Antimicrobial Fatty Acids from Green Alga Ulva rigida ...

Research ArticleAntimicrobial Fatty Acids from Green AlgaUlva rigida (Chlorophyta)

Amel Ismail 1 Leila Ktari1 Yosr Ben Redjem Romdhane1 Brahim Aoun1

Saloua Sadok1 Abdellatif Boudabous2 and Monia El Bour1

1Laboratory of Blue Biotechnology and Aquatic Bioproduction (B3 Aqua) National Institute of Marine Sciencesand Technologies (INSTM) 28 2 march street 1934 -2035 Salammbo Tunisia2Faculty of Mathematical Physical and Natural Sciences of Tunis El Manar University Tunisia

Correspondence should be addressed to Amel Ismail ismail mlyahoofr

Received 11 May 2018 Accepted 18 October 2018 Published 13 November 2018

Guest Editor Alladdin El Salabi

Copyright copy 2018 Amel Ismail et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study deals with the antimicrobial potential assessment ofUlva rigida in regard to collection period and sampling site Besideswe assess the chemical composition of bioactive compounds For this purpose Ulva rigida was seasonally collected from twonorthern sites in Tunisia Cap Zebib rocky shore (CZ) and Ghar El Melh lagoon (GEM) Crude organic extracts were preparedusing dichloromethane and dichloromethanemethanol and tested against 19 indicator microorganisms using the disk diffusionmethod and microdilution technique to determine the minimum inhibitory concentration (MIC) Silica gel column and thin layerchromatography were used for purification of active compounds Nuclear magnetic resonance (NMR) and gas chromatographywere used for compounds identification Samples of Ulva rigida collected from the two sites have uniform antimicrobial activitythroughout the year Algae collected from the lagoon showed the largest spectrumof activity andwere used for subsequent analysisBioguided purification of extracts from Ulva rigida collected at GEM leads to 16 active fractions with antibacterial effect mainlyagainst Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 These fractions were identified as fatty acidsmainly oleic (C18 1 w9) linoleic (C18 2 w6) palmitic (C16 0) and stearic (C14 0) MICs values ranged from 10 to 250 120583gml

1 Introduction

Seaweeds are a diverse group of marine organisms thathave developed complex biochemical pathways to survive ina highly competitive environment very different from theterrestrial one [1] Such situations require the productionof specific and potent bioactive substances that can leadto the development of new drugs and functional foods ornutraceuticals

From an economic point of view green algae (Chloro-phyta) are sustainable biomass feedstock for the food andbiotechnology industries including possibilities for inte-grated multitrophic aquaculture (IMTA) bioremediationand potential biofuel production [2 3] Ulva species are themost abundant representatives being ubiquitous in coastalbenthic communities around the world Ulvacean are con-sidered bioindicators species with increased importance incoastal ecosystem management mainly related to green tides

associated with eutrophication processes in shallow envi-ronments [3] In addition Ulva species represent untappedresources for food fuel and high value-added compoundsNevertheless the genus Ulva remains considerably under-studied [4]

In general algal chemical composition and therefore itsnutritional and biomedical value depend onmany factors thatinclude species and their development stages geographicalorigin collection period growth and environmental condi-tions [1 5] The green alga Ulva rigida is abundant on thecoast of Cap Zebib as well as in the lagoon of Ghar ElMelh two environments with very different hydrobiologicalcharacteristics TheGhar El Melh Lagoon is a shallow lagoonThe medium is hypereutrophic (low transparency low dis-solved oxygen concentration high nitrogen phosphorus andchlorophyll a) state generated by various land releases andamplified by water stagnation [6] Cap Zebib is a region inbeaten mode zone with presence of marine vegetation that

HindawiBioMed Research InternationalVolume 2018 Article ID 3069595 12 pageshttpsdoiorg10115520183069595

2 BioMed Research International

enriches environment by the oxygen [7] Therefore in thiswork U rigida was selected with the aim of studying theeffect of the collection period and the geographical site onthe production of bioactive secondary metabolites and theircharacterization

2 Materials and Methods

21 Alga Sampling and Identification U rigida C Agardhsampleswere collected seasonally from July 2006 to June 2007from the rocky shore of Cap Zebib (CZ) (37∘ 1621015840 N 10∘ 361015840E) and fromGhar ElMelh lagoon (GEM) (37∘ 1081015840N 10∘ 1681015840E) in the region of Bizerte (Northern coast of Tunisia) Algaesamples were collected by hand in shallow water (less than 2m depth) at low tide and kept on ice till their transfer to thelaboratory Algae were taxonomically identified according to[8ndash10] Specimen samples were conserved in 70 ethanol

22 Physicochemical Parameters Temperature salinity andpH were measured immediately after sampling using amultiparameter measuring device (HACH HQd field Case)The water quality of the two collection sites GEM and CZwas characterized seasonally through in situ measurementof temperature salinity dissolved oxygen and pH Analysisof nitrite (NO2

-) nitrate (NO3-) phosphate (PO4

3-) ammo-nium (NH4

+) total nitrogen (TN) and total phosphorus (TP)is done using spectrophotometric methods [11] Samples forchlorophyll a were filtered extracted in 90 acetone andquantified according to the method described by Stricklandand Parsons [11]

23 Extraction Procedure Fresh algae samples were rigor-ously washed three times with seawater and then with tap-water Subsequently they were dried in an oven at 40∘C orfor 15 days under ambient conditions in the shade The drybiomass was crushed until a powder was obtained whichwas kept at -20∘C for later analysis For algae crude extractpreparation 20 g of the dried algal biomass was extractedsuccessively by 2 organic solvents of increasing polaritydichloromethane (D) and dichloromethanemethanol (DM)(11 vv) These solvents are suitable to extract nonpolar andmoderately polar compounds Each extraction (24 h at roomtemperature) was repeated 3 times The extracts were pooledand filtered The filtrate was then concentrated in a rotaryevaporator to obtain crud extract which was stored at -20∘Cuntil use

24 Antimicrobial Test U rigida extracts and subsequentfractions (as described in purification fractionation andcharacterization analysis) were tested for antimicrobial activ-ity against indicators microorganisms The activity wasevaluated by the discs diffusion method 500 120583g of algalcrude extract was dissolved in dichloromethane (D) ordichloromethanemethanol (DM) (10 120583L) and placed onsterile filter paper discs (6 mm) After solvent evaporationdiscs were placed on Tryptone Soy Agar (TSA) plates alreadyinoculated with a test culture (106 bacteria mL-1) in TryptoneSoy Broth (TSB) Simultaneously a disc loaded with solventonly was used as a negative control Plates were incubated

overnight at 30∘C Inhibition diameters (mm)were measuredafter 24 h Antimicrobial activity tests were conducted intriplicate

25 Indicators Microorganisms A set of pathogenic bac-teria Gram+ve (Streptococcus agalactiae (Pasteur InstituteTunis) Staphylococcus aureus (Pasteur Institute Tunis) Saureus ATCC 25923 S aureus ATCC 6538 Enterococcusfaecalis ATCC 29212 Micrococcus sp (Pasteur InstituteTunis) and Gram-ve (Vibrio tapetis CECT4600 (Depart-ment of Microbiology and Parasitology University of San-tiago de Compostela Spain) V anguillarum ATCC 12964TV alginolyticus ATCC 17749T Escherichia coli O126-B16(ATTC 14948) E coli ATCC 25922 E coli ATCC 8739Pseudomonas cepacia (INSTM Tunisia) P fluorescens AH2(Danish Institute for Fisheries Research Denmark) P aerug-inosa ATCC 27853 Aeromonas salmonicida LMG3780 Ahydrophila B3 (RVAU-Denmark) Salmonella typhimuriumC52 (Laboratoire Hydrobiologie Marine et ContinentaleUniversite de Montpellier II France) and the yeast CandidaalbicansATCC10231 was used for testing antimicrobial activ-ity of seaweed extracts and fractions

26 Minimal Inhibition Concentration (MIC) The MIC wasdetermined for selective active extracts and fractions on TSBmedia according to Khan et al [12] and Ganiere et al [13]Assays were performed in sterile culture plates of 96 roundbottom wells Suspensions of indicator bacterial inoculumwere adjusted in the sterile broth medium TSB to the densityof 05 Standard McFarland (Corresponding to 0063 opticaldensity at 600 nm approximately 108 CFU mL-1) and thendiluted 10-fold twice to obtain a bacterial suspension densityof about 106 CFU mL-1 Microplates wells were inoculatedwith 180 120583L of the culture containing the inoculum 20 120583Lof each concentration of seaweed extract (diluted in dimethylsulfoxide (DMSO)) was added to the wells containing bacte-rial culture suspension The negative control contained 200120583L of culture medium only (without alga extract) Extracts(20 120583L) were adjusted to give a concentration range of 1600to 50 120583gmL (for alga crude extract) and 250 to 10 120583gmL (foralga fractions) Tests were performed in triplicate and plateswere incubated for 18-24 h at 37∘C Subsequently wells wereexamined by unaided eye for bacterial growth as indicated byturbidity [14] The last concentrations in the dilution seriesthat did not show visible growth (and showing only fewcolonies compared with other concentrations when spreadon agar plates) correspond to the MIC of the antimicrobialagent If difficulty is found to discern growth in some wellsMIC determination is then done with colony-forming unitscount

27 Fractionation Purification and Characterization Analy-sis In this study Thin Layer Chromatography (TLC) ana-lytic (TLCa) plates (Merck Fluka) were used The sol-vents system used for the fractions analysis is the n-HexEtOAcDCMMeOH with combinations and variablepercentages according to the fractions After their develop-ment chromatograms were revealed by chemical reactivesthe phosphomolybdic acid (PMA) and the liebermann For

BioMed Research International 3

Table 1 Nutrients and chlorophyll a concentration values at the collection sites

Locality Season N-NO2-

120583mol L-1N-NO3

-

120583mol L-1N-NH4

+

120583mol L-1P-PO4

3-

120583mol L-1TP120583mol L-1

TN120583mol L-1

Chl amgm-3

Cap Zebib

Winter 024 098 255 041 204 1625 326Spring - - - - - - -Summer 014 047 111 034 235 1023 313Autumn 022 076 470 056 377 1522 252

Ghar El Melh


TP total phosphorus and TN total nitrogen

Preparative Thin Layer Chromatography (TLCp) glass plates(20 x 20 cm) covered with silica gel (2 mm thickness)were used This technique allows the purification of smallproduct quantities (until sim100 mg) The band containingthe cleansed product is scratched and then the silica isextracted with a solvent The solvent system used in thisstudy is the n-hexaneEtOAc (13) The adsorption ColumnChromatography on Silica gel (CCS) (pore Size 60 A 0063-0200 mm (70-230 mesh) was used for U rigida extracts andsome fractions purification The solvent system used is the n-hexaneEtOAcDCMMeOH Sephadex LH-20 chromatog-raphy was used for separation of closely linked fractions(solvent system used was DCMMeOH)

Fractions obtained were analyzed for their fatty acidscomposition by gas chromatography Samples were homoge-nizedwith a chloroformmethanol (12 vv)mixture and incu-bated during 12 hours in darkness Residues were extracted 2-3 timeswith chloroform andmethanolThe phase containingthe chloroform was removed and vaporized Samples wereesterified in sulphuric acid (1) in absolute methanol andextracted with hexane by phase separation Samples wereanalyzed by means of a model leading chromatograph HP19091N-133 equipped with a polar column INNOWAX (30m of length 25 120583m of diameter thickness of the film is of025 120583m) mark Agilent Technology The oven temperaturewas from 150∘C to 240∘C with a gradient of 2∘Cmin Theinjector temperature is 220∘C that of the detector is 275∘Cflow 1mlmin and injection volume is 1120583l The chromatogrampeaks are identified comparedwith the retention time of stan-dards peaks (SUPELCO) injected in the same conditions

Nuclear magnetic resonance (NMR) was used for theactive fractions chemical characterization 1H NMR and13C NMR spectra were recorded on an AVANCE 300 MHzinstrument (Bruker) Extracts and fractions were solubilizedin CDCl3 Chemical shifts 120575 were expressed in parts permillion (ppm) coupling constants J was expressed in Hertz(Hz)The identification of mixture constituents by NMR 13Cwas realized by comparison of the chemical shifts of themixture with those of the reference compounds contained inone or several spectra databases

28 Statistical Analysis Analytical determinations were real-ized in triplicate and the average values were registered The

data were analyzed by using the IBM SPSS Statistics (v 20)and test Khi-2 used to determine significant variation of theactivity (Plt 005) Principal component analysis (PCA) wasused to determine correlations between antimicrobial activityand chemical composition of positive fractions

3 Results

31 Physicochemical Parameters Registration of physico-chemical parameters at GEMandCZ shows that the tempera-ture varies from 13∘C to 23∘C in CZ and of 15∘C to 24∘C inGEpH values salinity and dissolved oxygen are almost constantin both regions whereas values of nitrate ammonium totalphosphorus (TP) and chl a registered are clearly higher in thelagoon water (Table 1)

32 Antimicrobial Activity D and DM extracts of U rigidacollected fromGEMshowed significant antimicrobial activityduring the four seasons with a variable activity spectrum(Table 2) No significant seasonal variability of the antimicro-bial activity was detected The P value (calculated accordingto the Khi-2 test) was gt 005Themost sensitive bacteria wereA salmonicida S typhimurium Str agalactiae A hydrophilaP cepacia S aureus and E faecalis MIC values were 08mgmL against both P cepacia and A salmonicida The mostresistant strains were E coli Vibrio spp Pseudomonas sppMicrococcus sp and the yeast C albicans

Similarly forU rigida collected on CZ no seasonal effecton the antimicrobial activity was observed (pgt 005) Six of19 tested indicator bacteria were sensitive to the extracts ofU rigida (CZ) (Table 3) D and DM extracts show a strongactivity against S aureus ATCC 25923 and Str agalactiae Noactivity was detected against Gram-ve bacteria except on Asalmonicida for which the lowest MIC value (08 mgmL)was recorded Considering the activity spectrum U rigidacollected from the lagoon presented a more pronouncedantibacterial activity This difference is especially observedwith Gram-ve bacteria (58 of these Gram-ve bacteria wereinhibited by U rigida (GEM) while only 16 were inhibitedby U rigida (CZ))

Considering that U rigida from GEM showed the mostrelevant activity spectrum it was chosen for subsequentfractionation purification and chemical characterization


Table 2 Antimicrobial activity of U rigida collected seasonally from Ghar El Melh lagoon (data are in mm of inhibition diameter)

Dichloromethane DichloromethanemethanolWinter Spring Summer Autumn Winter Spring Summer Autumn

E coliO126 B16 - - - - 8plusmn0 113plusmn11 7plusmn0 8plusmn0V tapetis CECT 4600 - - - 12plusmn0 - - - 115plusmn05P cepacia - - 118plusmn02 126plusmn05 116plusmn05 96plusmn05 143plusmn05 12plusmn0P aeruginosa ATCC 27853 - 11plusmn0 7plusmn0 - - - - -Aeromonas salmonicida 14plusmn0 14plusmn0 14plusmn0 14plusmn0 12plusmn0 10plusmn0 11plusmn0 11plusmn0A hydrophilaB3 - 11plusmn0 - 156plusmn05 93plusmn11 10plusmn0 14plusmn0 15plusmn1S typhymurium 113plusmn11 - - 12plusmn0 12plusmn0 156plusmn05 116plusmn05 14plusmn0Str agalactiae 123plusmn05 116plusmn05 14plusmn0 146plusmn11 103plusmn05 86plusmn05 143plusmn15 146plusmn05S aureus 7plusmn0 7plusmn0 86plusmn05 10plusmn0 10plusmn0 126plusmn23 133plusmn11 16plusmn0S aureus ATCC 25923 13plusmn1 173plusmn11 17plusmn0 16plusmn0 10plusmn0 16plusmn0 18plusmn0 16plusmn0S aureus ATCC 6538 103plusmn05 11plusmn0 13plusmn0 103plusmn05 9plusmn0 10plusmn0 136plusmn05 11plusmn0E faecalis ATCC 29212 115plusmn08 113plusmn11 143plusmn05 156plusmn05 12plusmn0 113plusmn11 14plusmn0 17plusmn1Extracts were tested at concentrations of 500 120583gdisc +- represents the standard deviation the number of independent replicates was n=3

Table 3 Antimicrobial activity of U rigida collected seasonally from Cap Zebib shore (data are in mm of inhibition diameter)


V alginoliticus - 103plusmn05 8plusmn0 76plusmn05 - - - -A salmonicida 15plusmn0 128plusmn02 9plusmn0 - 116plusmn05 - 83plusmn05 8plusmn17Str agalactiae 15plusmn0 123plusmn11 15plusmn1 103plusmn01 96plusmn05 63plusmn05 163plusmn05 11plusmn0S aureus 96plusmn05 88plusmn02 103plusmn05 103plusmn05 83plusmn02 - 113plusmn05 116plusmn05S aureus ATCC 25923 128plusmn02 123plusmn05 126plusmn05 108plusmn02 91plusmn02 66plusmn1 163plusmn11 106plusmn05S aureus ATCC 6538 10plusmn0 106plusmn05 10plusmn02 106plusmn05 106plusmn05 11plusmn0 128plusmn02 10plusmn0Extracts were tested at concentrations of 500 120583gdisc +- represents the standard deviation the number of independent replicates was n=3

33 U rigida (Ghar El Melh) Crude Extract PurificationGiven that all U rigida (GEM) extracts independently of thecollection season gave a significant antibacterial activity theywere grouped in a single extract for a better purification Theelutionwas realized in gradientmode byCCSsNine fractions(FG1-FG9) were obtained and tested for their antibacterialeffect towards three indicator bacteria S aureus E faecalisand A Salmonicidawhich were the most sensitive bacteria topreviously tested U rigida (GEM) crude extracts (Table 4)The most active fraction FG1 was purified and a total of 27sub-fractions (G1-G27) were obtained (Figure 1) which werealso tested for their antibacterial potential

Results showed that G4-G9 G11 G14-G16 and G26fractions were active towards at least one of pathogenictested bacteria with low values of MIC (Table 5) TheTLC analysis and the PMA and LB revelation of G1 toG27 fractions show fatty acids (FA) characteristic spotsespecially for the G1 to G10 fractions (Figure 2) The G4G5 and G6 fractions contain FA in important quantityThese fractions were chosen for a final purification process(Figure 3) according to their higher antibacterial effect andlower MIC values In addition these fractions also showedsufficient weight for further purification Table 6 showsantibacterial activity results for G4 G5 and G6 fractionspresenting lower MIC values that ranged between 10 and40 120583gml

The successive purification of the U rigida crude extractand the chemical revelation (Figure 3) showed that activefractions (16 fractions G4-G9 G11 G13 G4 (4) G5 (5)B4 FX6 A B C and D) had characteristic blue spotsof FAs Figure 4 shows antibacterial activity of U rigidafractions against S aureus ATCC 25923 Therefore an NMR1H analysis was carried out to confirm the structure of activecompounds Subsequently gas chromatography was appliedto these fractions to determine their FAs composition TheNMR spectra of the G4 G5 FX6 B4 A B C and D fractionspossess typical NMR spectra of saturated fatty acids (SFA)and polyunsaturated fatty acids (PUFA) mixture Figure 5represents the NMR 1H and 13C spectra of G4 compound

FA composition of G4-G9 G11 G13 G4 (4) G5 (5) andB4 fractions (having a sufficient weight) is shown in Table 7Results showed that the fractions obtained contained satu-rated (SFA)monounsaturated (MUFA) and polyunsaturatedfatty acids (PUFA) with variable quantities according to thefraction Different fractions FA profiles showed that fractions(G4 G5 and G6) containing mainly SFAwere themost activewhile those containing low amounts of PUFAwere less active(G7 G8 G9 and G11)

In addition fractions having a high amount in palmiticacid were the most active (G4 G5 G6 G4 (4) G5 (5) andB4) Furthermore the increase in oleic acid amount in thefractions G4 and G5 is proportional to the increase of the


Table 4 Antibacterial activity of FG1-FG9 fractions obtained from U rigida (Ghar El Melh)

Fraction S aureus ATCC 25923lowast E feacalis ATCC 29212lowast A SalmonicidalowastID (mm) MIC (120583gml) ID (mm) MIC (120583gml) ID (mm) MIC (120583gml)

FG1 17plusmn1 250plusmn0 183plusmn05 250plusmn0 126plusmn05 500plusmn0FG2 136plusmn05 500plusmn0 136plusmn05 500plusmn0 66plusmn05 500plusmn0FG3 123plusmn2 500plusmn0 83plusmn05 500plusmn0 -FG4 86plusmn05 500plusmn0 - -FG5 - - -FG6 - - 14plusmn0 250plusmn0FG7 63plusmn05 500plusmn0 - 106plusmn05 500plusmn0FG8 9plusmn1 500plusmn0 - -FG9 - - -lowast concentration 500 120583gdisc ID inhibition diameter MIC minimal inhibition concentration and - not active

TLCa

U rigida crude extract117 g

64 fractions

27 sub-fractions (G1-G27)

CCS (n-HexEtOAcDCMMeOH)

TLCa Chemicalrevelation

FG9409 mg

FG1645 mg

G1 G4 G5 G6

(+)

Chemical revelations

9 fractions

---------------------

--------G27-------

NMR 1（ 13 GC

Figure 1 U rigida (Ghar El Melh) crude extracts purification stepsCCS column chromatography silica gel n-Hex n-hexane EtOAcethyl acetate DCM dichloromethane MeOH methanol TLCaanalytic thin layer chromatography GC gaz chromatography 1HNMR nuclear magnetic resonance of proton RMN13C nuclearmagnetic resonance of carbon (+) active against at least oneindicator microorganisms

activity in their subfractions G4 (4) B4 and G5 (5) Thisindicates that this FA is involved in the observed activityMoreover when comparing the FA profile of the G13 fraction(which is an inactive fraction) to those of the other active oneswe notice that the absence of the stearic acid and palmitoleicacid in G13 could partially explain the lack of activity

To determine the correlation rates between the observedantibacterial activity and the FA composition (SFA MUFA

Blues spots characteristicsof fatty acids

Phospho-molybdic acid revelation 82n-hex EtOAc

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10

Figure 2 Phosphomolybdic acid revealed TLC of U rigida (GEM)purified fractions (as explained in Figure 1)

and PUFA) a statistical analysis in principal components(PCA) was made (Figure 6) This representation allowsdistinguishing clearly 3 groups of fractions The first groupconsists of G4 G5 and G6 fractions which present an impor-tant activity towards S aureus and E feacalis and which arerich in SFAThe second group is composed of G7 G8 and G9fractions containing high amount in PUFA and MUFA andshowing low antibacterial activities The last group containsonly the G11 fraction Indeed G11 fraction is characterized byits activity against A salmonicida and relatively low amountof FAs (not exceeding the 55 ) compared to the otherfractions This fact suggests that observed activity againstA Salmonicida was probably caused by different non FAssubstances

4 Discussion

U rigida samples were collected from two different geo-graphic locations to determine the effect of geographical siteon the antimicrobial activity U rigida (CZ) is mainly activeagainst Gram+ve bacteria and only inhibits 16 of Gram-vebacteria whereas U rigida (GEM) has a broader spectrum ofactivity with an inhibitory effect against 5 of the 6 Gram+vebacteria and 58 inhibition of Gram-ve ones


(+)

--------

TLCp

G4(485 mg)

B4

5 fractions

(+)111 mg

(+)

------- ---

A B C D

7 fractions

G5(1847 mg)

TLCp

4 sub-fractions

(+)

(+)70 mg

(+)

6 fractions

12 fractions

G6(619 mg)


-----

FX1

(+)

(+)

(+)

FX6---------

16 mg

5 (1) 5 (7)G () 6 (1) 6 (12)G ()

NMR 1（

NMR 1（

（20

4 (1) 4 (5)G ()

（20

（20

NMR 1（

13

NMR 1（

13

NMR 1（

13

Figure 3 Purification steps of G4 G5 and G6 fractions obtained from U rigida CCS column chromatography silica gel TLCp preparativethin layer chromatography 1H NMR nuclear magnetic resonance of proton 13C NMR nuclear magnetic resonance of carbon (+) activeagainst at least one indicator microorganisms

Table 5 Antimicrobial activity of Ulva rigida (GEM) purified fractions (as explained in Figure 1)

Sub-fractions from FG1Indicator bacteria

S aureus ATCC 25923 E faecalis ATCC 29212 A salmonicidaID (mm) MIC (120583gml) ((120583gml) ID (mm) MIC (120583gml) (120583gml) ID (mm) MIC (120583gml) ((120583gml)

G4 216plusmn05 625plusmn0 22plusmn0 125plusmn0 - -G5 23plusmn15 625plusmn0 21plusmn15 250plusmn0 - -G6 18plusmn05 625plusmn0 163plusmn03 250plusmn0 - -G7 10plusmn0 250plusmn0 - - - -G8 96plusmn05 250plusmn0 - - - -G9 11plusmn0 250plusmn0 - - - -G10 - - - - 73plusmn05 250plusmn0G11 15plusmn1 250plusmn0 - - 73plusmn11 250plusmn0G12 - - - - 83plusmn05 250plusmn0G13 - - - - 10plusmn0 250plusmn0G14 24plusmn15 250plusmn0 - - 106plusmn05 1666plusmn72G15 22plusmn05 250plusmn0 nt nt nt ntG16 21plusmn05 250plusmn0 - - - -G23 73plusmn05 - nt nt nt ntG26 - - 96plusmn05 250plusmn0 - -MIC minimal inhibitory concentration nt nontested - no activity ID inhibition diameter of fractions tested at concentration of 250 120583gdisc

Gram-ve bacteria E coli was inhibited only by U rigida(GEM)This bacterium is known to be resistant to the major-ity of seaweed extracts andmost marine organisms in general[15 16] In addition to E coli the indicator bacteria V tapetisP cepacia P aeruginosa A hydrophila and S typhimurium

were also inhibited byU rigida (GEM)Theywere resistant tothe extracts of samples collected from CZ The susceptibilityof Gram-ve bacteria to U rigida (GEM) extracts can beexplained by the effect of factors related to the type andbiochemical characteristic of sediment and water of the


G4

G4 (1)G4 (3)

G4 (4)G4 (5)

T-

(a)

G5 G5 (7)

G5 (5)G5 (4)

G5 (6)

G5 (3)

G5 (1)G5 (2)

T-

(b)

A B

CD

T-

(c)

FX6

B4G4 (4)

T-

(d)

Figure 4 Antibacterial activity of U rigida G4 G5 and G6 fractions and subfractions against S aureus ATCC 25923

Table 6 Antibacterial activity against S aureus ATCC 25923 of sub-fractions obtained from G4 G5 and G6 of U rigida (Ghar El Melh)

Sub-fractions obtained by TLCp or LH20 Diametre (mm) MIC (120583gml)G4 (4) 173plusmn05 20G5 (5) 176plusmn05 40G6 (2) 18plusmn05 20B4 203plusmn05 20A 166plusmn05 10B 13plusmn0 40C 206plusmn05 10D 243plusmn11 20FX6 186plusmn05 10LH20 Liquid Sephadex chromatography TLCp preparative thin layer chromatography MIC minimal inhibitory concentration Fractions were tested at aconcentration of 40 120583gdisc +- represents the standard deviation the number of independent replicates was n=3

lagoon and other factors probably related to the interactionbetween seaweed and several micro and macroorganismsliving in the same environment

Physicochemical characteristics and hydrobiologicalproperties of the two collections sites are different inparticular the concentrations of ammonium nitrate totalnitrogen and chlorophyll a were markedly different in GEMand CZ Concentrations recorded from the lagoon water

were higher The water of the lagoon was concentrated withnitrate and ammonium when compared to the coast of CZThese nutrients (from agricultural sources or from urbanwastewater discharges) are indicators of environmentalpollution leading to eutrophication and causing the excessiveproliferation of green algae especially Ulva Moreoverit is also worth mentioning that GEM lagoon water wascharged with chlorophyll a The latter is considered as


ppm

(a)

ppm

(b)

Figure 5 NMR 1H (a) and 13C (b) spectra of G4 fraction in CDCl3 obtained from U rigida (Ghar El Melh) purification

an indicator of the abundance of microscopic algae Theantibacterial activity of U rigida (GEM) with regard toGram-ve bacteria can be explained by the fact that thealgae growing in a polluted environment (characterized bythe presence of unhealthy fish and invertebrates and a lowoxygen concentration) tend to defend themselves by theproduction of secondary metabolites that would not found inthe same specie collected from an unpolluted marine zone

It is conceivable that the geographical site plays an impor-tant role in the production of secondary metabolites Theseresults and observations support the hypothesis of the impactof collection site on the secondary metabolites produced byalgaeThis is confirmed byMartı et al [17]Marechal et al [5]and Salvador et al [18] who emphasized that the geographicalsite is among the factors affecting algae toxicityThis variation

related to the collection site might be due to the nature of thesite whether exposed to shear forces or quietmode in the seaor in protected bays Various biotic and abiotic environmentalfactorsmay impact the algae biology and physiology and thusinfluence their secondary metabolites productionMarti et al[17] have noted that also various ecological parameters suchas nutrients and photoperiod can determine the productionof secondary metabolites

Among the fractions obtained fromU rigida purification16 fractions contain FAs in high concentrations

FAs were previously incorporated into food with the aimto prevent the action of human pathogenic microorganismssuch as those of genus Salmonella Listeria and Staphylococ-cus [19] The antimicrobial effect of the FAs isolated fromU intestinalis was tested by Horincar et al [20] against four


G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA

Biplot (axes F1 et F2 9155 )

210 3 4minus2minus3minus4 minus1minus5F1 (7479 )

minus2

minus1

0

1

2

3

F2 (1

676

)

Figure 6 Correlations between fractions type fatty acids composition and antibacterial activity

Table 7 Fatty acids composition of U rigida (Ghar El Melh) fractions

Proportion ()STD STD G4 G5 G6 G7 G8 G9 G11 G13 G4 (4) G5 (5) B4SFA(C140) 770 299 339 117 121 086 116 144 077 096 287(C150) 040 127 209 125 092 369 079 088 143(C160) 1059 7545 7170 6876 3190 2164 2114 1489 2357 55 5332 6784(C180) 133 325 135 333 081 060 047 601 172 208 132MUFA(C161 w7) 1531 368 711 597 970 580 1203 725 851 641(C181 w9) 1058 1198 1232 292 1851 1443 1403 698 666 2052 2646 1728(C181 w7) 662 386 655(C201 w9) 236 137 376PUFA(C162 w4) 183 032 046(C182 w6) 100 135 041 338 346 377 214 042 091(C163 w4) 273 032 042(C183 w4) 085(C183 w3) 191 215 588 672 721 646 016(C184 w3) 282 604 756 805 888 341(C204 w6) 111(C204 w3) 187 037 044 046(C205 w3) 1681 024 025(C225 w3) 2816 071 089 091(C226 w3) 1079Total 9950 100 9905 8485 7575 6659 6282 5333 5710 8724 9222 9897PUFA 4457 135 106 215 164 1934 2067 1749 341 058 0 180MUFA 3489 1566 1943 816 2449 2413 1983 1901 1698 2777 3497 2369SFA 2003 8297 7855 7453 3486 2311 2230 1681 3327 583 5725 7347w3 3703 0 0 215 1301 1588 1689 1535 341 016 0 0w6 212 135 041 0 338 346 377 214 0 042 0 091


pathogenic bacteria (Bacillus cereus L monocytogenes E coliand S enteritidis) The MIC of the U intestinalis extractscontaining FAs was 38 mgml In the present work the MICof the active fractions containing a set of FAs is relativelylow (10-250 120583gmL) This clear difference could be explainedby a difference in the composition or the amounts of theactive FAs The activity can also be variable with the targetbacterium

Stabili et al [21] demonstrated that the alpha linolenicacid isolated from the green alga Cladophora rupestris col-lected from the Mediterranean Sea is the most dominantFA in April (Spring) which confirm its role in the observedactivity against Vibrio spp during this month with a MICvalue of 18 120583gml Antibacterial and antifungal propertieswere previously attributed to linoleic and oleic acids Thelatter is also known to have a bactericidal activity towardsseveral pathogenic microorganisms including S aureusHelicobacter pylori V Parahaemolyticus andMycobacterium[21ndash24]

In this study both G4 and G5 fractions are the mostactive compared to the other fractions (G7 G8 G9 and G11)obtained from the first purification of FG1G4 andG5 containmainly saturated fatty acids and have a low PUFA amountOn the other hand G7 G8 G9 and G11 which showedhigh PUFA proportions gave low antibacterial activity Thissuggests that G7 G8 G9 and G11 fractions contain besidesthe FAs other compoundswhichmay have antagonistic effecton these PUFA known to have a power interesting bioactiveeffect [25]This hypothesis also leans on the fact that in thesefractions the global proportion in FAdoes not exceed the 75contrary to the other fractions where the FA proportions arebetween 85 and 99 Thus this could explain that despitethe high quantity in PUFA in these fractions their inhibitionactivity was not remarkable

The correlation rates between the observed antibacterialactivity and the FA composition (SFA MUFA and PUFA)determined by CPA show that G11 fraction is characterizedby its activity on A salmonicida This fraction has relativelylow FA proportions compared to the other fractions Thisfraction is characterized by the fact that its total FA pro-portion does not exceed 55 suggesting that the observedactivity on A Salmonicida is caused by substances other thanFA Furthermore the G7 G8 G9 and G11 fractions beingcharacterized by their relatively high PUFA proportion (andlow FA proportion (between 53 and 75 )) compared tothe other fractions and a low activity towards S aureus Thissuggests that the observed activity on S aureus is probablydue to the effect of other substances which act by decreasingor by blocking the PUFA activity

We also noted that the oleic acid proportion (C18 1w9) in G4 and G5 fractions increased in their sub-fractionsG4 (4) and G5 (5) This increase is proportional with theantibacterial activity observed for these fractions This letsdeduce that the oleic acid is totally or partially responsiblefor the observed activity Although the G6 contains low oleicacid proportion this fraction showed significant inhibitioneffect This fraction may contain other active substancesthan FA Moreover the fractions having high palmitic acidproportion (G4 G5 G6 G4 (4) G5 (5) and B4) are the most

active fractions This suggests that the palmitic acid even notknown for its antibacterial properties could act in synergywith the oleic acid to give a bacterial inhibitive activityIn addition the stearic acid (C140) also seems to have arole in the observed activity especially towards S aureusATCC 25923

The PUFA C204 w3 C205 w3 and C225 w3 are knownto have antibacterial properties [25] In this study their effectwas not pronounced in the G7 G8 and G9 fractions sincethey are present in very small quantities (02 to 09 )Also alpha linolenic acid (C183 w3) and stearidonic acid(C184 w3) antibacterial affects were not observed in G7-G11fractions which are weakly active This could be explainedby the fact that the action of these FAs were inhibited bythe interference of others metabolites in the same fractionKnapp and Melly [26] demonstrated that the PUFA andMUFA are particularly active towards Gram+ve bacteriaThese authors indicated that the toxicity of the PUFA towardsS aureus depends on incubation time concentration and FAinsaturation

The antibacterial action of FAs is always attributed tolong chains of PUFA as the oleic linoleic and linolenic acidand their mechanism of action is to inhibit the synthesis ofbacterial FAs [25] FAs are known not to be able to inhibitthe Gram-ve bacteria such as E coli [27] This could be aconsequence of the external membrane impermeability of theGram-ve bacteria which acts as a barrier against hydrophobicsubstances [27] Even if relation between oleic acid structureand antimicrobial activity is not clear it seems that thenumber and the position of double bond as well as presenceof hydrophilic head and a hydrophobic tail can influence theantimicrobial activity affecting the bipolar membrane of thebacterial cell wall

5 Conclusions

U rigida collected from Tunisian coasts displayed antibac-terial activity throughout the year Algae collected from thelagoon possess the widest antibacterial activity spectrum ASalmonicida AHydrophila S typhimurium Str agalactiae Saureus and E feacalis pathogens are the most sensitive to Urigida collected from lagoon The difference between nitrateammonium total phosphorus and chlorophyll a values in thetwo collection sites seems to have an effect on antibacterialactivity variation of U rigida extracts Oleic palmitic andstearic acids seem to be responsible for the observed activityin the seaweed collected from the lagoon with low MIC val-ues Indicators pathogens inhibited by U rigida compoundspresent several resistances to antibiotics They are oftenassociated with many infections as the meningitis sepsisand endocarditis (the case of S aureus) A salmonicida andA hydrophila are responsible for furunculosis and ldquoMotileAeromonas Septicemiardquo affecting shellfish amphibians crus-taceans clams and various fish such as salmon and sea breamand are responsible for serious economic losses around theworld Therefore fatty acids from U rigida collected fromGhar El Melh lagoon might be potential source for use in thedevelopment of new antibacterial substances against humanand marine organisms-diseases


Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors wish to thank Pr Micheline Grignon DuboisUniversity of Bordeaux 1 for her help for NMR and GCanalysis Authors are grateful to Ms Amna Debbabi Facultyof Sciences of Monastir for her collaboration and for givingaccess to NMR facilities This study was supported by Cross-Border Project BIOVecQ PS13 08 cofinanced by the EU

References

[1] L Paiva E Lima A I NetoMMarcone and J Baptista ldquoNutri-tional and Functional Bioactivity Value of Selected AzoreanMacroalgae Ulva compressa Ulva rigida Gelidium microdonand Pterocladiella capillaceardquo Journal of Food Science vol 82no 7 pp 1757ndash1764 2017

[2] T Alsufyani Metabolite profiling of the chemosphere of themacroalga Ulva (Ulvales Chlorophyta) and its associated bacte-ria PhD Dissertation Friedrich Schiller University Jena JenaGermany 2014

[3] T Wichard ldquoExploring bacteria-induced growth and morpho-genesis in the green macroalga order Ulvales (Chlorophyta)rdquoFrontiers in Plant Science vol 6 2015

[4] T Wichard B Charrier F Mineur J H Bothwell O D Clerckand J C Coates ldquoThe green seaweed Ulva a model system tostudy morphogenesisrdquo Frontiers in Plant Science vol 6 2015

[5] J-P Marechal G Culioli C Hellio et al ldquoSeasonal variationin antifouling activity of crude extracts of the brown algaBifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanusamphitrite and the marine bacteria Cobetia marina and Pseu-doalteromonas haloplanktisrdquo Journal of Experimental MarineBiology and Ecology vol 313 no 1 pp 47ndash62 2004

[6] M Moussa L Baccar and R Ben Khemis ldquoGhar El Melhlagoon Ecological diagnoses and future hydraulic restoration[La lagune de Ghar El Melh Diagnostic ecologique et perspec-tives drsquoamenagement hydraulique]rdquo Revue des Sciences de lrsquoeauvol 18 pp 13ndash26 2005

[7] K BenMustapha and A Afli ldquoQuelques traits de la biodiversitemarine de Tunisie Proposition drsquoaires de conservation et degestionrdquo Report of the MedSudMed Expert Consultation onMarine Protected Areas and Fisheries Management Med SudMed Techniqueal Documents Rome Italy pp 32-55 2007

[8] G Hamel Pheophycees de France Wolf Press Paris France1931-1939

[9] M M Fisher L W Wilcox and L E Graham ldquoMolecularcharacterization of epiphytic bacterial communities on charo-phycean green algaerdquoApplied and Environmental Microbiologyvol 64 no 11 pp 4384ndash4389 1998

[10] J Cabioch J Y Floch A Le Toquin C F Boudouresque AMeinesz and M Verlaque Guide des algues des mers drsquoEuropeDelachaux et Niestle Press Paris France 2006

[11] J D H Strickland and T R Parsons ldquoA practical handbookof seawater analysisrdquo Bulletin of the Fisheries Research Board ofCanada vol 167 p 310 1972

[12] M A U Khan M K Ashfaq H S Zuberi M S Mahmoodand A H Gilani ldquoThe in vivo antifungal activity of the aqueousextract fromNigella sativa seedsrdquo Phytotherapy Research vol 17no 2 pp 183ndash186 2003

[13] J P Ganiere C Mangion and M Peridy ldquoDetermination desConcentrations Minimales Inhibitrices et Bactericides de lacefquinome la marbofloxacine la tylosine et la spiramycine ensolution dans du lait vis-a-vis de bacteries isolees de mammitesbovinesrdquo Revue de Medecine Veterinaire vol 155 no 8-9 pp411ndash416 2004

[14] CLSI Performance Standards for Antimicrobial SusceptibilityTesting CLSI supplement M100 Clinical and Laboratory Stan-dards Institute Wayne Pennsylvania USA 27th edition 2017

[15] A F Khaleafa M A M Kharboush AMetwalli A F Mohsenand A Serwi ldquoAntibiotic (Fungicidal) Action from Extracts ofSome Seaweedsrdquo Botanica Marina vol 18 no 3 pp 163ndash1661975

[16] Z Kamenarska M J Gasic M Zlatovic et al ldquoChemicalcomposition of the brown alga Padina pavonia (L) Gaill fromthe Adriatic seardquo Botanica Marina vol 45 no 4 pp 339ndash3452002

[17] R Martı M J Uriz and X Turon ldquoSeasonal and spatialvariation of species toxicity inMediterranean seaweed commu-nities Correlation to biotic and abiotic factorsrdquoMarine EcologyProgress Series vol 282 pp 73ndash85 2004

[18] N Salvador A Gomez Garreta L Lavelli and M A RiberaldquoAntimicrobial activity of Iberian macroalgaerdquo Scientia Marinavol 71 no 1 pp 101ndash113 2007

[19] B M Lekogo L Coroller A G Mathot P Mafart and ILeguerinel ldquoModelling the influence of palmitic palmitoleicstearic and oleic acids on apparent heat resistance of sporesof Bacillus cereus NTCC 11145 and Clostridium sporogenesrdquoInternational Journal of Food Microbiology vol 141 no 3 pp242ndash247 2010

[20] V B Horincar G Parfene A K Tyagi et al ldquoExtraction andcharacterization of volatile compounds and fatty acids from redand green macroalgae from the Romanian Black Sea in orderto obtain valuable bioadditives and biopreservativesrdquo Journal ofApplied Phycology vol 26 no 1 pp 551ndash559 2014

[21] L StabiliM I Acquaviva F Biandolino et al ldquoBiotechnologicalpotential of the seaweed Cladophora rupestris (ChlorophytaCladophorales) lipidic extractrdquo New Biotechnology vol 31 no5 pp 436ndash444 2014

[22] C Q Sun C J OrsquoConnor and A M Roberton ldquoAntibacterialactions of fatty acids and monoglycerides against Helicobacterpylorirdquo FEMS Immunology ampMedical Microbiology vol 36 no1-2 pp 9ndash17 2003

[23] V Seidel and P W Taylor ldquoIn vitro activity of extracts and con-stituents of Pelagonium against rapidly growing mycobacteriardquoInternational Journal of Antimicrobial Agents vol 23 no 6 pp613ndash619 2004

[24] MAAlamsjah K Ishibe D K Kim et al ldquoSelecive toxic effectsof polyunsaturated fatty acids derived from Ulva fasciata onred tide phyotoplanker speciesrdquo Bioscience Biotechnology andBiochemistry vol 71 no 1 pp 265ndash268 2014

[25] C J Zheng J-S Yoo T-G LeeH-Y ChoY-HKim andW-GKim ldquoFatty acid synthesis is a target for antibacterial activity ofunsaturated fatty acidsrdquo FEBS Letters vol 579 no 23 pp 5157ndash5162 2005


[26] H R Knapp and M A Melly ldquoBactericidal effects of polyun-saturated fatty acidsrdquoThe Journal of Infectious Diseases vol 154no 1 pp 84ndash94 1986

[27] J L Balcazar T Rojas-Luna and D P Cunningham ldquoEffect ofthe addition of four potential probiotic strains on the survivalof pacific white shrimp (Litopenaeus vannamei) followingimmersion challenge with Vibrio parahaemolyticusrdquo Journal ofInvertebrate Pathology vol 96 no 2 pp 147ndash150 2007

Hindawiwwwhindawicom

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Anatomy Research International

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Cell BiologyInternational Journal of



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MicrobiologyHindawiwwwhindawicom

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enriches environment by the oxygen [7] Therefore in thiswork U rigida was selected with the aim of studying theeffect of the collection period and the geographical site onthe production of bioactive secondary metabolites and theircharacterization

2 Materials and Methods

21 Alga Sampling and Identification U rigida C Agardhsampleswere collected seasonally from July 2006 to June 2007from the rocky shore of Cap Zebib (CZ) (37∘ 1621015840 N 10∘ 361015840E) and fromGhar ElMelh lagoon (GEM) (37∘ 1081015840N 10∘ 1681015840E) in the region of Bizerte (Northern coast of Tunisia) Algaesamples were collected by hand in shallow water (less than 2m depth) at low tide and kept on ice till their transfer to thelaboratory Algae were taxonomically identified according to[8ndash10] Specimen samples were conserved in 70 ethanol

22 Physicochemical Parameters Temperature salinity andpH were measured immediately after sampling using amultiparameter measuring device (HACH HQd field Case)The water quality of the two collection sites GEM and CZwas characterized seasonally through in situ measurementof temperature salinity dissolved oxygen and pH Analysisof nitrite (NO2

-) nitrate (NO3-) phosphate (PO4

3-) ammo-nium (NH4

+) total nitrogen (TN) and total phosphorus (TP)is done using spectrophotometric methods [11] Samples forchlorophyll a were filtered extracted in 90 acetone andquantified according to the method described by Stricklandand Parsons [11]

23 Extraction Procedure Fresh algae samples were rigor-ously washed three times with seawater and then with tap-water Subsequently they were dried in an oven at 40∘C orfor 15 days under ambient conditions in the shade The drybiomass was crushed until a powder was obtained whichwas kept at -20∘C for later analysis For algae crude extractpreparation 20 g of the dried algal biomass was extractedsuccessively by 2 organic solvents of increasing polaritydichloromethane (D) and dichloromethanemethanol (DM)(11 vv) These solvents are suitable to extract nonpolar andmoderately polar compounds Each extraction (24 h at roomtemperature) was repeated 3 times The extracts were pooledand filtered The filtrate was then concentrated in a rotaryevaporator to obtain crud extract which was stored at -20∘Cuntil use

24 Antimicrobial Test U rigida extracts and subsequentfractions (as described in purification fractionation andcharacterization analysis) were tested for antimicrobial activ-ity against indicators microorganisms The activity wasevaluated by the discs diffusion method 500 120583g of algalcrude extract was dissolved in dichloromethane (D) ordichloromethanemethanol (DM) (10 120583L) and placed onsterile filter paper discs (6 mm) After solvent evaporationdiscs were placed on Tryptone Soy Agar (TSA) plates alreadyinoculated with a test culture (106 bacteria mL-1) in TryptoneSoy Broth (TSB) Simultaneously a disc loaded with solventonly was used as a negative control Plates were incubated

overnight at 30∘C Inhibition diameters (mm)were measuredafter 24 h Antimicrobial activity tests were conducted intriplicate

25 Indicators Microorganisms A set of pathogenic bac-teria Gram+ve (Streptococcus agalactiae (Pasteur InstituteTunis) Staphylococcus aureus (Pasteur Institute Tunis) Saureus ATCC 25923 S aureus ATCC 6538 Enterococcusfaecalis ATCC 29212 Micrococcus sp (Pasteur InstituteTunis) and Gram-ve (Vibrio tapetis CECT4600 (Depart-ment of Microbiology and Parasitology University of San-tiago de Compostela Spain) V anguillarum ATCC 12964TV alginolyticus ATCC 17749T Escherichia coli O126-B16(ATTC 14948) E coli ATCC 25922 E coli ATCC 8739Pseudomonas cepacia (INSTM Tunisia) P fluorescens AH2(Danish Institute for Fisheries Research Denmark) P aerug-inosa ATCC 27853 Aeromonas salmonicida LMG3780 Ahydrophila B3 (RVAU-Denmark) Salmonella typhimuriumC52 (Laboratoire Hydrobiologie Marine et ContinentaleUniversite de Montpellier II France) and the yeast CandidaalbicansATCC10231 was used for testing antimicrobial activ-ity of seaweed extracts and fractions

26 Minimal Inhibition Concentration (MIC) The MIC wasdetermined for selective active extracts and fractions on TSBmedia according to Khan et al [12] and Ganiere et al [13]Assays were performed in sterile culture plates of 96 roundbottom wells Suspensions of indicator bacterial inoculumwere adjusted in the sterile broth medium TSB to the densityof 05 Standard McFarland (Corresponding to 0063 opticaldensity at 600 nm approximately 108 CFU mL-1) and thendiluted 10-fold twice to obtain a bacterial suspension densityof about 106 CFU mL-1 Microplates wells were inoculatedwith 180 120583L of the culture containing the inoculum 20 120583Lof each concentration of seaweed extract (diluted in dimethylsulfoxide (DMSO)) was added to the wells containing bacte-rial culture suspension The negative control contained 200120583L of culture medium only (without alga extract) Extracts(20 120583L) were adjusted to give a concentration range of 1600to 50 120583gmL (for alga crude extract) and 250 to 10 120583gmL (foralga fractions) Tests were performed in triplicate and plateswere incubated for 18-24 h at 37∘C Subsequently wells wereexamined by unaided eye for bacterial growth as indicated byturbidity [14] The last concentrations in the dilution seriesthat did not show visible growth (and showing only fewcolonies compared with other concentrations when spreadon agar plates) correspond to the MIC of the antimicrobialagent If difficulty is found to discern growth in some wellsMIC determination is then done with colony-forming unitscount

27 Fractionation Purification and Characterization Analy-sis In this study Thin Layer Chromatography (TLC) ana-lytic (TLCa) plates (Merck Fluka) were used The sol-vents system used for the fractions analysis is the n-HexEtOAcDCMMeOH with combinations and variablepercentages according to the fractions After their develop-ment chromatograms were revealed by chemical reactivesthe phosphomolybdic acid (PMA) and the liebermann For




120583mol L-1N-NO3

-

120583mol L-1N-NH4

+

120583mol L-1P-PO4

3-

120583mol L-1TP120583mol L-1

TN120583mol L-1

Chl amgm-3

Cap Zebib


Ghar El Melh








3 Results





















TLCa


64 fractions




FG9409 mg

FG1645 mg

G1 G4 G5 G6

(+)


9 fractions

---------------------

--------G27-------

NMR 1（ 13 GC






G1 G2 G3 G4 G5 G6 G7 G8 G9 G10



4 Discussion



(+)

--------

TLCp

G4(485 mg)

B4

5 fractions

(+)111 mg

(+)

------- ---

A B C D

7 fractions

G5(1847 mg)

TLCp

4 sub-fractions

(+)

(+)70 mg

(+)

6 fractions

12 fractions

G6(619 mg)


-----

FX1

(+)

(+)

(+)

FX6---------

16 mg

5 (1) 5 (7)G () 6 (1) 6 (12)G ()

NMR 1（

NMR 1（

（20

4 (1) 4 (5)G ()

（20

（20

NMR 1（

13

NMR 1（

13

NMR 1（

13









G4

G4 (1)G4 (3)

G4 (4)G4 (5)

T-

(a)

G5 G5 (7)

G5 (5)G5 (4)

G5 (6)

G5 (3)

G5 (1)G5 (2)

T-

(b)

A B

CD

T-

(c)

FX6

B4G4 (4)

T-

(d)








ppm

(a)

ppm

(b)








G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





120583mol L-1N-NO3

-

120583mol L-1N-NH4

+

120583mol L-1P-PO4

3-

120583mol L-1TP120583mol L-1

TN120583mol L-1

Chl amgm-3

Cap Zebib


Ghar El Melh








3 Results





















TLCa


64 fractions




FG9409 mg

FG1645 mg

G1 G4 G5 G6

(+)


9 fractions

---------------------

--------G27-------

NMR 1（ 13 GC






G1 G2 G3 G4 G5 G6 G7 G8 G9 G10



4 Discussion



(+)

--------

TLCp

G4(485 mg)

B4

5 fractions

(+)111 mg

(+)

------- ---

A B C D

7 fractions

G5(1847 mg)

TLCp

4 sub-fractions

(+)

(+)70 mg

(+)

6 fractions

12 fractions

G6(619 mg)


-----

FX1

(+)

(+)

(+)

FX6---------

16 mg

5 (1) 5 (7)G () 6 (1) 6 (12)G ()

NMR 1（

NMR 1（

（20

4 (1) 4 (5)G ()

（20

（20

NMR 1（

13

NMR 1（

13

NMR 1（

13









G4

G4 (1)G4 (3)

G4 (4)G4 (5)

T-

(a)

G5 G5 (7)

G5 (5)G5 (4)

G5 (6)

G5 (3)

G5 (1)G5 (2)

T-

(b)

A B

CD

T-

(c)

FX6

B4G4 (4)

T-

(d)








ppm

(a)

ppm

(b)








G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


















TLCa


64 fractions




FG9409 mg

FG1645 mg

G1 G4 G5 G6

(+)


9 fractions

---------------------

--------G27-------

NMR 1（ 13 GC






G1 G2 G3 G4 G5 G6 G7 G8 G9 G10



4 Discussion



(+)

--------

TLCp

G4(485 mg)

B4

5 fractions

(+)111 mg

(+)

------- ---

A B C D

7 fractions

G5(1847 mg)

TLCp

4 sub-fractions

(+)

(+)70 mg

(+)

6 fractions

12 fractions

G6(619 mg)


-----

FX1

(+)

(+)

(+)

FX6---------

16 mg

5 (1) 5 (7)G () 6 (1) 6 (12)G ()

NMR 1（

NMR 1（

（20

4 (1) 4 (5)G ()

（20

（20

NMR 1（

13

NMR 1（

13

NMR 1（

13









G4

G4 (1)G4 (3)

G4 (4)G4 (5)

T-

(a)

G5 G5 (7)

G5 (5)G5 (4)

G5 (6)

G5 (3)

G5 (1)G5 (2)

T-

(b)

A B

CD

T-

(c)

FX6

B4G4 (4)

T-

(d)








ppm

(a)

ppm

(b)








G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






TLCa


64 fractions




FG9409 mg

FG1645 mg

G1 G4 G5 G6

(+)


9 fractions

---------------------

--------G27-------

NMR 1（ 13 GC






G1 G2 G3 G4 G5 G6 G7 G8 G9 G10



4 Discussion



(+)

--------

TLCp

G4(485 mg)

B4

5 fractions

(+)111 mg

(+)

------- ---

A B C D

7 fractions

G5(1847 mg)

TLCp

4 sub-fractions

(+)

(+)70 mg

(+)

6 fractions

12 fractions

G6(619 mg)


-----

FX1

(+)

(+)

(+)

FX6---------

16 mg

5 (1) 5 (7)G () 6 (1) 6 (12)G ()

NMR 1（

NMR 1（

（20

4 (1) 4 (5)G ()

（20

（20

NMR 1（

13

NMR 1（

13

NMR 1（

13









G4

G4 (1)G4 (3)

G4 (4)G4 (5)

T-

(a)

G5 G5 (7)

G5 (5)G5 (4)

G5 (6)

G5 (3)

G5 (1)G5 (2)

T-

(b)

A B

CD

T-

(c)

FX6

B4G4 (4)

T-

(d)








ppm

(a)

ppm

(b)








G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



(+)

--------

TLCp

G4(485 mg)

B4

5 fractions

(+)111 mg

(+)

------- ---

A B C D

7 fractions

G5(1847 mg)

TLCp

4 sub-fractions

(+)

(+)70 mg

(+)

6 fractions

12 fractions

G6(619 mg)


-----

FX1

(+)

(+)

(+)

FX6---------

16 mg

5 (1) 5 (7)G () 6 (1) 6 (12)G ()

NMR 1（

NMR 1（

（20

4 (1) 4 (5)G ()

（20

（20

NMR 1（

13

NMR 1（

13

NMR 1（

13









G4

G4 (1)G4 (3)

G4 (4)G4 (5)

T-

(a)

G5 G5 (7)

G5 (5)G5 (4)

G5 (6)

G5 (3)

G5 (1)G5 (2)

T-

(b)

A B

CD

T-

(c)

FX6

B4G4 (4)

T-

(d)








ppm

(a)

ppm

(b)








G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



G4

G4 (1)G4 (3)

G4 (4)G4 (5)

T-

(a)

G5 G5 (7)

G5 (5)G5 (4)

G5 (6)

G5 (3)

G5 (1)G5 (2)

T-

(b)

A B

CD

T-

(c)

FX6

B4G4 (4)

T-

(d)








ppm

(a)

ppm

(b)








G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



ppm

(a)

ppm

(b)








G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



G4G5

G6

G7G8

G9

G11

S aureus

E faecalis

A salmonicida

FA

FASMUFA

PUFA



minus2

minus1

0

1

2

3

F2 (1

676

)













5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











5 Conclusions



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Data Availability




Acknowledgments


References































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018
