effect of processing conditions on phytochemical constituents of edible irish seaweed himanthalia...

EFFECT OF PROCESSING CONDITIONS ON PHYTOCHEMICALCONSTITUENTS OF EDIBLE IRISH SEAWEEDHIMANTHALIA ELONGATAjfpp_563 1..16

SABRINA COX, NISSREEN ABU-GHANNAM1 and SHILPI GUPTA

School of Food Science and Environmental Health, Dublin Institute of Technology, Cathal Brugha St., Dublin 1, Ireland

1Corresponding author. TEL: +35314027570;FAX: +35314023399; EMAIL:[email protected]

Accepted for Publication April 25, 2011

doi:10.1111/j.1745-4549.2011.00563.x

ABSTRACT

Seaweed is well recognized as an excellent source of phytochemicals. This study was apreliminary screening to investigate the effects of various food processing methodson the phytochemicals of Himanthalia elongata. Hydrothermal processing wascarried out until an edible texture was achieved. The total phenolic content (TPC) offresh H. elongata was 175.27 mg gallic acid equivalent (GAE)/100 g fresh weight(FW) while boiling significantly reduced the TPC to 25.4 mg GAE/100 g FW(P < 0.05). A drying pretreatment before boiling reduced the cooking time thereforeleading to less leaching of antioxidants upon boiling. In terms of extract, drying ofH. elongata followed by boiling had the most significant effect on the phytochemi-cals as TPC increased by 174%. Boiled extracts had the most effective 2, 2-diphenyl-1-picrylhydrazyl scavenging activity (EC50 of 12.5 mg/mL). As a comparison,seaweed subjected to the same treatments was studied in terms of antimicrobialactivity. Overall, extracts from fresh H. elongata achieved the highest inhibition.

PRACTICAL APPLICATIONS

Seaweeds contain various phytochemicals with potential health benefits and hencehave potential as functional ingredients. Processing of vegetables for consumptionexposes the phytochemicals present to detrimental factors that may lead to alter-ations in concentrations and health-related quality. Because seaweeds are consumedafter some form of processing, it is important to investigate the effect of such treat-ments on the bioactive compounds that are present. The effect of common process-ing conditions on the phytochemical constituents of an edible Irish seaweed,Himanthalia elongata, were investigated in terms of whole seaweed and as an extract.The nutritional quality of the samples varied with the different processing treat-ments. A drying pretreatment before boiling reduced the cooking time thereforeleading to less leaching of antioxidants upon boiling. These findings are promising assuch processing conditions could be applied in the development of new functionalfoods from whole H. elongata or its extracts.

INTRODUCTION

In recent years, many marine resources have attracted atten-tion in the search for bioactive compounds to develop newdrugs and health foods (Kuda et al. 2005). Seaweeds are aknown source of bioactive compounds as they are able toproduce a great variety of secondary metabolites character-ized by a broad spectrum of biological activities (Bansemir

et al. 2006). Compounds with antiviral, antifungal, antimi-crobial and antioxidant activities have been detected in green,brown and red algae (Vairappan et al. 2001; Bansemir et al.2006; Duan et al. 2006; Chandini et al. 2008; Cox et al. 2010).Antioxidant activity of marine algae may arise from pigmentssuch as chlorophylls and carotenoids, vitamins and vitaminprecursors including a-tocopherol, b-carotene, niacin, thia-mine and ascorbic acid, phenolics such as polyphenolics and

Journal of Food Processing and Preservation ISSN 1745-4549

1Journal of Food Processing and Preservation •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.

hydroquinones and flavonoids, phospholipids particularlyphosphatidylcholine, terpenoids, peptides, and other antioxi-dative substances, which directly or indirectly contribute tothe inhibition or suppression of oxidation processes (Shahidi2009). The environment in which seaweeds grow is harsh asthey are exposed to a combination of light and high oxygenconcentrations. These factors can lead to the formation of freeradicals and other strong oxidizing agents, but seaweedsseldom suffer from any serious photodynamic damageduring metabolism. This fact implies that their cells havesome protective antioxidative mechanisms and compounds(Matsukawa et al. 1997).

Reactive oxygen species (ROS) such as hydroxyl, superox-ide and peroxyl radicals are formed in human cells by endog-enous factors and result in extensive oxidative damage, whichcan lead to age-related degenerative conditions, cancer and awide range of other human diseases (Reaven and Witzum1996; Aruoma 1999). Phenolic compounds can act as antioxi-dants by chelating metal ions, preventing radical formationand improving the antioxidant endogenous system (Al-Azzawie and Mohamed-Saiel 2006). These phenolic com-pounds are commonly found in plants, including seaweeds(Duan et al. 2006). Polyphenols represent a diverse class ofcompounds including flavonoids (i.e., flavones, flavonols, fla-vanones, flavononols, chalcones and flavan-3-ols), lignins,tocopherols, tannins and phenolic acids (Shukla et al. 1997).

Interest in new sources of natural antioxidants hasincreased in recent years in order to reduce the use of syn-thetic forms such as butylated hydroxyanisole and butylatedhydroxytoluene. Natural antioxidants from plant origin canreact rapidly with these free radicals and retard or alleviate theextent of oxidative deterioration (Akoh and Min 1997). Fur-thermore, antioxidants from natural sources can also increasethe shelf life of foods. Phenolic phytochemicals inhibitautoxidation of unsaturated lipids, thus preventing the for-mation of oxidized low-density lipoprotein, which is consid-ered to induce cardiovascular diseases (Amic et al. 2003).Therefore, the consumption of foods with high levels of thesephytochemicals or addition of such extracts could protect thebody as well as the foods against these events (Chandini et al.2008).

Marine algae have been consumed in Asia since ancienttimes, and to a much lesser extent in the rest of the world.Many plant-based foods can be eaten raw or after cooking.Cooking can be performed in various ways, but for veg-etables, the most common are steaming, boiling and micro-waving. These cooking processes would bring about anumber of changes in the physical characteristics and chemi-cal composition of the vegetables (Zhang and Hamauzu2004). Reports on the effects of cooking on the antioxidantcompounds in vegetables have been inconclusive. There arereports demonstrating an enhancement or no change in anti-oxidant activity of vegetables (Gahler et al. 2003; Turkmen

et al. 2005), while others have indicated a deterioration ofactivity after thermal treatment (Ismail et al. 2004; Zhang andHamauzu 2004).

The presence and diversity of phytochemicals in vegetablesare important factors for human health. The phytochemicalcontents in untreated vegetables have been the most studied.Because a large part of ingested vegetables are generally ther-mally processed prior to consumption, it is also important toinvestigate how the processing affects the levels of these com-pounds (Volden et al. 2009). Processing of vegetables for con-sumption exposes the phytochemicals present to detrimentalfactors that may lead to alterations in concentrations andhealth-related quality. For example, wet-thermal treatmentcauses denaturation of enzymes that can catalyze the break-down of nutrients and phytochemicals. On the other hand,processing by heat can result in the reduction of constituentsby leaching or because of thermal destruction (Rungapam-estry et al. 2007). Turkmen et al. (2005) revealed that differentcooking methods (boiling, steaming and microwaving)caused losses of phenolics from squash, peas and leeks.However, under similar conditions, an increase in the phe-nolic content of vegetables such as green beans, peppers andbroccoli was reported (Turkmen et al. 2005). Watchtel-Galoret al. (2008) found that steaming and microwaving led tolosses in the total phenolic content (TPC) of broccoli, choy-sum and cabbage, although steaming had significantly lessloss than microwaved samples. Volden et al. (2009) alsoreported loss of phytochemicals in steamed cauliflower(19%).

The traditional process to preserve seaweeds is by sundry-ing (Lim and Murtijaya 2007) as several seaweeds are perish-able in their fresh state and could deteriorate within a few daysafter harvest. Drying is one of the most common food pro-cessing methods that can be used to extend the shelf life and toachieve the desired characteristics of a food product. Reduc-ing the water activity (aw) of food via this process can mini-mize deterioration from chemical reactions and microbialactivity (Chiewchan et al. 2010). Dried seaweeds are rehy-drated by various methods such as boiling before consump-tion; therefore, in the present study, drying was considered asa pretreatment before cooking.

Food poisoning is a concern for both consumers and thefood industry despite the use of various preservationmethods. Food processors, food safety regulators and regula-tory agencies are continuously concerned with the high andgrowing number of illness or outbreaks caused by somepathogenic and spoilage microorganisms in foods. Recently,consumers are demanding foods that are fresh, natural andminimally processed. Along with this, consumers are alsoconcerned about the safety of foods containing synthetic pre-servatives. This has put pressure on the food industry and hasfuelled research into the discovery of alternative natural anti-microbials (Shan et al. 2007).

EFFECT OF PROCESSING ON HIMANTHALIA ELONGATA S. COX, N. ABU-GHANNAM and S. GUPTA

2 Journal of Food Processing and Preservation •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.

Being rich in phytochemicals responsible for antioxidantand antimicrobial activity, there have been many studies con-ducted on seaweeds to quantify these compounds (Duan et al.2006; Chandini et al. 2008 and Cox et al. 2010); however, littleinformation is available on the effect of hydrothermal treat-ment on these phytochemicals in seaweeds. The purpose ofthis study was a preliminary screening to investigate the effectof different processing methods on the phytochemical con-stituents present in an Irish edible brown seaweed, Himantha-lia elongata. The effect was studied in terms of both the extractof H. elongata and as a whole food. The aim was, on one hand,to study the effect of common cooking treatments on the phy-tochemicals of the brown seaweed.At the same time, the effectof drying as a pretreatment on the cooking time and phy-tochemical content was also assessed. Moreover, the antimi-crobial properties of H. elongata extracts against commonfood pathogenic and food spoilage bacteria were also investi-gated after varied processing treatments.

METHODS

Chemicals

2, 2-Diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid,Folin–Ciocalteu’s phenol reagent, gallic acid, sodium carbon-ate (Na2CO3), sodium benzoate, vanillin, hydrochloric acid(HCl) (+)-catecin, aluminium chloride (AlCl3) and quercetinwere purchased from Sigma Aldrich Chemie (Steinheim,Germany). Tryptic soy broth (TSB) was purchased fromSparks (Dublin, Ireland).

Seaweed Material

H. elongata (Phaeophyta) was purchased from Quality SeaVeg., Co Donegal, Ireland. Samples were collected in Septem-ber and November 2009, washed thoroughly with freshwaterto remove epiphytes and stored at 4C until analysis.

Preparation of Samples

H. elongata was washed thoroughly with tap water, dried withabsorbent paper and then cut into 3 cm long pieces beforeprocessing. The effect of processing on H. elongata in termsof antioxidant and antimicrobial activity was evaluated bydrying, boiling, steaming, microwaving, and combinations ofdrying as a pretreatment before boiling and steaming until anedible texture was achieved as described in the next section.

Determination of Cooking Time andTexture Evaluation

Cooking time of seaweed was selected from preliminary experi-ments and was determined by the tactile method. To overcome

the subjectivity of the tactile method, a combination of tactileand instrumental textural methods were used in order to decidethecookingtimeof seaweed.Edible texturewasdeterminedbyasensory panel consisting of six judges. At 5-min cooking timeintervals,seaweed samples were removed to undergo tactile andinstrumental texture analysis.Shear tests were performed usingan Instron Universal Testing Machine (Model 4301, Canton,MA) attached to Bluehill 2 version 2.14 analysis software formaterials testing.AWarner Bratzler cutter was used in the sheartests.An aluminum plate with dimensions of 10 ¥ 6 cm2, thick-ness 1.3 cm with an opening of 3 mm in the center was sup-ported in the Instron base. Seaweed samples (5 g) were shearedat a speed of 200 mm/min. The cutting implement was allowedto travel the depth of the seaweed, cutting through the sampleand seaweed hardness was defined as the peak of force-deformation curve recorded in Newtons per mm (N/mm). Tenreplications of each sample were carried out.

Drying pretreatment. Seaweed samples were placed in5-g lots on a drying tray in a single layer. Drying of seaweedwas investigated in a drier (Innova 42, Mason Technology,Dublin, Ireland) at 25C air drying temperature over a periodof 12–24 h. Air velocity was 2.0 � 0.1 m/s measured withVWR Enviro-meter digital anemometer (VWR, Ireland).

Boiling. The seaweed samples (dried or fresh) were boiled byimmersion in 2 L of distilled water kept at the specified boilingtemperatures (80 and 100C) using a water bath (Lauda, Aqual-ineAL5,Mason Technology,Ireland) until an edible texture wasachieved (30–32 N/mm) as described in section on determina-tion of cooking time and texture evaluation. After boiling, thecooked seaweeds were drained using a wire mesh strainer andplaced on ice to cool before the extraction procedure.

Steaming. Regular steaming was performed on dried andfresh seaweeds using an atmospheric steam cooker (FS360,Kenwood, Havant, U.K.). The seaweed samples (5 g), wereplaced in the center tray of the steam cooker, covered with thelid and steamed over 2 L of boiling water. Steaming time wasselected according to preliminary experiments, in whichsteaming time was determined when an edible texture wasachieved (30–32 N/mm) as described in section on determi-nation of cooking time and texture evaluation. After thesteaming process, the cooked seaweeds were drained andplaced on ice to cool before the extraction procedure.

Microwaving. Fresh seaweed samples (5 g) were placed in apyrexbowl,coveredwithaplasticfilmtopreventwater lossandmicrowaved in a domestic microwave oven (Sharp PlatinumCollection R-957,Sharp,Uxbridge,U.K.) at 450 and 900 W for30 and 20 s respectively. Cooking time was selected according

S. COX, N. ABU-GHANNAM and S. GUPTA EFFECT OF PROCESSING ON HIMANTHALIA ELONGATA


to preliminary experiments in which microwaving time wasdetermined when an edible texture was achieved (30–32 N/mm) as described in section on determination of cooking timeand texture evaluation. After microwaving, the seaweeds wereplaced on ice to cool before the extraction procedure.

Extraction of Phytochemicals

Seaweed samples after respective processing (5 g originalweight) were powdered in liquid nitrogen using a mortar andpestle, and were then extracted with 50 mL of methanol(60%) under nitrogen atmosphere for 2 h. Liquid nitrogenwas used as it can reduce the particle size of a large amount ofseaweed in a short period of time. The extraction was carriedout at 40C at 100 rpm in a shaker incubator (Innova 42,Mason Technology) as optimized in our previous work (Coxet al. 2010). Samples were filtered using Whatman Number 1filter papers (Sigma Aldrich Chemie) and centrifuged at10,000 rpm for 15 min (Sigma 2K15, Mason Technology).Resulting extracts were evaporated to dryness using vacuumpolyevaporator (Buchi Syncore Polyvap, Mason Technology)at 60C. A pressure gradient program was designed for evapo-ration of the solvents with vacuum conditions of 337 and72 mbar for methanol and water respectively.

TPC

The TPC of seaweed samples (concentration 1 mg/mL ofextract in water) was measured using the Folin–Ciocalteaumethod as reported by Taga et al. (1984). The TPCs of thewhole seaweeds were expressed as mg gallic acid equivalentper 100 g fresh weight (mg GAE/100 g FW) and as mg GAE/gfor extracts.

DPPH Radical-Scavenging Activity

Free radical-scavenging activity was measured by DPPHaccording to the method of Yen and Chen (1995) with somemodifications. Briefly, a 100 mL aliquot of test sample wasplaced in a 96-well microtitre plate and 100 mL of 0.16 mMDPPH methanolic solution was added. The mixture wasshaken and incubated for 30 min in darkness at 25C. Changesin the absorbance of the samples were measured at 517 nmusing a microplate reader (Powerwave, Biotek, VT).

The ability to scavenge the DPPH radical was calculatedusing the following equation given by Duan et al. (2006):

Scavenging effect (%) sample sampleblank

control

= −−⎛

⎝⎜⎞⎠⎟1

A A

A⎡⎡⎣⎢

⎤⎦⎥

×100

(1)

Where: Acontrol is the absorbance of the control (DPPH solu-tion without sample), Asample is the absorbance of the testsample (DPPH solution plus test sample) and Asample blank is the

absorbance of the sample only (sample without any DPPHsolution). DPPH results were interpreted as the“efficient con-centration” or EC50 value, which is the concentration of sub-strate that causes 50% loss of the DPPH activity.

Total Flavonoid Content (TFC)

TFCs were determined according to the method of Zhishenet al. (1999). Quercetin was used to prepare the standardcurve and results were expressed as mg quercetin equivalents(QE)/100 g FW (mg QE/100 g FW) for whole seaweeds and asmg QE/g for extracts.

Total Condensed Tannin Content (TTC)

TTCs were determined according to the method of Julkunen-Titto (1985). (+)-Catechin was used to prepare the standardcurve and results were expressed as mg catechin equivalents(CE)/100 g FW (mg CE/100 g FW) for whole seaweeds and asmg CE/g for extracts.

Antimicrobial Activity

Microbial Culture. Two species of common food patho-gens and two species of food spoilage bacteria selected for thisstudy were Listeria monocytogenes American Type CultureCollection (ATCC) 19115, Salmonella abony National Collec-tion of Type Cultures 6017, Enterococcus faecalis ATCC 7080and Pseudomonas aeruginosa ATCC 27853 respectively(Medical Supply Company, Dublin, Ireland). All cultureswere maintained at -70C in 20% glycerol stocks and grown inTSB at 37C; apart from P. aeruginosa, which was incubated at30C to obtain subcultures. Working cultures were preparedfrom subcultures and grown at optimal conditions for eachbacterium for 18 h before analysis. Bacterial suspensions werethen prepared in saline solution (NaCl 0.85%, BioMérieux,Marcy L’Etoile, France) equivalent to a McFarland standardof 0.5, using the Densimat photometer (BioMérieux Inc) toobtain a concentration of 1 ¥ 108 colony-forming units (cfu)/mL. This suspension was then diluted in TSB to obtain aworking concentration of 1 ¥ 106 cfu/mL.

Antimicrobial Activity Assay. The influence of varyingconcentrations of extract on efficacy was assessed againstL. monocytogenes, S. abony, E. faecalis and P. aeruginosa using96-well microtitre plates (Sarstedt Ltd, Bath, U.K.). Seaweedextracts after respective processing (5 g original seaweedweight) were dissolved in 2.5 mL of TSB and 200 mL wasadded to the first row of each plate. All other wells were filledwith 100 mL of TSB and 100 mL from the first well was serialdiluted two-folds along each column. Finally, 100 mL of bac-terial suspension containing 1 ¥ 106 cfu/mL was added to the



wells. The last column was used for bacterium and media con-trols, and sample blanks were prepared for all of the extracts.Absorbance readings were then taken at 0 and 24 h at 600 nmusing a microplate spectrophotometer (Powerwave, Biotek)with 20 s agitation before each optical density (OD) measure-ment. Analysis of growth over time was also performed onmost effective extracts. OD measurements were taken every3 h for 24 h. Sodium benzoate and sodium nitrite were usedas controls. Percentage inhibition was calculated as follows:

Bacterial inhibition (%) = −⎛⎝

⎞⎠

⎡⎣⎢

⎤⎦⎥

×O E

O100 (2)

Where: O is (OD of the organism at 24 h – OD of theorganism at 0 h) and E is (OD of the extract at 24 h – blank at24 h) – (OD of the extract at 0 h – blank at 0 h). Results wereinterpreted by categorizing percentage inhibitions based oninhibition intensity according to Dubber and Harder (2008).

Statistical Analysis. All experiments were performed intriplicate and replicated twice. All statistical analyses werecarried out using STATGRAPHICS Centurion XV (StatPointTechnologies, Inc., Warrenton, VA). Statistical differencesbetween different processing treatments were determinedusing analysis of variance followed by least significant differ-ence testing. Differences were considered statistically signifi-cant when P < 0.05.

RESULTS AND DISCUSSION

Effect of Processing on TPC

Increased intake of vegetables is generally associated with areduced risk of cancer and cardiovascular disease (Kris-Etherton et al. 2002). Processing and preparation of veg-etables, especially thermal treatment, which are appliedprior to consumption may affect the phytochemicals. Heatapplications such as boiling, steaming or microwaving arecommon practices in the processing of food products inorder to render them palatable and microbiologically safe.Because seaweed would need to undergo some heat treat-ment prior to usage, it was relevant to assess the effects ofheat treatment on the stability of seaweed antioxidant prop-erties. In the present study, cooking of H. elongata wascarried out by three commonly used procedures and theantioxidant properties of the cooked product were evaluatedand compared with that of the fresh product. It is a well-known fact that cooking time as well as cooked texture,appearance and flavor are important cooking quality char-acteristics (Xu and Chang 2008). Firmness or softness is oneof the most important criteria in determining the accept-ability of foods. Acceptable texture parameters are outlinedin the literature for various foods such as legumes (Xu and

Chang 2008), but there are no such values previously out-lined for seaweed. Because sensory evaluation is based onhuman senses, which detect myriad characteristics of mate-rial properties simultaneously, it is difficult to find a goodcorrelation between orally perceived texture and instrumen-tally measured texture (Nishinari 2004). Therefore, in thepresent study, the cooking time of fresh seaweed boiled at100C was calculated using a tactile and instrumental texturemeasurement based on the length of time it took theseaweed to become edible. Samples were taken every 5 minand edibility was judged based on the hardness and chewi-ness of the samples until an acceptable edible texture wasachieved. From these methods, it was found that softeningof fresh H. elongata from 45 N/mm to 30–32 N/mm was anacceptable edible texture as can be seen in Table 1.

The TPC of processed H. elongata can be seen in Fig. 1A.In order to achieve an edible texture, it was necessary thatH. elongata was boiled for 40 and 35 min at 80C and 100Crespectively. This resulted in almost 82.31 and 85.5% loss inTPC respectively as compared with fresh seaweed. A reduc-tion of TPC from 175.27 to 25.4 mg GAE/100 g FW wasobserved. Similar results were obtained with steaming,although it was not as detrimental as boiling. The seaweedwas steamed for 45 min, which resulted in a loss of 32.06%TPC. The reason for such high reduction during boilingcould be due to leaching of nutrients in water, which wasalso proved by a reduction in the amount of extractobtained. Fresh H. elongata had a 3.14% yield of extract,whereas steaming or boiling resulted in a significant(P < 0.05) reduction in the yield of extract to as low as0.26% (Table 2). The reduction of vitamin levels duringprocessing and cooking can vary largely depending on thecooking method and type of food (Leskova et al. 2006).

TABLE 1. INSTRUMENTAL AND SENSORY TEXTURE EVALUATION TODETERMINE EDIBLE TEXTURE LEVEL OF COOKED HIMANTHALIAELONGATA

Cookingtime (min)

Instrumentaltexture (N/mm)

Sensory paneljudgments*

0 45.33 � 1.30 Too hard and chewy5 40.47 � 2.31 Too hard and chewy

10 37.38 � 1.93 Too hard and chewy15 35.89 � 0.98 Too hard and chewy20 34.62 � 1.61 Too hard and chewy25 33.98 � 0.79 Too hard and chewy30 32.40 � 0.15 Edible35 32.11 � 0.74 Edible40 30.42 � 1.05 Edible45 30.22 � 0.95 Edible50 30.09 � 1.02 Edible

Each value is presented as mean � standard deviation (n = 6).Seaweeds were boiled at 100C.* Sensory panel judgments were based on the hardness and ease ofchew of the seaweed.



Studies have shown that phenolic compounds are sensitiveto heat, whereby boiling of vegetables for a few minutescould cause a significant loss of phenolic content, which canleach into the boiling water (Amin et al. 2006). Xu andChang (2008) reported a 40–50% loss in the TPC oflegumes because of phenolics leaching into the boilingwater, whereas Oboh (2005) found up to 200% increase inthe phenolic content of boiled tropical green leafy veg-etables. Reduction in TPC was also found in other veg-etables such as broccoli, kale and spinach (Ismail et al. 2004;Zhang and Hamauzu 2004). These authors stated the prob-able reason was the dissolution of polyphenols into thecooking water, which could be the case with H. elongata as itrequires long cooking times to become edible. Althoughmicrowave cooking has been reported to be the mostdeleterious with respect to the antioxidant properties ofvegetables (Sultana et al. 2008), the results in the present

study were encouraging as microwaving increased the TPCby 22.49% (450 W) and 36.58% (900 W), as compared withfresh.

In order to reduce the cooking time and eventual loss ofphytochemicals, drying was used as a pretreatment. Theprocess of drying in itself was not detrimental as a dryingperiod of 12 and 24 h retained 80.1 and 85.96% of the origi-nal phenolic content as compared with fresh seaweed. Pos-sible losses could be attributed to stressing the plant duringthe drying process because of loss of water through the cellwalls. Moreover, drying for 12 and 24 h before boiling notonly reduce the cooking time but also the loss in TPC.Drying for 24 h before boiling at 100C decreased cookingtime by 15 min and reduced the loss of total phenols by8.83%, as compared with boiling at 100C without a dryingpretreatment. Drying as a pretreatment was not effective forsteaming as 44.19% reduction in TPC was seen when

A

B

FIG. 1. (A) TOTAL PHENOLIC CONTENT OFPROCESSED HIMANTHALIA ELONGATA (MGGALLIC ACID EQUIVALENTS/100 G FRESHWEIGHT). (B) TOTAL PHENOLIC CONTENT OFPROCESSED H. ELONGATA EXTRACT (MGGALLIC ACID EQUIVALENTS/G EXTRACT)Each value is presented as mean � standarddeviation (n = 6).Means above each bar with different lettersdiffer significantly (P < 0.05).Where each abbreviation is as follows: Fr,fresh; D 12h, dried 12 h; D 24h, dried 24 h; B80C, boiled 80C; B 100C, boiled 100C; D 12hB 80C, dried 12 h and boiled 80C; D 12h B100C, dried 12 h and boiled 100C; D 24h B80C, dried 24 h and boiled 80C; D 24h B100C, dried 24 h and boiled 100C; St,steamed; D 12h St, dried 12 h and steamed;D 24h St, dried 24 h and steamed; M 450w,microwaved at 450 W; M 900w, microwavedat 900 W.



seaweed was dried for 24 h followed by steaming (P < 0.05).Microwaving was not carried out on dried seaweeds as itwas not a hydrothermal process and therefore re-hydrationwould not take place.

Although heat processing seriously degraded the qualityof seaweed, an interesting observation was an increase in thepotency of the extract (Fig. 1B). When the activity of theheat-processed extract was compared in terms of per gramdried extract, it was found that boiling at 80 and 100Cresulted in an increase of 104.03 and 71% TPC/g of driedextract respectively as compared with fresh extract(P < 0.05). Fresh H. elongata contained 55.75 mg GAE/g ofextract. Drying for 12 and 24 h led to a significant decreasein TPC of up to 22.42% as compared with fresh (P < 0.05).Drying pretreatment for 12 h followed by boiling at 80 and100C for 30 and 25 min respectively had a 161.43 and125.11% increase per gram of extract as compared withfresh samples (P < 0.05). Samples pretreated by drying for24 h followed by boiling at 100C for 20 min increased theTPC by 165.32% as compared with fresh. Drying of H. elon-gata for 24 h followed by boiling at 80C for 30 min had themost significant effect (P < 0.05) on the TPC of all treat-ments resulting in 173.99% increase in TPC. Steaming for45 min had a 36.62% decrease in TPC/g of extract, while adrying pretreatment before steaming for 50 min had a 40%decrease. Microwaving at 450 W caused no significantincrease in the TPC per g of extract as compared with fresh(P < 0.05), while microwaving at 900 W increased the TPCto 82 mg GAE/g of extract (47.08% increase). As the TPC ofH. elongata per gram of extract was increased because ofprocessing, a lower concentration of the extract would be

required to have a potential effect on preventing oxidationin food products, which is a promising finding.

Effect of Processing on DPPHRadical-Scavenging Activity

The results of the DPPH free radical-scavenging ability of theseaweed processed under different conditions are shown inTable 2. DPPH reagent has been used extensively for investi-gating the free radical-scavenging activities of compounds(Shon et al. 2003). The results indicated that free radical-scavenging ability of the processed seaweeds ranged from52.51 to 100% (concentration 100 mg/mL extract) withextracts from seaweed dried for 12 h followed by boiling at100C being most effective. Significant differences (P < 0.05)in DPPH values were found for all processing treatments.At 100 mg/mL extract concentration, drying led to slightdecrease in DPPH radical-scavenging activity from 75.5 to74.69% (12 h) and 67.87% (24 h) while boiling led to a sig-nificant increase (P < 0.05). Boiling at 100C increased DPPHscavenging by 23.89%, from 75.5 to 93.54%. Drying ofH. elongata for 12 h followed by boiling at 100C had the mostsignificant increase in antioxidant activity as 100% inhibitionof the DPPH radical was achieved with 100 mL/mL of extract.Steaming significantly reduced the DPPH radical-scavengingactivity (P < 0.05) to 52.51%. Extracts from H. elongata givendrying pretreatments followed by steaming had 53.79%(12 h) and 53.5% (24 h) scavenging of the DPPH radical.Seaweed microwaved at 450 W had 76.29% activity againstDPPH radical while microwaving at 900 W had 75.35%activity.

TABLE 2. YIELD OF TOTAL EXTRACT (AS % W/W OF SEAWEED ON FRESH WEIGHT BASIS), DPPH RADICAL-SCAVENGING ACTIVITY (%) OFPROCESSED HIMANTHALIA ELONGATA EXTRACTS (CONCENTRATION 100 mG/ML) AND EC50 (mG/ML)* OF EACH EXTRACT

Processing treatmentTotal methanolextract (%)

DPPH radical-scavengingactivity (%) EC50 (mg/mL)

Fresh 3.14 � 0.19a 75.50 � 2.30a 25.00 � 1.81a

Dried 12 h 3.25 � 0.20a 74.69 � 2.31b 50.00 � 3.31b

Dried 24 h 3.48 � 0.29ab 67.87 � 1.05c 50.00 � 2.55b

Boiled 80C – 40 min 0.27 � 0.09c 92.96 � 1.55d 12.50 � 1.12c

Boiled 100C – 35 min 0.26 � 0.06c 93.54 � 1.24e 12.50 � 2.00c

Dried 12 h and boiled 80C – 30 min 0.28 � 0.05c 95.33 � 0.80f 12.50 � 1.00c

Dried 12 h and boiled 100C – 25 min 0.28 � 0.06c 100.00 � 0.00g 12.50 � 1.02c

Dried 24 h and boiled 80C – 30 min 0.24 � 0.02c 88.50 � 1.24h 12.50 � 1.21c

Dried 24 h and boiled 100C – 20 min 0.27 � 0.02c 89.04 � 1.85i 12.50 � 1.00c

Steamed – 45 min 3.37 � 0.21a 52.51 � 0.56j 100.00 � 0.98d

Dried 12Vh and steamed – 50 min 3.31 � 0.19a 53.79 � 1.88k 100.00 � 1.99d

Dried 24Vh and steamed – 50 min 3.30 � 0.20a 53.50 � 0.97l 100.00 � 0.96d

Microwaved 450 W – 30 s 3.75 � 0.55b 76.29 � 1.57m 25.00 � 1.52a

Microwaved 900 W – 20 s 4.34 � 0.56d 75.35 � 2.00n 25.00 � 1.88a

Each value is presented as mean � standard deviation (n = 6).Means within each column with different letters (a–n) differ significantly (P < 0.05).* EC50 value is defined as the amount of extract necessary to decrease the initial DPPH radical concentration by 50%.DPPH, 2, 2-diphenyl-1-picrylhydrazyl.



DPPH results are often interpreted as the “efficient con-centration” or EC50 value, which is defined as the concentra-tion of substrate that causes 50% loss of the DPPH activity(Molyneux 2004). The EC50 values of DPPH radical-scavenging activity from dried methanolic extracts of sea-weeds are also presented in Table 2. Processing ofH. elongata resulted in significantly different EC50 values(P < 0.05), depending upon the treatment. The EC50 levelsranged from 12.5 to 100 mg/mL of extract with all treat-ments in which boiling was found to have the most effectiveEC50 values (12.5 mg/mL of extract). Extracts from freshseaweed had an EC50 of 25 mg/mL. Drying of seaweed led toa significant (P < 0.05) reduction in the DPPH radical-scavenging activity of the extract to 50 mg/mL (12 and 24 h).Steaming had the most detrimental effect on the DPPHradical-scavenging activity of the extract as 100 mg/mL wasrequired to reduce the DPPH radical by 50% and activity at100 mg/mL concentration was almost half that of the mosteffective processed seaweed (53.5 and 100% respectively).

There was no significant difference between microwavedseaweed extracts compared with fresh (P > 0.05) as all hadan EC50 value of 25 mg/mL.

Effect of Processing on TFC

The bioavailability of phytochemicals is influenced by thematrix and microstructure of the food they occur in, thestorage conditions (light, oxygen and temperature regime)and thermal processing they are subjected. As a consequence,knowledge of the content and stability of phytochemicals infoods after processing is essential to evaluate the nutritionalvalue of foods rich in these phytochemicals, like seaweed forexample (Parada and Aguilera 2007).

The TFC of processed whole H. elongata is presented inFig. 2A. The TFC of fresh H. elongata was 53.18 mgQE/100 g FW. Drying for 12 and 24 h had no significanteffect on the TFC as there was only a slight increase of 0.72and 0.25% respectively (P > 0.05). All treatments, which

A

B

FIG. 2. (A) TOTAL FLAVANOID CONTENT OFPROCESSED HIMANTHALIA ELONGATA (MGQUERCETIN EQUIVILANTS/100 G FRESHWEIGHT). (B) TOTAL FLAVANOID CONTENTOF PROCESSED H. ELONGATA EXTRACT(MG QUERCETIN EQUIVALENTS/G EXTRACT)Each value is presented as mean � standarddeviation (n = 6).Means above each bar with different lettersdiffer significantly (P < 0.05).Where each abbreviation is as follows: Fr,fresh; D 12h, dried 12 h; D 24h, dried 24 h; B80C, boiled 80C; B 100C, boiled 100C; D 12hB 80C, dried 12 h and boiled 80C; D 12h B100C, dried 12 h and boiled 100C; D 24h B80C, dried 24 h and boiled 80C; D 24h B100C, dried 24 h and boiled 100C; St,steamed; D 12h St, dried 12 h and steamed;D 24h St, dried 24 h and steamed; M 450w,microwaved at 450 W; M 900w, microwavedat 900 W.



included boiling, significantly reduced the TFC, within arange of 88.86–90.18%. This highest reduction was seen infresh seaweed boiled at 100C, which led to a 90.18% reduc-tion in TFC (P < 0.05). Flavonoids commonly accumulate inepidermal cells of plant organs, being found as glycosidesand in nonglycosidic forms (aglycones) (Sakihama et al.2002). Release of flavonoids and increased chemical extrac-tion of these compounds could be induced by the effect ofboiling (Olivera et al. 2008). This release of flavonoidscoupled with contact and leaching into water could haveresulted in high reduction in TFC for boiled samples. Theresults of the present study are similar to Olivera et al.(2008) who found that boiling decreased TFC in brusselssprouts. Steaming also led to a significant reduction in TFCcompared with fresh but this was significantly less as com-pared with boiled seaweeds (P < 0.05). Steaming retained17.4% more TFC than boiled samples as compared withfresh samples. This could be due to the fact that steamedseaweeds were not in direct contact with water, whichresulted in considerably less leaching of flavonoids. Micro-waving at 450 W had a 12.69% increase in TFC while micro-waving at 900 W raised the TFC by 10.65% in wholeH. elongata (P < 0.05). These results are in line with Fran-cisco et al. (2010) and Rodrigues et al. (2009) who alsoreported significant losses of flavonoids up to 67% incooked conventional vegetables.

TFC of processed H. elongata extracts are presented inFig. 2B. Extracts from fresh H. elongata contained 42.29 mgQE/g of extract. All treatments significantly changed theTFC content as compared with fresh (P < 0.05). Simpledrying for 12 and 24 h significantly reduced (P < 0.05) theTFC in the range of 2.45–9.35%, whereas boiling at 80 and100C resulted in an increase up to 15.76%. However, a com-bination of drying pretreatment followed by boiling had themost significant effect on the TFC of H. elongata. Dryingfor 12 h followed by boiling at 80 and 100C resulted in18.72 and 21.67% increase in TFC respectively (P < 0.05).Drying for 24 h followed by boiling at 100C for 20 min hada 26.6% increase in TFC. The most significant increase of32.02% was seen in samples dried for 24 h followed byboiling at 80C for 30 min. Steaming alone and in combina-tion with 12 and 24 h drying pretreatments resulted in 14,11.43 and 11.98% increase in TFC respectively. The increasein the case of microwaved samples ranged from 8.72 to14.29%.

Effect of Processing on TTC

Phlorotannins are a group of phenolic compounds that arerestricted to polymers of phloroglucinol and have beenidentified from several brown algae. Many studies haveshown that phlorotannins are the only phenolic groupdetected in brown algae (Jormalainen and Honkanen 2004;

Koivikko et al. 2007). TTC of processed H. elongata canbe seen in Fig. 3A. Condensed tannins of the studiedseaweeds ranged from 70.61 to 5.5 mg QE/100 g FW.Fresh H. elongata contained 70.05 mg CE/100 g FW whiledrying for 12 and 24 h reduced the TTC by 2.92 and 4.73%respectively.

Similarly to TFCs, TTC was significantly reduced uponboiling (P < 0.05). Boiling at 80C for 40 min significantlyreduced the TTC from 70.05 to 6.22 mg CE/100 g FW(91.11% reduction). The most significant reduction of92.13% in TTC was seen in H. elongata boiled at 100C for35 min (P < 0.05). Similar to TFC, steaming had a lowerreduction of TTC as compared with boiling. Steamingretained 40.5% more TTC than boiled samples as comparedwith fresh (P < 0.05). Microwaving at 450 and 900 W had a20.27 and 22.54% reduction in TTC respectively (P < 0.05).The basis of the significant decrease in cooked seaweeds couldalso be attributed to the possible breakdown of tanninspresent in the seaweed to simple phenol (Akindahunsi andOboh 1999). Khandelwal et al. (2010) and Somsub et al.(2008) also found decreases in tannin levels of cookedlegumes and vegetables.

In contrast to TPC and TFC, a significant reduction in thetotal condensed tannins of processed H. elongata extracts wasobserved as compared with fresh extracts (Fig. 3B). Extractsfrom fresh H. elongata contained 55.7 mg CE/g, while dryingfor 12 and 24 h had 7.73 and 8.65% reduction in TTC respec-tively. Boiling at 80C for 40 min led to a 58.97% reduction inTTC, while 62.89% reduction was seen in H. elongata boiledat 100C for 35 min. Drying pretreatment followed by boilingalso had significant losses of TTC but less than that of boiledseaweed. Drying for 12 h before boiling at 80 and 100C had53.52 and 53.78% reduction respectively. Drying for 24 h fol-lowed by boiling at 80C for 30 min caused a loss of TTC by55.71%, while drying for 24 h in combination with boiling at100C for 20 min had a 55.91% reduction from 55.7 to24.55 mg CE/g of extract. Steaming resulted in 18.91% reduc-tion while in combination with a drying pretreatment therewas a 28% reduction as compared with extracts from freshH. elongata. Microwaving at 450 and 900 W for 30 and 20 shad a 19.14 and 16.88% reduction in TTC respectively(P < 0.05).

Antimicrobial Activity of ProcessedH. elongata Extracts AgainstL. monocytogenes

Consumers are concerned about the safety of foods contain-ing synthetic preservatives. This has put pressure on thefood industry and has fuelled research into the discovery ofalternative natural antimicrobials (Shan et al. 2007). Antimi-crobial activity of processed seaweed extracts was studied inorder to analyze the effects of food processing on their activ-



ity. The entire yield of extract from 5 g original weight ofeach of the processed seaweeds were dissolved in 2.5 mLTSB and utilized in the assay. Therefore, different concentra-tions were achieved at each dilution level as can be seenin Table 3. In the present study, antimicrobial activitywas tested against common food spoilage (E. faecalis andP. aeruginosa) and food pathogenic (L. monocytogenesand S. abony) bacteria. These organisms were studiedafter discussions with the Food Safety Authority ofIreland because they have been identified as being problem-atic in the Irish food industry. The entire spectrum ofinhibitory effects is reported as outlined by Dubber andHarder (2008).

L. monocytogenes is a Gram-positive pathogenic bacte-rium commonly isolated from foods in many countriesincluding Ireland (Chitlapilly-Dass et al. 2010). The per-centage inhibition of the processed seaweed extracts againstL. monocytogenes are presented in Table 4 and the concen-trations of extract for each dilution of processed H. elongata

are outlined in Table 3. At the highest extract concentra-tions, extract from fresh seaweed and those dried for 12 and24 h (31.44, 32.47 and 34.77 mg/mL respectively) had 100%inhibition against L. monocytogenes. Any processing treat-ment that included boiling of H. elongata had weak activityagainst L. monocytogenes (<25% inhibition); however,extract concentrations were lower most likely because of theleaching of phytochemicals during the boiling procedure.Extracts of steamed seaweed had strong activity againstL. monocytogenes at the highest dilution tested (96.34%inhibition at 34 mg/mL). However, when drying was used asa pretreatment before steaming there was less than half theinhibition against L. monocytogenes; 43.25% (31 mg/mL)and 43.85% (29.3 mg/mL) for 12 and 24 h dried samplesrespectively. Microwaving at 450 and 900 W producedextracts with strong inhibition against L. monocytogenes inthe first dilution (97.24 and 97.26% inhibition at 37.56 and43.37 mg/mL extract respectively). As the yield ofmicrowaved H. elongata extract was higher than other

A

B

FIG. 3. (A) TOTAL CONDENSED TANNINCONTENT OF PROCESSED HIMANTHALIAELONGATA (MG CATECHINEQUIVILANTS/100 G FRESH WEIGHT).(B) TOTAL CONDENSED TANNIN CONTENTOF PROCESSED H. ELONGATA EXTRACT(MG CATECHIN EQUIVALENTS/G EXTRACT)Each value is presented as mean � standarddeviation (n = 6).Means above each bar with different lettersdiffer significantly (P < 0.05).Where each abbreviation is as follows: Fr,fresh; D 12h, dried 12 h; D 24h, dried 24 h; B80C, boiled 80C; B 100C, boiled 100C; D 12hB 80C, dried 12 h and boiled 80C; D 12h B100C, dried 12 h and boiled 100C; D 24h B80C, dried 24 h and boiled 80C; D 24h B100C, dried 24 h and boiled 100C; St,steamed; D 12h St, dried 12 h and steamed;D 24h St, dried 24 h and steamed; M 450w,microwaved at 450 W; M 900w, microwavedat 900 W.



treatments, this would suggest that the extract is in factslightly less potent than those of fresh seaweed. Extractsfrom fresh H. elongata at the first concentration tested(31.44 mg/mL) were the most effective of the processed sea-weeds overall.

There was no significant difference (P > 0.05) betweenthe first and second dilutions of fresh H. elongata extracttested against L. monocytogenes (15.72 mg/mL), which againhad 100% inhibition in the second dilution. An inhibitionactivity of 89.88–92.99% was obtained by dried extracts,whereas extracts from boiled seaweeds had completely lostthe antimicrobial activity. Drying pretreatment beforeboiling seemed to maintain weak antimicrobial activity ofthe extracts in the range of 9.81–15.89% in the second

dilution as compared with 0%. Extracts from steamedseaweed at the second dilution of 17 mg/mL had strongactivity against L. monocytogenes giving 96.24% inhibition.Seaweeds that received a drying pretreatment (12 and 24 h)before steaming had significantly less antimicrobial activityagainst L. monocytogenes in the second dilution with inhibi-tion levels of 34.14 and 34.15% at 15.5 and 14.65 mg/mLrespectively (P > 0.05). Extracts from the second dilution ofmicrowaved seaweed had 82.9% (450 W) and 81.45%(900 W) inhibition against L. monocytogenes at 18.78 and21.74 mg/mL extract concentrations respectively. At thethird and fourth dilutions, although fresh seaweeds still had98% activity, all processed seaweeds had significantly lessinhibition (P < 0.05).

TABLE 3. CONCENTRATION OF HIMANTHALIA ELONGATA EXTRACTS (MG/ML) FROM DIFFERENT PROCESSED SEAWEEDS (5 G ORIGINAL SEAWEED)FOR EACH DILUTION TESTED

Processing treatment Dilution 1 (mg/mL) Dilution 2 (mg/mL) Dilution 3 (mg/mL) Dilution 4 (mg/mL)

Fresh 31.44 15.72 7.86 3.93Dried 12 h 32.47 16.23 8.12 4.06Dried 24 h 34.77 17.39 8.69 4.35Boiled 80C – 40 min 2.73 1.36 0.68 0.34Boiled 100C – 35 min 2.67 1.33 0.67 0.33Dried 12 h and boiled 80C – 30 min 2.80 1.40 0.70 0.35Dried 12 h and boiled 100C – 25 min 2.82 1.41 0.70 0.35Dried 24 h and boiled 80C – 30 min 2.42 1.21 0.61 0.30Dried 24 h and boiled 100C – 20 min 2.77 1.38 0.69 0.35Steamed – 45 min 34.00 17.00 8.50 4.25Dried 12 h and steamed – 50 min 31.00 15.50 7.75 3.88Dried 24 h and steamed – 50 min 29.30 14.65 7.33 3.66Microwaved 450 W – 30 s 37.56 18.78 9.39 4.69Microwaved 900 W – 20 s 43.47 21.74 10.87 5.43

TABLE 4. PERCENTAGE INHIBITION OF METHANOLIC EXTRACTS OF HIMANTHALIA ELONGATA PROCESSED UNDER DIFFERENT CONDITIONSAGAINST LISTERIA MONOCYTOGENES

Processing treatment Dilution 1 Dilution 2 Dilution 3 Dilution 4

Fresh 100.00 � 0.0aw 100.00 � 0.00aw 99.37 � 1.24ax 98.09 � 3.81ay

Dried 12 h 100.00 � 0.0aw 89.88 � 1.70bx 43.89 � 1.43by 31.68 � 2.08bz

Dried 24 h 100.00 � 0.0aw 92.99 � 8.16cx 29.84 � 2.19cy 26.98 � 6.72cz

Boiled 80C – 40 min 27.05 � 0.60bw 0.00 � 0.00dx 0.00 � 0.00dx 0.00 � 0.00dx

Boiled 100C – 35 min 46.45 � 3.42cw 0.00 � 0.00dx 0.00 � 0.00dx 0.00 � 0.00dx

Dried 12 h and boiled 80C – 30 min 24.95 � 3.51dw 10.00 � 1.71ex 0.00 � 0.00dy 0.00 � 0.00dy

Dried 12 h and boiled 100C – 25 min 21.02 � 2.54ew 14.00 � 0.25fx 0.00 � 0.00dy 0.00 � 0.00dy

Dried 24 h and boiled 80C – 30 min 10.48 � 3.23fw 9.81 � 1.15gx 0.00 � 0.00dy 0.00 � 0.00dy

Dried 24 h and boiled 100C – 20 min 23.20 � 2.54gw 15.89 � 1.68hx 0.00 � 0.00dy 0.00 � 0.00dy

Steamed – 45 min 96.34 � 3.64hw 96.24 � 1.56ix 81.54 � 5.56ey 77.88 � 5.54ez

Dried 12 h and steamed – 50 min 43.25 � 3.23iw 35.14 � 0.25jx 29.57 � 1.77fy 20.45 � 0.78fz

Dried 24 h and steamed – 50 min 43.85 � 0.23jw 34.15 � 1.12kx 28.89 � 1.89gy 19.55 � 0.98gz

Microwaved 450 w – 30 s 97.24 � 0.05kw 82.90 � 5.90lx 48.73 � 1.57hy 29.41 � 5.45hz

Microwaved 900 w – 20 s 97.26 � 1.25ew 81.45 � 3.07mx 74.20 � 1.84iy 72.86 � 3.10iz

Each value is presented as mean � standard deviation (n = 6).Means within each column with different letters (a–m) differ significantly (P < 0.05).Means within each row with different letters (w–z) differ significantly (P < 0.05).



Antimicrobial Activity of ProcessedH. elongata Extracts Against S. abony

Salmonella is a Gram-negative food-borne pathogenic bacte-rium and has become one of the most important causes ofacute enterocolitis throughout the world. Salmonellosis iscaused by any of over 2,300 serovars of Salmonella (Wonget al. 2000; Lee et al. 2001). Fresh H. elongata had the highestactivity against S. abony as can be seen in Table 5. However,overall, the processed extracts were least effective againstS. abony, and no processing treatment achieved 100% inhibi-tion of the bacteria.A potential cause is that S. abony is Gram-negative, as there are significant differences in the outer layersof Gram-negative and Gram-positive bacteria. Gram-negative bacteria possess an outer membrane and a uniqueperiplasmic space, which is not found in Gram-positive bac-teria (Nikaido 1996; Duffy and Power 2001). Antibacterialsubstances can easily destroy the bacterial cell wall and cyto-plasmic membrane and result in a leakage of the cytoplasmand its coagulation (Kalemba and Kunicka 2003).

Fresh H. elongata extracts had activity, which ranged from87.03 to 37.18% in the first to fourth dilutions. Seaweed thathad been dried for 12 and 24 h had moderate activity againstS. abony in the first dilution of extract but had weak activityfrom the second dilution onward (<50% inhibition). Anyprocessing treatment that included boiling had weak activityagainst S. abony with a maximum of 22.11% inhibition.Extracts from steamed H. elongata had strong activity in thefirst and second dilutions (>90%) and moderate activitythereafter. Drying of seaweed before steaming led to a signifi-cant reduction (P < 0.05) in antimicrobial activity as com-pared with steaming alone against S. abony (44% inhibition)

in the first dilution and weak activity in subsequent dilutions.Activity of microwaved H. elongata extracts at 450 and900 W ranged from strong (�94.4%) to weak (<50%) againstS. abony.

Antimicrobial Activity of ProcessedH. elongata Extracts Against E. faecalis

The Gram-positive bacterium E. faecalis is a natural memberof the human and animal gastrointestinal flora. This bacte-rium is an indicator of fecal contamination and has beendetected in food, milk and drinking water (Rincé et al. 2003).Antimicrobial activity of processed H. elongata extractsagainst E. faecalis are outlined in Table 6. Fresh H. elongataextracts had more than 99% antimicrobial activity until thesecond dilution and moderate activity in the third and fourthdilutions (87.03 and 78.02% respectively). Dried seaweedextracts had very strong (100%) and strong (95.4%) activityin the first dilution for 12 and 24 h respectively; however, thislevel was halved in the second dilution with 39.24% (12 h)and 53.01% inhibition (24 h). Extracts from boiled seaweedshad less than 10% activity in the first two dilutions and wascompletely lost in lower dilutions. A drying pretreatmentbefore boiling retained more of the bioactivity (P < 0.05) ofthe extract, for example extracts of H. elongata processed bydrying for 12 h followed by boiling at 80C for 30 min had51.45% inhibition. Extracts from steamed H. elongata hadstrong activity in the first and second dilutions (�95.21%)and moderate activity thereafter. Drying of seaweed beforesteaming led to a significant reduction (P < 0.05) in antimi-crobial activity as compared with steaming alone againstE. faecalis also (�44.57% inhibition). Extracts of microwaved

TABLE 5. PERCENTAGE INHIBITION OF METHANOLIC EXTRACTS OF HIMANTHALIA ELONGATA PROCESSED UNDER DIFFERENT CONDITIONSAGAINST SALMONELLA ABONY


Fresh 87.03 � 3.91aw 78.80 � 1.41ax 65.13 � 4.60ay 37.18 � 5.71az

Dried 12 h 77.29 � 1.26bw 39.24 � 2.27bx 26.83 � 3.75by 11.01 � 2.06bz

Dried 24 h 83.52 � 4.23cw 49.01 � 3.08cx 27.17 � 4.02cy 10.99 � 1.07cz

Boiled 80C – 40 min 10.37 � 3.97dw 5.50 � 0.12dx 0.00 � 0.00dy 0.00 � 0.00dy

Boiled 100C – 35 min 16.56 � 4.11ew 0.00 � 0.00ex 0.00 � 0.00dx 0.00 � 0.00dx

Dried 12 h and boiled 80C – 30 min 13.73 � 1.09fw 10.34 � 0.89fx 0.00 � 0.00dy 0.00 � 0.00dy

Dried 12 h and boiled 100C – 25 min 13.87 � 1.69gw 6.01 � 1.29gx 0.00 � 0.00dy 0.00 � 0.00dy

Dried 24 h and boiled 80C – 30 min 22.11 � 2.98hw 10.61 � 1.68gx 0.00 � 0.00dy 0.00 � 0.00dy

Dried 24 h and boiled 100C – 20 min 15.54 � 1.75iw 11.02 � 4.87hx 0.00 � 0.00dy 0.00 � 0.00dy

Steamed – 45 min 93.23 � 2.51jw 93.04 � 2.51ix 82.45 � 4.25ey 77.12 � 4.45ez

Dried 12 h and steamed – 50 min 44.21 � 3.78kw 35.78 � 0.29jx 29.87 � 2.51fy 20.12 � 2.58fz

Dried 24 h and steamed – 50 min 44.12 � 2.53kw 36.15 � 1.25kx 28.78 � 1.88gy 19.70 � 1.80gz

Microwaved 450 W – 30 s 93.00 � 0.98lw 58.65 � 1.29lx 48.62 � 1.58hy 30.64 � 1.35hz

Microwaved 900 W – 20 s 94.40 � 1.58mw 64.09 � 2.26mx 53.07 � 5.98iy 31.28 � 3.78iz




H. elongata had strong activity in the first dilution(�95.81%) while extracts from the second dilution onwardwere significantly less effective with activity below 53.07%.

Antimicrobial Activity of ProcessedH. elongata Extracts Against P. aeruginosa

P. aeruginosa is a ubiquitous Gram-negative food spoilagebacterium with great adaptability and metabolic versatility.P. aeruginosa can attach onto a variety of surfaces and in avariety of niches including the food-processing environmentsby forming biofilms, which are more resistant to environmen-

tal stresses, host-mediated responses, sanitizing agents andantimicrobial agents (Bremer et al. 2001). Antimicrobialactivity of processed H. elongata extracts against P. aerugi-nosa are presented in Table 7. Similar to Gram-negativeS. abony, there was also no processed seaweed extract thatgave 100% inhibition against P. aeruginosa. Antimicrobialactivity of extracts from fresh H. elongata were strong in thefirst dilution with 96.39% inhibition and moderate in thesubsequent dilutions. Seaweed that had been dried for 12 and24 h achieved a maximum activity of 94.05%. Boiling led to asignificant reduction in antimicrobial activity of extractsagainst P. aeruginosa. Activity of steamed H. elongata extracts

TABLE 6. PERCENTAGE INHIBITION OF METHANOLIC EXTRACTS OF HIMANTHALIA ELONGATA PROCESSED UNDER DIFFERENT CONDITIONSAGAINST ENTEROCOCCUS FAECALIS


Fresh 100.00 � 0.00aw 99.61 � 0.66ax 87.03 � 1.41ay 78.02 � 0.98az

Dried 12 h 100.00 � 0.00aw 39.24 � 2.27bx 36.30 � 6.50by 33.89 � 5.10bz

Dried 24 h 95.40 � 0.72bw 53.01 � 3.08cx 42.28 � 0.08cy 29.96 � 1.38cz

Boiled 80C – 40 min 7.31 � 1.25cw 5.50 � 0.12dx 0.00 � 0.00dy 0.00 � 0.00dy

Boiled 100C – 35 min 10.52 � 2.54dw 0.00 � 0.00ex 0.00 � 0.00dx 0.00 � 0.00dx

Dried 12 h and boiled 80C – 30 min 49.45 � 1.26ew 10.34 � 0.89fx 0.00 � 0.00dy 0.00 � 0.00dy

Dried 12 h and boiled 100C – 25 min 22.11 � 4.32fw 6.01 � 1.29dx 0.00 � 0.00dy 0.00 � 0.00dy


Dried 24 h and boiled 100C – 20 min 33.72 � 3.92hw 11.02 � 4.87gx 0.00 � 0.00dy 0.00 � 0.00dy

Steamed – 45 min 95.21 � 1.29iw 93.54 � 2.51hx 81.47 � 1.58ey 76.42 � 2.57ez

Dried 12 h and steamed – 50 min 42.32 � 5.29jw 35.78 � 0.29ix 28.74 � 2.50fy 19.99 � 1.54fz

Dried 24 h and steamed – 50 min 44.57 � 0.98kw 36.15 � 1.25jx 29.87 � 1.75gy 18.78 � 1.24gz

Microwaved 450 W – 30 s 90.12 � 2.54lw 48.62 � 1.29kx 31.13 � 2.01hy 23.48 � 2.06hz

Microwaved 900 W – 20 s 95.81 � 4.58mw 53.07 � 2.26cx 43.61 � 3.39iy 24.42 � 5.14iz


TABLE 7. PERCENTAGE INHIBITION OF METHANOLIC EXTRACTS OF HIMANTHALIA ELONGATA PROCESSED UNDER DIFFERENT CONDITIONSAGAINST PSEUDOMONAS AERUGINOSA


Fresh 96.39 � 2.55aw 89.11 � 3.31ax 74.63 � 6.64ay 51.11 � 5.84az

Dried 12 h 72.80 � 5.22bw 48.77 � 5.26bx 17.71 � 3.02by 1.93 � 0.72bz

Dried 24 h 94.05 � 4.03cw 41.19 � 5.8cx 22.88 � 7.57cy 12.16 � 0.18cz

Boiled 80C – 40 min 6.45 � 2.00dw 5.46 � 1.23dx 0.00 � 0.00dy 0.00 � 0.00dy

Boiled 100C – 35 min 34.74 � 1.52ew 5.03 � 5.27ex 0.00 � 0.00dy 0.00 � 0.00dy

Dried 12 h and boiled 80C – 30 min 22.94 � 2.34fw 22.92 � 1.56fx 0.00 � 0.00dy 0.00 � 0.00dy


Dried 24 h and boiled 80C – 30 min 24.21 � 1.60hw 10.21 � 1.07hx 0.00 � 0.00dy 0.00 � 0.00dy

Dried 24 h and boiled 100C – 20 min 34.12 � 3.24iw 20.98 � 3.24ix 0.00 � 0.00dy 0.00 � 0.00dy

Steamed – 45 min 95.32 � 2.99jw 85.66 � 1.58jx 83.45 � 1.89ey 77.54 � 3.45ex

Dried 12 h and steamed – 50 min 44.14 � 4.54kw 34.21 � 0.98kx 27.89 � 2.14fy 19.78 � 2.54fz

Dried 24 h and steamed – 50 min 43.25 � 1.24lw 36.47 � 0.16lx 28.77 � 2.12gy 19.10 � 2.88gz

Microwaved 450 W – 30 s 93.44 � 5.48mw 70.25 � 7.30mx 46.73 � 3.56hy 33.16 � 2.31hz

Microwaved 900 W – 20 s 95.73 � 4.84nw 70.21 � 1.77mx 46.85 � 5.84iy 37.73 � 4.11iz

Each value is presented as mean � standard deviation (n = 6).Means within each column with different letters (a–n) differ significantly (P < 0.05).Means within each row with different letters (w–z) differ significantly (P < 0.05).



ranged from 95.32 to 77.54% in the first to fourth dilutions.Extracts from seaweed given a drying pretreatment followedby steaming had less than half the level of activity as com-pared with steaming alone. The antimicrobial activity ofmicrowaved extracts ranged from strong to weak (95.73–33.16%). In the present study, extracts from fresh seaweedshad strong antimicrobial activity at a concentration as low as3.93 mg/mL. In the majority of reports on antimicrobialactivities of seaweed extracts, bacterial growth-inhibitingactivities were investigated using standard agar disc diffusionassays (Bansemir et al. 2006; Kuda et al. 2007; Shan-mughapriya et al. 2008). There have been few quantitativereports on antimicrobial activity of seaweed extracts;however, from those available, the results of the present studyhave been shown to be more effective than reported byDubber and Harder (2008). These authors found extracts ofCeramium rebrum and Laminaria digitata had strong activityat 10 and 31 mg/mL respectively, which is less potent thanthose of fresh seaweeds in the present study.

CONCLUSION

The findings of the present study indicate that the method ofprocessing significantly influences the concentrations of phy-tochemicals, antioxidant and antimicrobial parameters inH. elongata. Consumption of H. elongata is dependant onsome heat treatment in order to achieve an edible texture.Because cooking invariably leads to a loss of antioxidantproperties, a compromise must be reached between palatabil-ity and nutrition. It was found that a combination of dryingfollowed by boiling reduced cooking time and led to lessleaching of phytochemicals. In terms of antioxidant activityof extract, a drying pretreatment followed by boilingenhanced the phenolic content per gram of extract and as aresult less amount of the extract would be required to have asignificant effect in food products. Processing significantlyaffects the antimicrobial activity of extracts from H. elongata.Extracts from fresh H. elongata had the highest antimicrobialactivity against L. monocytogenes, S. abony, E. faecalis andP. aeruginosa with good inhibition as low as 4.16 mg/mLextract. A better knowledge of how these processing condi-tions affects the phytochemical compounds of interest is ofpivotal importance. Reduction in the moisture content andcooking time also could have benefits in reducing transportand energy costs. Losses of health-related phytochemicals arethus likely to be a function of drying and cooking parameterssuch as time, temperature and degree of wounding stress tothe plant during these processes.

ACKNOWLEDGMENTS

The authors acknowledge funding from the Dublin Instituteof Technology under the ABBEST Programme. The authors

thank Denis Benson and Noel Grace from the Dublin Insti-tute of Technology for their technical support and Manus McGonagle for the supply of fresh seaweed.

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effect of processing conditions on phytochemical constituents of edible irish seaweed himanthalia...

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