup
Rz: acyl group
+
quantities, 12 and 4% in C/M/W extract and phosphate buffer extract , respecti vely. Presence of these active substances was also detected in the ex udates col lected from the glass padding of the desiccation chamber. Detection of substances was done based on Rr va lues of compounds in ex udates and isolated pure active compounds, developed on silica gel F~5.J TLC and visuali zed using specific sprays such as molybdo(VI) phosphoric acid and orcinol-H 2S04 reagent l6, as menti oned earli er.
Discussion Net loss in algal weight could be attributed to the
loss of moi sture during desiccation process. Simi lar findings ha ve al so been reported by Gardner and Mitchel1 23
, and Va irappan and SUZLIki 2.J during their studi es on seaweed dry ing However, since thi s investigation in volved monitoring of surface bacteri a that li ves on mi cro-s tructured alga l thallus. a mere moisture determinati on was not sufficient. The algal surface with deposits of organi c matters and algal ex udates gives ri se to a micro-structured surface wi th minute water capill aries within these layers. A mere moisture monitoring would not give much in fo rmation pertain ing to the forces that affect the surface bac teri a which fl ouri sh on thi s mi crolayed algal surface. Rather, water activity index (a".)
appears to be the most stabl e water content indi ca torl ~ . The water activity index wou ld give a rea li st ic fi gure of the moisture co ntent ava il able to the surface bacteri a and could be defined as the readil y ava il able moisture in the alga l thallus. In thi s study, solutes are NaCl , alga l exudates, di ssolved organi c
d 1 . 1 . ~ matter an ot ler mlnera s present In seawater-. Complex ity of interactions between solutes and difficu lties faced in quantifying these indi vidual solutes rules out any poss ibility of determining the water ac ti vity index (a".) through the usage of GibbsDuhen's integration equati on as suggested by Bone" et alR. Hence, water ac ti vity index (a",) has proved to be a more appropriate parameter. Relat ionship between internal moi sture content and its water ac tivity index of S. slOlonifolium in thi s study was comparable to those reported by Troll er and Cl . . 19 d V . d S k·24 lrI stlan an alrappan an uzu ' I .
Comparison of water ac tivity index (aw ) with algal weight decrement gave good indication of the alga l total moisture content. Gradual decl ine in water acti vity index also suggested the algal ability to harbor bacteria and sustain their growth. Water activity index decreased from 0.99 to 0.88 at the end
of des icca tion. Even then , water activ ity index was 0.88 a", and still ideal for the growth of moderately h 1 h' I' b . IY ?5 a op I IC ac tena '- .
Initi al increase in bacteri al count could be attributed to increase in avai lable surface for bacteri al growth . This phenomenon was a l. 0 illustrated by Zobell and Upham26 Drop in total bac teri al count and bacterial species count after 71h day of desiccation was drasti c and could not be due to dehydrati on. Salin ity has been regarded as one of the factors bes ides alga l ex udates, organi c matters (di ssolved) and minerals that govern the water ac ti vity index (aw ) of desiccated seaweed fronds17 However, water acti vity index (0".)
was still idea l for bacterial growth . Influence of salinity has been taken into considera ti on based on Gibbs-Duhen eq uati onS and was ruled out as the causati ve factor for the drop in the bacterial count s.
Calculated ratios of alginic ac id increment and moisture decrement also gave va lu able in formation pertaining to dynamics of surface bacteria. Since the ratios were almost sa me, it became apparent that surface bac teri a were not utili zing alginic acid as their carbon source although Aeronw nCis pIIllclala, Micrococclis sp. and Vibrio alginolyticus are known
. liP Z 11 d to produce ex tracellul ar alglllase . -. obe an Upham26 have suggested that alginase produc ing marine bacteri a, particularl y bacteria from the genus Vibrio, are able to digest and utili ze alginic acid as its carbon source. Hence, at thi s juncture, it is reasonab le to suggest that drasti c drop in surface bacteri al counrs can not be attributed to sa linity, water ac ti vity index Caw) or lack of carbon source. Therefore, drop in the bacterial cou nt could be due to the release of intracellul ar antibacterial compounds as algal th al lus cell s rupture.
The presence of exudates was also observed on glass padding of desiccation chamber during first two weeks of des iccation. However, ex udates could not be observed in SEM mi crographs since the specimens were p" eserved in fi xative and were dehydrated prior to coating. Direct freeze-drying of samples without use of fixat ive resulted in appearance of excess salt crystal s and failed to produce good SEM images.
SEM micrograph also suggested that algal ex udates could have leaked from the crac ked algal thalli as shown in Fig . 4. There were two morphologicall y different bacterial ce ll s in the SEM micrograph -( 1) VB-normal bacteri al ce lls; and (2) DB-deteriorated bacteri al cell s. Since the whole specimen was processed in the same manner, thi s implies that the differences in the bacteri al ce ll morphology refer to
844 INDIAN J EXP BIOL, AUGUST 2003
dead and viable bacteri al cel ls prior to process ing. SEM spec imen processing protocol could not have caused these effects because the specimens were fi xed prior to dehydrati on. Therefore, it has been suggested that most of the surface bacteri a were affected by the ex udates that could have caused the drop in bacteri al counts.
Further investi gation, using in vitro antibacterial bioassay of algal ex trac ts aga inst it s surface bac teri a, confirmed the presence of such an inherentl y avai lable antibacteria l potential. Results obtained from in vit ro antibacterial bioassay test of four difTerent extracts with varying solvent polarity suggested that antibac teri al mechani sm could be operating on a multi-compound basis. Thi s was obvious since ex tracts of different polarity showed inhibition towards different bac teri a. Compounds could be bacterium specifi c and C/M/W ex tract showed the best activity. Bioassay guided separation methodology and purifi ca tion of acti ve metabolites yielded 6 active compounds. Active metabolites isolated in thi s study have been show n to ex hibit biological activities in other brown algae. In an effort to verify the antibacterial activities of fatty acids, pure commerciall y available corresponding fatty ac ids were teo ted aga inst the sa me range of bacteri a. Tested fa tty ac ids ex hibited anti-bacterial acti vity at various intensiti es. In vestigation conducted by Rose ll and Srivastava2H have also reported saturated and unsat urated free fatty ac ids as antibacterial principal in nine spec ies of brown algae harvested from Vancouver Island, Brit ish Co lumbia. MGDGs occur widely throughout the pl ant kingdom and it constitutes a major class of lipids in photosy ntheti c ti ssues of terrestri al and marine pl ants. Studies on runner-bean leaves have shown that MGDGs are acti vely utili zed as a means of defense response when mechanical damage is inflicted to the plant membrane2
'J. MGDGs isolated from Pliormidium tel/Ile have also shown to exhibit autolytic activity towards algal cells22
. Recently , researchers are discovering biological activities of glycolipids, and its importance is not limited as a constituent of plant ce ll
30·12 component . . In conc lusion, findin gs of thi s in vestigation suggest
that seaweeds have inherently available antibacterial protecti on ab i lity to protect themselves against in trusion of surface/environmental bacteria. Since isolated active metabolites consisted of glycolipids and fatty ac ids, it is suggested that seaweed's defense is located in its ce ll membrane where glycolipids form
the major component. This study could be considered as another milestone in our effort to fully understand alga l intricate defense mechani sm aga inst marine microorgani sms. Hence, it has become apparent th at des iccati on of S. stoloniJolium upon harvest does not contribute to fermentation or other processes that would red uce algal's alginate quantity .
Acknowledgment The author wishes to th ank Dr Suzuki Minoru ,
Graduate School of Environmental Earth Science, Hokkaido University , Japan , for va luable adv ice and sugges tions in spectroscopi c data analys is.
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