Antioxidant potential and antimicrobial efficacy of seaweed(Himanthalia elongata ) extract in model food systems

Sabrina Cox & Grace Hamilton Turley &

Gaurav Rajauria & Nissreen Abu-Ghannam &

Amit Kumar Jaiswal

Received: 26 August 2013 /Revised and accepted: 20 November 2013# Springer Science+Business Media Dordrecht 2013

Abstract Extracts from marine sources exhibit antimicrobialand antioxidant properties in vitro and there has been greatinterest within the food industry to move towards naturalmethods of food preservation. Natural extracts from seaweedscould potentially have a multiple functionality within the foodindustry to increase safety and enhance the quality of foodproducts. The present study is aimed to assess the antimicrobialactivity of a hydrophilic extract from the fucoid brown algaHimanthalia elongata in model food systems. Carbohydrateand protein model food systems (CMFS and PMFS, respec-tively) were studied at varying concentrations (1 %, 5 % and10 %) and bacterial inhibition of the extract was investigatedagainst Salmonella abony and Listeria monocytogenes . Theextract provided up to 100 % inhibition of the bacteria with abactericidal effect in CMFS, while a bacteriostatic effect wasseen in PMFS. In general, there was a significant difference (P<0.05) between the efficacies of the extract against S. abony ascompared to L. monocytogenes with higher inhibition for S.abony. In terms of antioxidants; the extract had a total phenoliccontent of 34.0 mg GAE g−1 of extract and a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of139.8 mg AAE g of extract. The results of the present study

are promising as it provides an insight for the inclusion ofseaweed extracts into real food systems.

Keywords Seaweeds . Antimicrobials . Foodmodelsystems . Antioxidants .Himanthalia elongata . Phaeophyta

Introduction

The problem of oxidation and microbial contamination aremost common aspects of food preservation, especially whenthe products develop undesirable flavours, unpleasant taste,rancid odours, discoloration and other forms of spoilage re-sponsible for the loss of quality, safety and shortening of shelflife. Synthetic antimicrobial agents such as sodium benzoate,sodium nitrite and sorbic acid; synthetic antioxidants such asbutylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT) and tert-butyl-hydroxyquinone (TBHQ) are widelyused in the food industry for preserving the safety and qualityof foods. However, these synthetic antioxidants have beenreported to be toxic and exerting a carcinogenic effect (Chenet al. 1992). In recent times, there has been an increasingtendency towards the use of natural substances instead of thesynthetic ones. With the increase in the price of raw materials,the problem of cost benefits for chemical production is becom-ing more considerable.

Many secondary metabolites found in plants have a role indefence against herbivores, pests and pathogens. The defen-sive forces of these secondary metabolites, are in the form ofantimicrobial agents such as alkaloids, flavanols and phenoliccompounds (Lopez-Malo et al. 2002). Many marine plants,including seaweeds, often carry significantly less macro andmicroepibionts on their thalli compared to co-occurringbiofilms on inanimate substrata (Hellio et al. 2001; Lam andHarder 2007). Therefore, it has been assumed that seaweeds

S. Cox :G. Hamilton Turley :G. Rajauria :N. Abu-Ghannam (*) :A. K. JaiswalSchool of Food Science and Environmental Health, College ofSciences and Health, Dublin Institute of Technology, Cathal BrughaStreet, Dublin 1, Dublin, Irelande-mail: nissreen.abughannam@dit.ie

S. Coxe-mail: sabrina.cox@dit.ie

G. Hamilton Turleye-mail: gaurav.rajauria@dit.ie

G. Rajauriae-mail: amit.jaiswal@dit.ie

J Appl PhycolDOI 10.1007/s10811-013-0215-0

defend themselves against bacterial fouling by production ofsecondary metabolites which prevent attachment and growthof bacterial colonizers (Maximilien et al. 1998). Similarly,antioxidant activity of marine algae may arise from pigmentssuch as chlorophylls and carotenoids, vitamins and vitaminprecursors, phenolics and other antioxidative substances,which directly or indirectly contribute to the inhibition orsuppression of oxidation processes (Shahidi 2009). The envi-ronment in which seaweeds grow is harsh as they are exposedto a combination of light and high oxygen concentrations.These factors can lead to the formation of free radicals andother strong oxidizing agents, but seaweeds seldom sufferfrom any serious photodynamic damage during metabolism.This fact implies that their cells have some protective antiox-idative mechanisms and compounds (Matsukawa et al. 1997).Research findings suggest that seaweeds are also rich inantioxidants metabolites, which may be used to improve foodquality by increasing shelf life of food products (Bergmanet al. 2003; Sabeena Farvin and Jacobsen 2013; O’Sullivanet al. 2013).

Over the last two decades, great deal of efforts have beendirected toward identifying low-cost natural products fromplant, animal and microbial origins. In particular, 'natural' prod-ucts such as polyphenols have been reported to have a variety ofbiological effects, including antioxidant, anticarcinogenic, anti-inflammatory and antimicrobial activities (Xia et al. 2010).Phenolic extracts from different plant origins, such as rosemary,cocoa, olive oil (Bubonja-Sonje et al. 2011), onion, garlic(Benkeblia 2004), essential oils (Gutierrez et al. 2009), mango(Kaur et al. 2010), almond skins (Mandalari et al. 2010) andseaweeds (Bansemir et al. 2006; Dubber and Harder 2008; Coxet al. 2010) have demonstrated their antimicrobial activityagainst numerous spoilage and pathogenic bacteria. Other stud-ies carried out within food products such as fresh and cookedpork mince (Moroney et al. 2013), chicken products (Kanattet al. 2010), and cod protein (Wang et al. 2010) have demon-strated the potential application of plant extracts as antimicro-bial and antioxidant agents in order to prolong the shelf life ofthe final products.

However, to our knowledge, reports on the antimicro-bial activity of seaweed extracts within model food sys-tems are limited. Therefore, the aim of the present studywas to investigate the antimicrobial efficacy of an Irishseaweed extract in protein and carbohydrate model foodsystems (CMFS) and also to evaluate its antioxidantpotential.

Materials and methods

Himanthalia elongata was purchased from Quality Sea Veg.(Co Donegal, Ireland). The seaweeds were collected in Octo-ber 2012 and stored at −18 °C until further use.

Sequential extraction and purification

H. elongata samples (5 g) were crushed with liquid nitrogenusing a mortar and pestle, and extracted sequentially withsolvents having different polarity. In the first phase of extrac-tion, the powdered samples were mixed with equal-volumemedium to low polarity solvents mixture (50 mL) of chloro-form, diethyl ether and n-hexane. The samples were flushedwith liquid nitrogen andwere kept at 100 rpm, 40 °C in a shakerincubator. After 1.5 h, the supernatant was collected and theresidue of the seaweed samples was extracted again with highpolarity solvents mixture of 60% (v/v) aqueousmethanol underthe same extraction conditions. After the extraction, both theliquid extracts obtained from medium to low polarity and highpolarity solvents were pooled and was filtered together usingWhatman #1 filter paper.

The crude extract obtained from sequential extraction waspartially purified with liquid–liquid partition approach. Abiphasic solvent system of water and ethyl acetate (1:1, v/v)was then prepared and left for 30 min before being utilized inthe purification of the supernatants. The equal amount ofbiphasic solvent and the supernatant were mixed and stirredfor 1.5 h. The mixture was transferred in separatory funnelsand left overnight in order to get a clear separation of com-pounds between the layers. The separatory funnel were cov-ered with tin foil and kept in the dark to avoid the degradationof photosensitive compounds in the extract. The separatedaqueous and ethyl acetate layers were then collected afterrepeated washing with water and filtered separately. The sol-vents were evaporated to dryness using a multi-evaporator(Büchi Syncore Polyvap, Mason Technology, Ireland) at37 °C with a pressure ranging from 470 to 150 mbar and thedried extracts obtained from aqueous and ethyl acetate frac-tions were dissolved in water and methanol, respectively. Boththe extracts were submitted for the estimation of total phenoliccontent (TPC), antioxidant potential and extraction yield andbased on the results, only dried aqueous fraction was testedagainst different food model systems.

Preparation of protein food systems

Bovine serum albumin (BSA; Sigma-Aldrich, Ireland) solu-tions were prepared at 1 %, 5 % and 10 % w/v concentrationsby dissolving suitable quantities of BSA in sterile phosphatebuffered saline (PBS) according to Simpson and Gilmour(1997). The solutions were then filter-sterilised using 0.2-μmNalgene syringe filters.

Preparation of carbohydrate food systems

Carbohydrate solutions were prepared at 1 %, 5 % and 10 %(w/v) concentrations by dissolving glucose in sterile PBSstock solution according to Simpson and Gilmour (1997).

J Appl Phycol

The solutions were then filter-sterilised using 0.2-μmNalgenesyringe filters.

Antimicrobial activity

Microbial culture

Two species of common food pathogenic bacteria were used inthis study; Listeria monocytogenes ATCC 19115 and Salmo-nella abony NCTC 6017 (Medical Supply Company, Dublin,Ireland). All cultures were maintained at −70 °C in 20 %glycerol stocks and grown in Tryptic Soy Broth (TSB) at37 °C overnight to obtain sub-cultures. Working cultures wereprepared from sub-cultures and grown at optimal conditions foreach bacterium for 18 h before analysis. Bacterial suspensionswere then prepared in saline solution (NaCl 0.85 %;BioMérieux, France) equivalent to a McFarland standard of0.5, using the Densimat photometer (BioMérieux Inc, France)to obtain a concentration of 1×108 colony forming units (CFU)mL1. This suspension was then diluted in TSB to obtain aworking concentration of 1×106 CFU mL−1.

Antimicrobial activity assay

This assay was carried out according to the procedure of Coxet al. (2010), with some modifications. The influence of varyingconcentrations (8, 4, 2, 1, 0.5, 0.25 and 0.125 mg mL−1) ofhydrophilic extract on efficacy in food model systems wereassessed against L. monocytogenes and S. abony using 96-wellmicrotitre plates. The dried seaweed extract was dissolved inpeptone water and an aliquot of 200 μL of extract solutions wereadded to the first horizontal row of each plate in quadruplicate.All other wells were filled with 100 μL of model food system.Then, 100 μL from the first horizontal row was serial diluted 2-fold along each section. Bacterial suspension (100 μL) wasadded to the first, second and third vertical rows of each section.The fourth vertical row was used for sample blanks, to whichpeptone water was added (100 μL), while the negative controlsand control blanks resided in the eighth horizontal row of eachsection. Absorbance readings were taken at 0 and 24 h at 600 nmusing a microplate spectrophotometer (Powerwave, Biotek),with 20 s of agitation before each optical density (OD) reading.Sodium benzoate was used as a positive control. Percentageinhibition was calculated according to the following equation:

Bacterial inhibition %ð Þ ¼ O−EO

� �� �� 100;

where O is (OD of the organism at 24 h−OD of the organismat 0 h) and E is (OD of the extract at 24 h−blank at 24 h)−(OD of the eExtract at 0 h−blank at 0 h).

Enumeration of bacteria

Aliquots of sample (100 μL) from wells of the microtitreplates were selectively spread plated in duplicate. Plate countagar (PCA) was used as a growth medium and spread platingwas carried out under aseptic conditions. After incubation for24 h at 37 °C, the plates were observed for the presence orabsence of bacterial growth.

Total phenolic content

The total phenolic concentration was measured using theFolin–Ciocalteau method as outlined by Cox et al. (2012).The TPCs were expressed as mg gallic acid equivalent pergram of extract (mg GAE g−1).

DPPH radical scavenging activity

Free radical scavenging activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the methoddescribed by Jaiswal et al. (2012). The antioxidant potentialwas expressed as mg ascorbic acid equivalent per gram ofextract (mg AAE g−1).

Statistical analysis

All experiments were performed in triplicate and replicatedtwice. All statistical analyses were carried out using STATGRAPHICS Centurion XV software (StatPoint Technologies,Inc., USA). Statistical differences were determined usingANOVA followed by least significant difference (LSD) test-ing. Differences were considered statistically significant whenP< 0.05.

Results

Initial studies were carried out to extract both hydro-philic and lipophilic compounds from the H. elongata.However, preliminary results showed a very low yieldof lipophilic fraction (0.03 %) so based on these find-ings further studies were carried out with hydrophilicfraction.

Extract concentration and efficacy

Efficacy of seaweed extracts in carbohydrate model foodsystems

The results of efficacy of hydrophilic seaweed extracts atconcentrations of 8, 4, 2, 1, 0.5, 0.25 and 0.125 mg mL−1,which were tested against S. abony and L. monocytogenes inCMFS (1 %, 5 % and 10 %) are shown in Tables 1 and 2. The

J Appl Phycol

results highlight that higher hydrophilic extract concentrationswere positively correlated with higher percentage inhibitionsof the food-borne pathogens tested. Between 8 and 2mgmL−1

extract concentration, bacterial inhibition against S. abonywas detected in the range of 100 to 48.4 % (10 % CMFS),however at concentrations less than 1 mg mL−1, no bacterialinhibition was observed. Percentage inhibition results againstL. monocytogenes were similar with no bacterial inhibitiondetected below 1 mg mL−1. There was no significant differ-ence between 1 and 5 % CMFS inoculated with S. abony at8 mg mL−1 hydrophilic seaweed extract, or between 5 and10 % CMFS (P >0.05). However, there was a significantdifference (P< 0.05) between 1 and 10 % CMFS for agiven concentration of extract. Concentration dependentantibacterial activity was detected as the extract concen-trations decreased and bacterial inhibition also declined.When the efficacy of the extract against S. abony and L.monocytogenes were compared; a significant differencewas found between the two food-borne pathogens. Inorder to determine if the extract exerted bacteriostatic orbactericidal effect spread plating was carried out. Theextract had a potential bactericidal effect in CMFS withbacterial cell death being most probable as there were fewcolonies or no growth observed on the plates.

Efficacy of seaweed extracts in protein model food Systems(PMFS)

Similarly, as in the case of the extract incorporated intoCMFS, the greater the concentration of extract, thehigher the bacterial inhibition that was observed forthe PMFS. However, while no inhibition was detectedbelow 1 mg mL−1 concentration in CMFS, bacterialinhibition was exhibited at all extract concentrations inPMFS (8, 4, 2, 1, 0.5, 0.25 and 0.125 mg mL−1).Bacterial inhibition was high up to 100 % in 1 and5 % PMFS against both pathogens (Tables 3 and 4). Aswith CMFS, spread plating was carried out to assess thepotential mode of action of the extract in PMFS. Thequalitative spread plating method confirmed that theextract worked in a bacteriostatic manner in PMFS, asit exhibited an inhibitory effect preventing furthergrowth of the bacteria. With regard to both S. abonyand L. monocytogenes , all of the plates exhibited alawn of growth. It was evident, however, that8 mg mL−1 extract had a stronger bacteriostatic effectagainst L. monocytogenes compared to 2 mg mL−1 asthe lawn of bacterial growth appeared thinner, in partic-ular when compared to control plates.

Table 1 Percentage inhibition (%) of seaweed extract against S. abony in carbohydrate model food system (CMFS) at varying concentrations

Extract concentration (mg mL−1)

CMFS (%) 8 4 2 1 0.5 0.25 0.125

1 94.9±1.4ay 90.2±0.5ay 74.6±0.9az – – – –

5 96.5±4.4ax 78.3±3.3by 11.5±5.1bz – – – –

10 100±0.0bx 92.2±1.1ay 48.4±1.1cz – – – –

Each value is presented as mean±SD (n=6)

Means within each column with different letters (a–c) differ significantly (P <0.05)

Means within each row with different letters (x–z) differ significantly (P<0.05)

Table 2 Percentage inhibition (%) of seaweed extract against L. monocytogenes in carbohydrate model food system (CMFS) at varying concentrations

Extract concentration (mg mL−1)

CMFS (%) 8 4 2 1 0.5 0.25 0.125

1 78.6±3.4ax 68.9±1.2ay 67.3±0.7ay 14.2±0.1az – – –

5 81.9±2.6ax 58.0±2.8by 23.9±8.9bz – – – –

10 90.8±1.6bx 71.6±1.9 cy 50.9±7.7cz – – – –

Each value is presented as mean±SD (n=6)

Means within each column with different letters (a–c) differ significantly (P <0.05)

Means within each row with different letters (x–z) differ significantly (P<0.05)

J Appl Phycol

Comparison of efficacy of extract against sodium benzoate

Sodium benzoate, a bacteriostatic preservative used in thefood industry, was used as a positive control in 5 % CMFSand 5 % PMFS (Table 5). Spread plating confirmed thatsodium benzoate was bacteriostatic due to lawns of bacterialgrowth after incubation, which is due to the bacteria growthbeing inhibited rather than killing. The results were similar tothose produced by the extract incorporated into PMFS. Sodi-um benzoate also had a bacteriostatic effect in CMFS whilethe extract had a bactericidal effect against the two pathogens.At 8 and 4 mg mL−1, the extract exhibited greater bacterialinhibition and no L. monocytogenes inhibition was noted bysodium benzoate at 4 mg mL−1 or less. Sodium benzoate,however, exhibited greater inhibition at 2–0.5 mg mL−1 com-pared to the extract (Table 5). In contrast, the extract andsodium benzoate had a bacteriostatic effect against the patho-gens in PMFS; however, the extract had a much strongerbacteriostatic effect compared to the artificial preservative.

At the conditions used in this study, the hydrophilic extractexhibited a greater effect in CMFS and PMFS against both ofthe studied bacteria as compared to sodium benzoate and therewas a significant difference (P< 0.05) between them. It wasalso found that sodium benzoate had a stronger efficacyagainst S. abony as compared to L. monocytogenes. At

8 mg mL−1 extract concentration (5 % PMFS), bacterialinhibition of 100 % was displayed against S. abony and L.monocytogenes (Tables 3 and 4), while sodium benzoate onlyinhibited the pathogens by 67 and 30 %, respectively(Table 5).

Total phenolic content and antioxidant potential of extract

The results from Table 6 indicate that the values of TPC,antioxidant potential and extraction yield (%) was significant-ly higher (P <0.05) in the hydrophilic fraction as compared tothe lipophilic fraction of seaweed extract. The extraction yield(%) of the hydrophilic fraction was 1.92 % while the TPC ofthe same fraction was 34.0 mg GAE g−1 of extract. The DPPHradical scavenging activity of the same fraction was 139.8 mgAAE g−1 of extract.

Discussion

Recent studies have shown that the crude seaweed extractfrom H. elongata possessed excellent antimicrobial and anti-oxidant properties (Rajauria et al. 2013; Cox et al. 2010). Thepresent study used a sequential extraction and purificationmethod in order to obtain hydrophilic and lipophilic extracts.

Table 3 Percentage inhibition (%) of seaweed extract against S. abony in protein model food system (PMFS) at varying concentrations

Extract concentration (mg mL−1)

PMFS (%) 8 4 2 1 0.5 0.25 0.125

1 100.0±0.0av 100.0±0.0ay 100.0±0.0av 85.5±5.7aw 67.0±1.8ax 59.9±1.5ay 44.2±1.4az

5 100.0±0.0aw 100.0±0.0by 100.0±0.0aw 70.9±0.7bx 62.3±3.8by 44.6±3.6bz 44.1±0.9az

10 77.6±0.2bv 76.1±3.5cy 67.3±6.8bw 28.6±3.5cx 25.5±3.5cx 6.7±4.0cy 0.0±0.0bz

Each value is presented as mean±SD (n=6)

Means within each column with different letters (a–c) differ significantly (P <0.05)

Means within each row with different letters (v–z) differ significantly (P<0.05)

Table 4 Percentage inhibition (%) of seaweed extract against L. monocytogenes in protein model food system (PMFS) at varying concentrations

Extract concentration (mg mL−1)

PMFS (%) 8 4 2 1 0.5 0.25 0.125

1 100.0±0.0av 83.7±7.8aw 81.8±3.2aw 67.8±3.2ax 44.4±3.5ay 40.2±1.8az 39.1±1.5az

5 100.0±0.0av 100.0±0.0bv 100.0±0.0bv 80.1±5.1bw 44.6±0.8ax 30.9±4.7by 25.9±2.1bz

10 98.5±1.3av 62.6±9.9cw 61.9±9.4cw 58.6±1.9cx 56.3±5.3bx 39.9±3.3cy 32.0±4.0cz

Each value is presented as mean±SD (n=6)

Means within each column with different letters (a–c) differ significantly (P <0.05)

Means within each row with different letters (v–z) differ significantly (P<0.05)

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The results show that the values of TPC and antioxidantpotential of hydrophilic fractions were 2.3 and 5.8 timeshigher than the lipophilic fraction, respectively. Also, the sameextract showed a higher yield; therefore, based on these find-ings, the hydrophilic extract was focussed on as an antimicro-bial for the present study.

As expected, higher extract concentrations resulted inhigher percentage inhibitions of bacteria. There was a greaterantimicrobial efficacy of the extract at 8 and 4 mg mL−1

concentrations when applied to a 10 % CMFS compared to1 %, meaning the extract can perform better with increasingconcentrations of glucose which may be related to a synergis-tic effect. Glucose causes a reduction in water activity (aw)which in turn restricts microbial growth; therefore the CMFSin combination with extract would have a potential synergisticeffect.

With regard to extract concentrations of 8, 4 and2 mg mL−1, the microtitre assay showed no significant differ-ence (P >0.05) between the inhibition of S. abony and L.monocytogenes in 5 % PMFS, as 100 % inhibition wasexhibited. Therefore, if the extract was applied it would bemore economical to use the lower concentration of2 mgmL−1, as it produces the same effect as the higher extractconcentrations. In general, it was found that the extract had a

greater antibacterial effect against S. abony compared to L.monocytogenes .

Proteins are more structurally diverse than carbohydrates.The PMFS, made up of BSA at varying concentrations, i.e.,1 %, 5 % and 10 %, may have had a protective effect over thepathogens. It is probable, based on the evidence obtained fromthe microtitre assay and subsequent spread plating, that theextract was able to penetrate the bacterial cells enough tocause a bacteriostatic effect, i.e., inhibition of any furthergrowth. BSA is made up of three homologous domains whichare split into nine groups by 17 disulfide bonds and it alsocontains tryptophan residues (Bourassa et al. 2011). Despiteits complexity compared to carbohydrates, Bourassa et al.(2011) indicated that BSA can act as a transporter for com-pounds such as drugs.

In reported studies investigating the antimicrobial potencyof plant extracts, model food systems tend to decrease extractefficacy (Cerrutti and Alzamora 1996; Del Campo et al.2000). However, comparing results to the control (TSB inplace of model system) in the present study, results showedthat CMFS enhanced the overall antimicrobial potential.Comparing the two bacteria tested, there was a greater per-centage bacterial inhibition against S. abony (Gram-negative)as compared with L. monocytogenes (Gram-positive). This

Table 5 Percentage inhibition (%) of sodium benzoate against L. monocytogenes and S. abony in 5 % protein model food system (PMFS) andcarbohydrate model food system (CMFS)

Sodium benzoate concentration (mg mL−1)

8 4 2 1 0.5

5 % CMFS

L. monocytogenes 16.0±0.5a – – – –

S. abony 62.2±2.8bv 52.1±2.3aw 47.5±1.3ax 34.9±3.1ay 26.9±4.5az

5 % PMFS

L. monocytogenes 30.2±4.1c – – – –

S. abony 67.3±2.0dv 17.6±1.6bw 15.4±4.1bx – –

Each value is presented as mean±SD (n=6)

Means within each column with different letters (a–c) differ significantly (P <0.05)

Means within each row with different letters (v–z) differ significantly (P<0.05)

Table 6 Total phenolic content, antioxidant potential and extraction yield of hydrophilic and lipophilic fraction of H. elongata seaweed extract

Extract Extraction yield (%) TPC (mg GAE g−1) Antioxidant potential (mg AAE g−1)

Aqueous fraction 1.92±0.07a 34.0±1.1a 139.8±2.7a

Ethyl acetate fraction 0.03±0.01b 14.7±0.2b 24.1±0.7b

Each value is presented as mean±SD (n=6)

Means within each column with different letters (a–b) differ significantly (P<0.05)

Extraction yield (%) is calculated in terms of g of dry extracts/100 g of fresh weight. TPC and antioxidant potential are expressed as mg gallic acidequivalents/g (dw) and mg ascorbic acid equivalents/g (dw), respectively

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finding is of interest because the literature generally indicatesthat Gram-positive bacterial species are more susceptible toantimicrobial agents than Gram-negative (Rang et al. 2012).According to Rang et al. (2012) and Russell and Chopra(1996), the outer membrane of Gram-negative microbes pos-sesses porins, which are essentially pores in the membranecomposed of proteins through which hydrophilic substancescan move freely. The brown seaweeds are known to containphlorotannins such as eckol, dieckol, and phloroglucinolwhich are hydrophilic in nature. These compounds have beenreported to show a strong antibacterial activity against numberof microorganisms (Eoma et al. 2012). It is anticipated that thepartial purified aqueous extract could have enriched withphlorotannins and showed strong antimicrobial activityagainst tested bacteria. Furthermore, the peptidoglycan layerin Gram-negative species is much thinner compared to Gram-positive species (Golan et al. 2008). Therefore, within a 24 hincubation period at 37 °C, it is possible that the extracttravelled faster across the Gram-negative outer membraneand cell wall of S. abony more easily than the thick peptido-glycan layer of L. monocytogenes .

It is known that food preservatives can either act in abacteriostatic or a bactericidal manner. In order to determinewhich effect the extract adopted in carbohydrate and proteinconditions, a qualitative spread plating study was carried out.With regard to extract incorporated into CMFS, a potentialbactericidal effect was observedwith bacterial cell death beingmost probable as there were few colonies or no growth ob-served on the plates. The current findings are also in line withAhn et al. (2004), who reported that phlorotannins frombrown seaweed showed a strong bactericidal effect againstfood-borne bacteria.

Proteins are more structurally diverse than carbohydrates.The PMFS, made up of BSA at varying concentrations, i.e.,1 %, 5 % and 10 %, may have had a protective effect over thepathogens. It is probable, based on the evidence obtained fromthe microtitre assay and subsequent spread plating, that theextract was able to penetrate the bacterial cells enough tocause a bacteriostatic effect, i.e., inhibition of any furthergrowth. BSA is made up of three homologous domains whichare split into nine groups by 17 disulfide bonds and it alsocontains tryptophan residues (Bourassa et al. 2011). Despiteits complexity compared to carbohydrates, Bourassa et al.(2011) indicated that BSA can act as a transporter for com-pounds such as drugs.

There have beenmany studies carried out on the efficacy ofantimicrobials of plants in model food systems. However,there have not been previous studies published in literaturein relation to marine extracts incorporated into model foodsystems to challenge their antimicrobial efficacy. Gutierrezet al. (2009) reported antimicrobial activity of essential oils(EO) of oregano, thyme and lemon balm against L.monocytogenes in lettuce leaf and beef model media. They

have reported that higher antimicrobial activity in the lettuceleaf model compared to the beef model is due to the fewernutrients present in the lettuce media.

In a previous study, Moroney et al. (2013) examined theeffects which a spray-dried extract from brown seaweed(Laminaria digitata) would have on the safety and qualityaspects of fresh and cooked minced pork. The results showedno antimicrobial effect by the extract; however, the highestconcentration of extract (0.5 % w/w) exhibited antioxidantpotential against lipid oxidation to a slightly greater extent incooked patties compared to fresh and were significantly dif-ferent (P< 0.05) to the control. However, lack of antimicrobialactivity could be due to the lower level of polyphenoliccompound present in L. digitata. Furthermore, the main com-ponents responsible for the antibacterial effects may be differ-ent in L. digitata as compared to H. elongata . Such extractsmay have a potential use as antioxidant agents in productssuch as salad dressings which potentially enhance the shelflife of the product.

Food spoilage due to the presence of bacteria causes eco-nomic losses on a global scale. The results of the present studyshow that the seaweed extract could have potential as a sourcefor new antimicrobial agents equal to that of commerciallyapplied synthetic antibacterial agents. This would have poten-tial commercial interest as Salmonella and Listeria infectionshave a large impact on human health. The ubiquitous presenceof these microorganisms in the environment can lead to theiroccurrence in food-processing environments and thus in thefood chain. As antioxidants are known to enhance the safetyof foods by preventing lipid oxidation, the total phenoliccontent (TPC) and antioxidant potential of the extract wasevaluated. The seaweed extract had a TPC of 34.0 mg GAEg−1 of extract. These results are in line with Chandini et al.(2008), who reported that brown seaweed extracts had aphenolic content of 24.61 and 49.16 mg GAE g−1 seaweedextract. Wang et al. (2009) reported the TPC in differentIcelandic seaweeds to be ranging from 4 to 242 mgphloroglucinol equivalants (PGE) g−1 extract. The DPPH rad-ical scavenging activity of the extract was 139.8 mg AAE g−1

extract. In terms of percentage inhibition, at a concentration of250 μg mL−1 extract, the DPPH free radical was scavenged by47.7 %. At 500 μg mL−1 extract, the DPPH free radical wasscavenged by 64.2%which is significantly different (P< 0.05).The results of the present study are promising as algal polyphe-nolic compounds are effective antioxidants in delaying oilrancidity, therefore the seaweed extracts could have potentialin food applications (Yan et al. 1996). A recent study in Icelandby Wang et al. (2010) incorporated extract from a brownseaweed (Fucus vesiculosus) into washed cod muscle systemsand cod protein isolates in order to evaluate its efficacy againstlipid oxidation of cod during ice storage. The results concludedthat the seaweed extract could potentially inhibit the lipidperoxidation when used at higher amount compared to the

J Appl Phycol

synthetic preservatives. In the current study, the hydrophilicextract of tested seaweed showed strong antioxidant and anti-microbial efficacy against different model food systems. Thus,the study provides promising findings with potential to utilisethe extract in food or drink products, as antioxidants to enhancefood quality, and also as antimicrobial agents, increasing thesafety and shelf life of the products.

In conclusion, higher extract concentrations were positive-ly correlated with the percentage inhibition of the food-bornepathogens with up to 100 % inhibition of bacteria detected. Asignificant difference was noted between the inhibitions of thepathogens, as it was found overall that the antimicrobialproperties of the extract was more effective on S. abony. Incertain circumstances, there was no significant difference inthe efficacy of the extract at concentrations between 8 and2 mg mL−1. It can be concluded that hydrophilic seaweedextract could potentially have a multi-purpose functionalitywithin foodstuffs to increase safety and enhance quality at lowconcentrations.

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