Functional Ingredients from Algae for Foods and Nutraceuticals || Antimicrobial activity of compounds isolated from algae

© Woodhead Publishing Limited, 2013

8

Antimicrobial activity of compounds isolated from algae N. Abu-Ghannam and G. Rajauria , College of Sciences and Health, Dublin Institute of Technology, Ireland

DOI : 10.1533/9780857098689.2.287

Abstract : Micro and macro algae (seaweeds) represent a vastly under-utilized renewable resource characterized as being rich in a range of biological activities. In this chapter the focus will be primarily on antimicrobials extracted from seaweeds for possible applications in the food industry to enhance food safety and quality. The chapter initially outlines the effects of environmental and extraction conditions on infl uencing the effi cacy of seaweed antimicrobials and in this regard highlights the importance of standardization of the methodologies employed. It then describes various antimicrobial susceptibility tests and the effi cacy of seaweed extract on bacteria associated with food deterioration.

Key words : marine algae, seaweeds, antimicrobial activity, lipophilic extracts, thin layer chromatography (TLC)-bioautography.

8.1 Introduction

The industrial applications of seaweeds have until recently been focused on the farming of edible species, or on the production of agar, carrageenan and alginate. In particular, hydrocolloids attained considerable commercial sig-nifi cance due to their physical properties such as gelling, water-retention and emulsifi cation capacity. In recent years attention has shifted to the exploita-tion of potential biological activities or pharmacological properties of sea-weeds commonly referred to as secondary metabolites. This new approach for exploiting seaweeds is motivated by ecological observations and mechanisms to cope with environmental pressures (Haefner, 2003). The environment in which seaweeds grow is harsh since they are exposed to a combination of light and high oxygen concentrations, which can lead to the formation of

288 Functional ingredients from algae for foods and nutraceuticals


free radicals and other oxidizing agents. However, seaweeds seldom suffer any serious photodynamic damage during metabolism, implying the presence of some protective mechanisms and compounds (Matsukawa et al., 1997). The current thinking is towards exploitation of such properties for identifying new active compounds from seaweeds such as antimicrobials. Seaweed-based antimicrobials represent a vast untapped source for pharmaceutical, environ-mental and food applications, thus mitigating many of the side-effects often associated with synthetic antimicrobials (Newman et al. , 2003).

The synergistic effects of seaweed antimicrobial extracts in association with antibiotics can provide effective therapy against drug resistant bacteria, thus providing the pharmaceutical sector with novel products characterized by multiple mechanisms of action. Food-borne diseases and food spoilage have been also recognized as main challenges for the food industry in addition to consumer resistance and concern regarding the incorporation of synthetic antimicrobials in food. Therefore, the need for identifying new natural antimi-crobial agents is considered public health priority for the pharmaceutical and food industries (Newman et al. , 2003).

8.1.1 Potential antimicrobial compounds in seaweeds Seaweeds are exceptionally rich in a wide range of structurally diverse secondary metabolites including polyphenols, alkaloids, terpenes and stil-benes with many of these compounds being halogenated (Watson and Cruz-Rivera, 2003). Functions attributed to seaweed secondary metabo-lites include defence against herbivores, fouling organisms and pathogens in addition to protection from UV radiation and allelophatic agents. Chemical defence mechanisms that inhibit bio-fi lm development are a common occur-rence in seaweeds, with many secondary metabolites in seaweeds having bacteriocidal or bacteriostatic properties (Steinberg et al. , 1997). Harder (1917) was the pioneer in observing the antimicrobial potentials of sea-weeds. Secondary metabolites with potential antimicrobial activity in sea-weeds have been attributed to a wide range of compounds including fatty acids, halogenated compounds such as haloforms, halogenated alkanes and alkenes, alcohols, aldehydes, hydroquinones and ketones, carbonyls, sul-phur-containing heterocyclic compounds (sulphated polysaccharides) and phlorotannins (Lincoln et al. , 1991). Compounds such as sterols, hetero-cyclic and phenolic compounds, including quinones, fl avones, fl avonoids, fl avonols and tannins, have exhibited some antibiotic activity with possible exploitation as antiseptics and cleansing agents; however, their antibiotic activity in vivo can be achieved only at toxic concentrations (Lincoln et al. , 1991). Secondary metabolite production in seaweeds is a function of life history, with different stages having different physiological properties. In addition, the life cycle stage of the seaweed, its geographical location and seasonal variations have also been identifi ed as contributing factors.

Antimicrobial activity of compounds isolated from algae 289


8.1.2 Mechanism of algal antimicrobial action Although, terrestrial biodiversity has been well recognized as a source of anti-microbial agents for applications in the food and pharmaceutical industries, the ocean also has enormous biodiversity and potential for providing novel anti-microbial compounds with commercial value (Amaro et al. , 2011; Sultanbawa, 2011). Antimicrobial agents extracted from terrestrial plants affect microbial cells by various antimicrobial mechanisms, including attacking the phospho-lipid bilayer of the cell membrane, disrupting enzyme systems, compromis-ing the genetic material of bacteria and forming fatty acid hydroperoxidases through the oxygenation of unsaturated fatty acids (Proestos et al. , 2008). The literature acknowledges the antimicrobial potential of many algal extracts, but it is only recently that the implied phytochemicals were characterized. Chlorellin (a mixture of fatty acids), the fi rst antimicrobial compound isolated from Chlorella algae, was found to be effective against both Gram positive and negative bacteria (Pratt et al. , 1944). Most of the compounds responsible for the antimicrobial activity of seaweed are primarily phenolic and polysac-charide components, and their mechanism of action could be stasis (growth inhibition of microorganisms) or cidal (direct destruction of microorganisms) (Davidson and Naidu, 2000). It is suggested that the antimicrobial action of polyphenols may be due to their ability to alter microbial cell permeability, thereby permitting the loss of macromolecules from the interior or they could interfere with the membrane function and cause a loss of cellular integrity and eventual cell death (Bajpai et al. , 2008). Different phenolic compounds show different antimicrobial action which could be attributed to the number of phenolic rings and the substituent attached to the aromatic ring. Algal poly-phenols, also called phlorotannins, differ from terrestrial plant polyphenols because they contain an extremely heterogeneous group of molecules which provides them a wide range of potential biological activities (Glombitza and Keusgen, 1995). A number of studies have indicated that phlorotannins are the only phenolic group detected in seaweeds (Koivikko et al. , 2007). Compared to terrestrial sources, algal polyphenols have up to eight interconnected rings making them more potent microbial inhibitors than other polyphenols derived from terrestrial plants, which only have three to four rings (Hemat, 2007). Although the exact mechanism of algal polyphenol action is still unclear, it is expected that the interactions of phlorotannins with bacterial proteins may play a crucial role in the bactericidal action (Nagayama et al. , 2002). With respect to algal polysaccharides, their antimicrobial nature is attributed to the presence of glycoprotein- receptors in the cell-surface of bacteria which is capa-ble of recognizing and binding to the charged compounds associated with the polysaccharide molecules (Rostand and Esko 1997). Recently, the antimicro-bial activity of algal polysaccharides has been attributed to their distribution, molecular weight, charge density, sulphate content (in the case of sulphated polysaccharides) and most importantly their structural and conformation aspects (Amorim et al. , 2012). In the case of carotenoids, the antimicrobial



mechanism is not as clear as that of the polyphenols and polysaccharides and the proposed mechanism of action is that carotenoids could lead to the accumulation of lysozyme, an immune enzyme that digests bacterial cell walls, therefore generating the antimicrobial activity (Cucco et al. , 2007).

8.2 Factors affecting the efficacy of antimicrobial compounds extracted from seaweeds

Antimicrobial activity from natural resources is not usually attributed to a sin-gle compound but could be related to a combination of metabolites. Standard criteria for the evaluation of seaweed antimicrobial activity are lacking and in some cases there are signifi cant variations in the reported results which could be due to the infl uence of natural factors such as climate, salinity, reproduc-tive state, geographical location and seasonality in which the seaweeds grow. However, other factors such as choice of extraction method/solvents, antimi-crobial analysis methodologies and microorganisms tested also infl uence the antimicrobial effi cacy of seaweeds. Therefore, sampling throughout the year, standardization of extraction methods and antimicrobial testing techniques are essential to facilitate proper interpretation and comparison of results obtained worldwide.

8.2.1 Natural factors The concentration of secondary metabolites in seaweeds could vary due to changes in environmental conditions, mainly water temperature. Consequently, the effect of seasonal variations on the antimicrobial activity of seaweeds has been questioned. Due to global variations in ocean temperatures and the presence of thousands of species of seaweeds worldwide reacting differently to a range of environmental conditions including salinity, temperature and pollution, it is quite diffi cult to provide a generalized conclusion on the effects of seasonal variations on seaweed microbial activity, thus emphasizing the importance, when running a screening programme, to make multiple collec-tions refl ecting different seasonal times. A study by Stirk et al. (2007) reported that seasonal variations were observed for a number of seaweed species col-lected at Rocky Bay on the east coast of South Africa. Extracts generally had antibacterial activity in late winter and early spring collections but no activity in the summer collections. Similar fi ndings were reported by Tan et al. (2012) where antimicrobial activity from seaweeds collected in Ireland showed the lowest activity in spring/summer and the highest in autumn/winter. Trigui et al. (2012) reported that the biological properties of Ulva rigida collected over 12 months from Sidi Mansour Sfax (Tunis) revealed strong antimicrobial and antioxidant properties for samples collected during spring.

Similar to seasonality, the geographical location of seaweeds also infl u-ence their antimicrobial effi cacy. For instance, methanolic extracts of Ulva



lactuca collected from the coast of New York, USA and Morocco exhibited different antimicrobial activity against E. coli , suggesting the presence of dif-ferent bioactive compounds infl uenced by the geographical location effect (Tan et al. , 2012). Similarly, the life stage and maturity (age) of seaweeds are also contributing factors in altering the concentration or structure of sec-ondary metabolites and their associated biological activities. Paul (1992) sug-gested that Halimeda species of seaweed accumulate higher concentrations of defence chemicals in areas of new growth as compared to older parts. In a recent study by Chowdhury et al. (2011), the distribution of phlorotan-nins in mature thalli of E. cava was found to be 1.5-fold higher than young thalli. The previous study also reported that amounts of phlorotannins were higher in the blade tissue of the same algae than the stipe or holdfast. Thus, the variations in the activity of algal derived compounds could be attributed to different quantities and distributions of a single compound, or to the syn-thesis of a variety of compounds due to different environmental and growth conditions.

8.2.2 Processing factors Secondary metabolites are ubiquitous but not uniformly distributed within plants at the tissue, cellular or sub-cellular levels (Naczk and Shahidi, 2006). These metabolites are present in matrices as a complex mixture of compounds that provide a cocktail of many active components present either free or in the bound states as conjugates with sugars, fatty acids or proteins (Shahidi and Naczk, 1995). These complexes could be quite insoluble, their solubil-ity being affected by the chemical nature of the plant sample in addition to the polarity of solvent(s) used in the extraction procedures. Therefore, the extraction of these active ingredients from plant matrices is the key step in utilizing them for food or pharmaceutical applications. Thus, it is of critical importance to select an effi cient extraction procedure/method to maintain the stability of the compounds of interest. This usually involves the use of selec-tive solvents and the application of the appropriate extraction technology. However, the quality of the extract could be affected by a number of other variables such as the seaweed species, particle size, polarity of the extractant and the extraction procedure.

Extraction method In recent years, a number of novel extraction techniques such as microwave or ultrasound-assisted extractions, subcritical water extraction, supercriti-cal or pressurized fl uid extraction and accelerated solvent extraction have been applied for the extraction of active ingredients from seaweed materi-als (Herrero et al. , 2006; Ibáñez et al. , 2012). Mendiola et al. (2008) have extracted antimicrobial compounds from the green alga Dunaliella salina using supercritical fl uid extraction. Similarly, Santoyo et al. (2009) extracted



antimicrobial compounds from Haematococcus pluvialis alga using pressur-ized liquid extraction. Recently, Klejdus et al. (2010) developed a new hyphen-ated technique (involving sample pretreatment using sonication, followed by supercritical fl uid extraction) for the extraction and determination of fl a-vonoids from various seaweeds. Despite the development of various modern techniques, solid-liquid extraction (or leaching) is the oldest and most com-monly used procedure to prepare extracts from seaweed materials.

Choice of solvent The biological activity of extracted compounds from plant-based resources depends signifi cantly on the type of solvent used in the extraction procedure. Typical properties of good solvents include low toxicity so that any remaining residues will not interfere with the bioassay, ease of evaporation at low tem-perature, promotion of a rapid physiologic absorption of the extract, preserva-tive action and inability to cause the extract to complex or dissociate (Hughes, 2002). In general, most antimicrobial active compounds are not water soluble and thus organic solvent extracts have been found to be more potent. The most commonly used solvents in the extraction of antimicrobials are methanol, ethanol, acetone, diethyl ether, chloroform, hexane ethyl acetate and water. The application of serial exhaustive extractions involving successive extraction with solvents of increasing polarity from a non-polar (e.g. hexane) to a more polar solvent (e.g. methanol or water) in order to ensure that a wide polarity range of compounds could be extracted (Green, 2004) has been successfully applied to terrestrial plants, and it is worth being investigated in the case of seaweed extracts. During extraction, solvents diffuse into the seaweed material and solubilize material with similar polarity (Green, 2004).

The choice of solvent has also an important impact on the extraction yield which directly infl uences the antimicrobial effect. The secondary metabolites present in seaweeds are not a qualitative or quantitative absolute, but vary according to the extraction solvent used. In a reported study by Rajauria et al. (2012), extraction using 60% aqueous methanolic extract for Himanthalia elongata produced a much higher yield (6.8%) as compared to 100% meth-anolic extract (1.2%). Herrero et al. (2005) also suggested that the polarity of solvents signifi cantly affects the yield of Spirulina microalgae wherein the highest yield was recorded with ethanol as compared with other higher and lower polarity solvents. Similarly, Santoyo et al. (2009) considered ethanol to be the best solvent for the extraction of antimicrobial compounds from Haematococcus pluvialis algae. Rosell and Srivastava (1987) investigated the antimicrobial activity of kelp from British Columbia. Several solvents were used for the extraction process including acetone, chloroform, diethyl ether, ethyl acetate, methanol and water. The effi ciency in extracting antimicrobial compounds using organic solvents was comparable; however, water extraction was less effi cient. The effect of different lengths of time on the extraction rate at room temperature did not signifi cantly improve the antimicrobial activity. Using gas-liquid chromatography (GLC) either alone or in combination with



mass spectrometry suggested that the algal compounds responsible for anti-microbial activity are organic with a lipophilic nature.

Extraction process In addition to the importance of selecting appropriate solvents and extraction procedures, the recovery of phytochemicals from seaweed materials is also infl uenced by other factors such as the length of the extraction period, tem-perature, pressure, pH, solvent-to-sample ratio, particle size of the seaweed tissue, sample preparation and related factors. For instance, longer extrac-tion times increase the chance of oxidation of phenolics causing a decrease in the extracts yielded (Naczk and Shahidi, 2006). However, an increase in the extraction temperature and decrease in the viscosity and the surface ten-sion of the extraction solvents helps the solvents to reach the sample matri-ces and promote extraction rate by increasing both mass transfer rate and solubility of the analyte (Naczk and Shahidi, 2006). Herrero et al. (2006) and Rodríguez-Meizoso et al. (2010) reported that a much higher extraction yield from algae was obtained when using pressurized liquid extraction within the temperature range of 100–200°C; however, the higher yield obtained did not have a signifi cant infl uence on the antimicrobial activity. Similar to tempera-ture, the extraction pressure also shows an impact on yield and the biological activity of the extract. In Hypnea charoides , high pressure enhanced the con-tent of lipid and unsaturated fatty acids and ultimately improved the overall extraction yields (Cheung, 1999).

The type of sample (fresh, frozen or dried), preparation or preprocess-ing is also a major factor affecting extraction conditions. Prior to extraction, samples are generally treated by milling, grinding and homogenization, which may be preceded by air-drying or freeze-drying (Abascal et al. , 2005). These preprocessing conditions not only affect the content of phytochemicals but may also cause some undesirable effects on the constituent profi les of plant samples. In a recent study, E. cava seaweed was washed using tap water fol-lowed by drying, using either sun-drying, shadow-drying, oven-drying or lyo-philization prior to submission to extraction procedures. The results indicate that pretreatment methods such as drying signifi cantly change the content of phlorotannins in the crude extract as the highest and the lowest amounts of crude phlorotannin were recorded from natural non-washed lyophilized and sun-dried tissues, respectively. Also the study reported that non-washed seaweeds have higher phlorotannin content than tap water-washed seaweeds (Chowdhury et al. , 2011; Gupta et al. , 2010).

Grinding the seaweed material to fi ner particles with the aid of liquid nitro-gen increases the surface area for extraction, thereby increasing the rate of extraction, and also shortens the extraction period. Crushing the raw mate-rial improved the carotenoid extraction in Spirulina plantesis algae (Mendiola et al. , 2008). Shaking the plant material-solvent mixture also increases the extraction rate (Eloff, 1998). In terrestrial plant systems, Nostro et al. (2000) reported that acidifi ed aqueous environments promoted easier extraction.



It is important to note that there is a wide diversifi cation in the usage of solvents for antimicrobial extraction from seaweeds, and there is a need for standardizing extraction methods and solvent systems to minimize the vari-ability in the antimicrobial effi cacy reported particularly for comparison purposes.

8.3 Antimicrobial susceptibility testing

Antimicrobial susceptibility test (AST) is used to determine the effi cacy of potential antimicrobials from biological extracts against a number of differ-ent microbial species. With the increase in natural compounds characterized by antimicrobial activity, there is always a need for new bioassay techniques with high levels of sensitivity to detect small amounts of biologically active chemicals (Lampinen, 2005). The overall essential criteria for susceptibility tests include ease of use, reproducibility, sensitivity and specifi city (Struelens et al. , 1995). Standardization to minimize the signifi cant infl uence of differ-ent in vitro tests is essential when screening extracts of natural origin. In the case of crude extracts, results obtained from antimicrobial effi cacy are often diffi cult to compare with published results due to the infl uence of different antibacterial tests employed and microorganisms tested, in addition to other natural and physical variables associated with seaweeds.

AST standard methods can be broadly categorized into diffusion methods (including agar well or disc diffusion and bioautography tests) and dilution methods (including agar dilution and broth micro- or macro-dilution tests) (Rios et al. , 1988). Both diffusion and bioautography are qualitative measure-ments that indicate the presence or absence of antimicrobial agents, while the broth dilution method is a quantitative assay used for the assessment of the minimum inhibitory concentration (MIC) (Rios et al. , 1988).

The diffusion methods are simple and practical and are the most com-monly used methods for antimicrobial screening as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). These methods are widely used to screen seaweed extracts for antimicrobial activ-ity (Rajauria et al. , 2012; Trigui et al. , 2012; Pierre et al. , 2011; Vijayabaskar and Shiyamala, 2011). However, diffusion techniques are suitable for prelim-inary screening and ideally can only be used for antimicrobial susceptibil-ity testing of pure substances rather than mixtures containing constituents which tend to exhibit different diffusion rates thus causing possible unreli-ability of the obtained results (Silva et al. , 2005). On the other hand, bio-autography provides a more comprehensive investigation of the extract through a combination of sub-fractionation coupled with visualization of antibacterial effi cacy of the fractions. In this method the analyte is adsorbed onto a thin layer chromatography (TLC) plate thus providing a simplifi ed approach for the isolation of possible antimicrobial components from crude



extracts by using very small amounts of the extract and additionally allow-ing the determination of the polarity of the active compounds. Both sepa-ration and antimicrobial activity detection of separated compounds are performed on the same TLC plate, by spraying the developed plate, usually with tetrazolium salts (Silva et al. , 2005). Tan et al. (2012) applied the bio-autography method to fractionate the extract obtained from the macroalga U. lactuca and reported that it was a more appropriate assay compared to the disc diffusion method.

The broth dilution or the microtiter plate method is a robust and useful method in providing a quantitative assessment of MIC for a large number of test samples. MIC is important in diagnostic laboratories to confi rm resis-tance of microorganisms to an antimicrobial agent and to monitor the activ-ity of new antimicrobial agents. Gupta et al. (2010) applied the microtiter plate method to investigate the antimicrobial activity of three brown Irish edible seaweeds, Himanthalia elongta, Laminaria saccharina and Laminaria digitata, against a range of food pathogenic and spoilage bacteria. Similarly, Rajauria et al. (2012) screened the antimicrobial effi cacy of H. elongta extracts using the disc diffusion and broth dilution methods against a num-ber of food grade bacteria. A series of quantitative analyses with respect to percentage inhibition of the most effective concentration for a range of seaweed extracts were obtained using the microtiter method which allowed for a more comprehensive and accurate comparison of antimicrobial effi cacy between the different seaweed species extracts with respect to the studied microorganisms.

8.4 Efficacy of hydrophilic and lipophilic extracts on bacteria associated with food safety and quality

The incorporation of seaweed antimicrobials in perishable food products, such as refrigerated ready-to-eat foods, could open up opportunities for extending food shelf-life, reducing or eliminating pathogenic bacteria and increasing the overall acceptability of processed food by substituting synthetic with natural antimicrobial agents.

Using the microtiter plate method, Cox et al. (2010) evaluated the anti-microbial activity of six edible species of Irish seaweeds: L. digitata, L. sac-charina , H. elongata , Palmaria palmata , Chondrus crispus and Enteromorpha spirulina . In this reported study, the methanolic extracts, using in vitro test-ing, inhibited the growth of typical food spoilage and pathogenic bacteria tested including: Listeria monocytogenes , Salmonella abony , Enterococcus faecalis and Pseudomonas aeruginosa . While red and green seaweeds showed a signifi cant reduction in microbial growth, the brown species had the higher antimicrobial activity with up to 100% inhibition in most cases. It is suggested that phlorotannins are responsible for this observed activity. In contrast to



terrestrial tannins, phlorotannins are tannin compounds which have been found only in marine algae with a unique molecular skeleton composed of up to eight interconnected rings while terrestrial tannins have only three to four rings (Hemat, 2007). In the study by Cox et al. (2010), there was a signifi cant increase ( p < 0.05) in the activity of green and red seaweeds when ethanol and acetone were used as extraction solvents suggesting other possible antimicro-bial components of lipophilic nature being extracted such as fucoxanthin, which is one of the most abundant carotenoids present in seaweeds. Table 8.1 provides a summary of studies on a range of algal extracts tested in vitro against food pathogenic and spoilage bacteria.

The antimicrobial activity of the lipophilic components of brown sea-weed crude extracts was further investigated. A range of concentrations (7.5, 15, 30 mg/mL) of crude extract corresponding to (0.75%, 1.5%, 3%) of lipophilic extract were obtained using a mix of solvents (n-hexane, diethyl ether and chloroform) and were tested for their antibacterial activity against selected microorganisms associated with food safety and quality, as seen in Fig. 8.1. Irrespective of the type of microorganism tested, all seaweed extracts exhibited a signifi cantly higher antibacterial activity in the range of 90.45–98.03% at the 3% lipophilic extract concentration in comparison with synthetic antimicrobial agents such as sodium benzoate and sodium nitrite. Among all the seaweeds studied, the 3% lipophilic extract as obtained from H. elongata was the most potent against the tested microorganisms. At the same concentration, extracts from L. saccharina were more effective against L. monocytogenes and P. aeruginosa (Gram positive) whereas extracts of L. digitata were more effi cient against S. abony and E. faecalis (Gram negative).

The antimicrobial activity for lipophilic and hydrophilic (60% methanol) extracts alongside those for sodium benzoate and sodium nitrite are illus-trated in Fig. 8.2 for comparison purposes. At the concentration of 3.0%, all the extracts showed signifi cantly higher ( p < 0.05 ) antimicrobial activity com-pared to the synthetic antimicrobials examined. The potency of the lipophilic extract was such that at a concentration of 0.75% its antimicrobial activity was comparable to the synthetic reference compounds as seen in Fig. 8.2.

The antimicrobial activity exhibited by 3.0% concentration of lipophilic extract was similar to or higher than the activity showed by 6.0% concentra-tion of the hydrophilic extract. In the case of L. saccharina , at the concentra-tion of 3.0%, the lipophilic extract exhibited 90.5% ( E. faecalis ), 93.7% ( S. abony ), 97.4% ( L. monocytogenes ) and 95.9% ( P. aeruginosa ) inhibition which was 1.5, 1.2, 1.3 and 1.2-fold higher than the activity showed by the hydro-philic extract at 6%, respectively. Similarly, in the case of L. digitata , the lipo-philic extract exhibited 90.7% and 93.0% inhibition against P. aeruginosa and E. faecalis which was 2.0-fold and 1.6-fold higher than the activity showed by the hydrophilic extract, at the same concentration of extract, respectively, as seen in Fig. 8.2.

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Table 8.1 Algal extracts tested in vitro against food pathogenic and spoilage bacteria

Algal species Extract Bacterial species Methods Active compounds References

Ulva lactuca n-Hexane chloroform ethyl acetate

dichloromethane acetone ethanol methanol water

Staphylococcus aureus Listeria innocua Bacillus subtilis Enterococcus faecalis

Cronobacter sakazakii Pseudomonas aeruginosa Escherichia coli Salmonella typhimurium

Disc diffusion TLC-

bioautography

Semi-purifi ed fractions

Tan et al . , 2012

Himanthalia elongata 0, 20, 40, 60, 80 and 100% methanol in water

Pseudomonas aeruginosa Listeria monocytogenes Enterococcus faecalis Salmonella abony

Broth micro-dilution

Disc diffusion

Polyphenols Rajauria et al ., 2012

Ulva rigida 80% ethanol–water methanol

Bacillus subtilis Staphylococcus aureus

Escherichia coli Pseudomonas aeruginosa Listeria monocytogenes Enterococcus faecalis

Agar well diffusion

Polyphenols Trigui et al . , 2012

Gracilaria ornata Water Bacillus subtilis Staphylococcus aureus Enterobacter aerogens Escherichia coli Pseudomonas aeruginosa Salmonela choleraesuis Salmonela typhi

Disc diffusion Sulphated polysaccharide

Amorim et al . , 2012

(Continued)

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Sargassum wightii Turbinaria ornata

Methanol Bacillus subtilis, Escherichia coli Enterococcus faecalis,

Pseudomonas aeruginosa, Aeromonas hydrophila,

Proteus vulgaris, Klebsiella pneumoniae, Shigella fl exneri

Staphylococcus aureus

Well diffusion Polyphenols Vijayabaskar and Shiyamala, 2011

Chaetomorpha aerea Chloroform/ hexane Staphylococcus aureus Salmonella enteretidis P. aeruginosa Enterococcus faecalis Bacillus subtilis Micrococcus luteus

Disc diffusion Sulphated galactan Pierre et al . , 2011

Kappaphycus alvarezii Padina boergessenii

Diethyl ether methanol cetylpyridinium

chloride

Escherichia coli (12 antibiotic resistant

strain)


Sulphated polysaccharides and polyphenols

Kumaran et al . , 2010

Ulva fasciata CH2Cl2/CH3OH Vibrio parahaemolyticus Vibrio alginolyticus Vibrio vulnifi cus

Disc diffusion Labdane diterpenoids

Chakraborty et al . , 2010

Himanthalia elongata Laminaria saccharina Laminaria digitata Enteromorpha spirulina Chondrus crispas palmaria palmata

60% methanol in water

60% ethanol in water

60% acetone in water



Polyphenols Cox et al . , (2010)

Table 8.1 Continued

Algal species Extract Bacterial species Methods Active compounds References

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Himanthalia elongata Laminaria saccharina Laminaria digitata

60% methanol in water



Polyphenols Gupta et al . , 2010

Haematococcus pluvialis Hexane and ethanol Staphylococcus aureus Escherichia coli


Fatty acids, alkanes, polyphenols

Santoyo et al . , 2009

Odonthalia corymbifera Staphylococcus aureus Bacillus subtilis Micrococcus luteus Proteus vulgaris Salmonella typhimurium Escherichia coli


Bromophenols Oh et al . , 2008

Gelidiella acerosa Gracilaria edulis

Turbinaria conoides Padina gymnospora

Chondrococcus hornemanni

Hypnea pannosa Dictyota dichotoma Jania rubens Sargassum wightii Haligra sps.

Methanol Staphylococcusaureus Bacillus cereus Vibrio vulnifi cans Salmonella typhi Listeria monocytogenes Enterococcus faecalis E.coli

Disc diffusion Polyphenols Devi et al . , 2008

Sargassum sp. methanol Staphylococcus aureus Escherichia coli Bacillus subtilis

Disc diffusion Unknown Patra et al . , 2008

Dunaliella salina SFE with CO2 Escherichia coli Staphylococcus aureus Candida albicans Aspergillus niger

Indolic compounds PUFAs b-ionone neophytadiene

Mendiola et al . 2008



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Fig. 8.2 Antimicrobial activity of crude hydrophilic and lipophilic extract of H. elongata seaweeds against food spoilage and pathogenic bacteria (LM: Listeria mono-cytogenes; SA: Salmonella abony; PA: Pseudomonas aeruginosa; EF: Enterococcus

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Sodium benzoateSodium nitrite

Fig. 8.1 Antimicrobial activity of crude lipophilic extract of brown seaweeds against food spoilage and pathogenic bacteria.



Generally, the natural antimicrobial compounds that have been identifi ed are water insoluble and the extracts from organic solvents have been found to give more consistent antimicrobial activity compared to aqueous extracts (Parekh et al. , 2005). Thus, a combination of medium to low polarity solvents would be more selective, compared to the aqueous methanol, in order to extract more potent antimicrobial compounds from seaweed (Tan et al. , 2012).

8.5 Screening and purification of antimicrobial crude seaweed extracts using thin layer chromatography (TLC)-bioautography

Screening of algal extracts for their potential antimicrobial activity dates back to 1917, where a range of green, red and brown seaweeds were examined against both Gram positive and Gram negative bacteria including pathogenic and non-pathogenic strains (Harder, 1917; Pratt et al. , 1944; Rajauria et al. , 2012). So far, many of the reported studies on the antimicrobial properties of seaweed extracts have focused on utilizing the crude extract, which means that the minimum MIC tends to be quite high (about 60 mg/mL), as reported by Gupta et al. (2010), thus placing seaweed extracts at a disadvantage when compared to commercial antimicrobials where signifi cantly lower concentrations are used. This highlights the critical importance of identifying relevant techniques and assays to purify the extracts to produce more potent compounds with effi cacies comparable to established antimicrobial agents.

Following on from the previous section, the lipophilic extract from H. elongata was further purifi ed and tested on L. monocytogenes due to its high potency towards this bacterium, as seen in Fig. 8.1. The compounds in the lipophilic extract were separated on a TLC plate and the antimicrobial effi cacy of each separated band was screened using TLC-bioautography. The most active band was purifi ed using preparative TLC and the compound of interest was scratched from the TLC plate. The purifi ed compound was characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Furthermore, the antimicrobial activity of the same purifi ed scratched compound was tested in vitro using conventional disc diffusion bioassay against L. monocytogenes and com-pared with the activity of the crude extract (methanolic extract) and the commercial fucoxanthin compound. The same amount (25 μ L) though using a different concentration of the crude extract (10 mg/mL; 250 μ g per disc) and the purifi ed compound and fucoxanthin standard (1 mg/mL; 25 μ g/disc) were impregnated on a paper disc, as illustrated in Fig. 8.3. Results indi-cated that despite the application of a very low amount of the purifi ed com-pound (10 times less than the crude extract) on the paper disc, the inhibition zone was statistically ( p < 0.05) bigger in the case of the purifi ed compound (10.72 mm) than the crude extract (9.95 mm). In addition it was observed



that both the purifi ed compound and fucoxanthin standard exhibited similar antimicrobial activity against the tested bacteria. These fi ndings suggest that the activity shown by the purifi ed compound against L. monocytogenes was almost 10 times higher than the crude extract, advocating the role of purifi -cation in enhancing the overall biological activity. Recently, Tan et al. (2012) observed similar fi ndings for U. lactuca seaweed where the crude extract was purifi ed by preparative TLC and column chromatography. The crude extract and the collected fractions/sub-fractions were tested against Staphylococcus aureus , Bacillus subtilis and methicillin-resistant S. aureus using TLC bio-autography indicating that very small amounts of the purifi ed sub-fractions (6–10 μ g) were enough to inhibit the growth of the tested microorganisms when compared to the crude extract of 300 μ g. Previously, the crude extracts and the purifi ed fractions of E. kurome brown algae were also tested against a number of Gram positive and Gram negative bacteria and showed that the purifi ed fractions exhibited much higher bactericidal activity than the crude extracts (Nagayama et al. , 2002).

Due to the non-specifi c nature of the extraction methods and/or solvents, crude extracts are always a mixture of some unwanted substances such as sugars, organic acids, chlorophyll pigments and other cell wall components. Additionally, extracted compounds may also exist as complexes with carbohy-drates, proteins and other plant components, resulting in possible interference with the bioactivity of the targeted compounds (Shahidi and Naczk, 1995). In most cases, the purifi cation step brings the target analytes to a concentration level more suitable for separation and detection. Therefore, optimization of

Fig. 8.3 Antimicrobial activity analysis of reference compound, purifi ed com-pound and crude lipophilic extract of H. elongata seaweed, against L. monocytogenes bacteria. (fucoxanthin standard (1a and 1b); purifi ed compound (2a and 2b); crude

extract (3a and 3b).)



appropriate purifi cation techniques not only enhances the process of isolation and identifi cation but also improves the biological activity associated with the analytes.

8.6 Conclusions

Marine algae offer a substantial opportunity as sources of antimicrobial agents; however, optimization of extraction and purifi cation methodolo-gies is essential in order to capture their real antimicrobial activity. Crude extracts are always a mixture of some unwanted substances such as sugars, organic acids and cell wall components which interfere with the bioactivity of the targeted compounds. In this chapter an application example of the antimicrobial activity of seaweeds obtained using different extraction sol-vents was tested against typical microorganisms that compromise the shelf-life and safety of food products, in an attempt to establish the antimicrobial potential of seaweeds in comparison to synthetic antimicrobials. The out-come indicated a comparable or even stronger effi cacy, particularly when using lipophilic extracts. The next step would be the incorporation of such extracts in real food systems under typical processing and storage condi-tions in order to assess whether similar effi cacies could be sustained.

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Functional Ingredients from Algae for Foods and Nutraceuticals || Antimicrobial activity of compounds isolated from algae