Investigation of Anti-bacterial Activity against Food-borne Pathogens among Korean Domestic Algae

The aim of this study is to explore algal species with anti-bacterial activity against six food-borne pathogens. Among 51 marine algae, Laurencia okamurae Yamada and Dictyopteris undulata Holmes was elucidated to have a potent anti-bacterial activity against food-borne pathogens. Laurencia okamurae Yamada showed the clear zone around agar well on B. cereus, S. aureusand L. monocytogenes-spreading agar plate. Dictyopteris undulata Holmes had the anti-bacterial activity against S. chorelaesuis, B. cereus, S. aureus and L. monocytogenes on bacterial spreading agar plates. Antibacterial activity of L. okamurae Yamada and D. undulata Holmes had specifically susceptibility for B. cereus, S. aureus and L. monocytogenes and were superior to streptomycin, the authentic antibiotics. It is anticipated that new food preservatives can be explored and developed on the basis of this study.


INTRODUCTION
Foodborne diseases are still a major concern in the world.In 2005, it was reported that 1.8 million people died from diarrhoeal diseases, largely attributable to contaminated food and drinking water by World Health Organization (WHO).This is not just an underdeveloped world problem.About 76 million cases of food-borne diseases, resulting in 325,000 hospitalizations and 5000 deaths, are estimated to occur each year in the United States of America (USA) alone (Mead et al., 1999).According to the reports regarding foodborne microorganisms outbreak, Salmonella enterica, Staphylococcus aureus, Bacillus cereus and Clostridium botulinum were found in eggs, beef, chickenand dairy products in the U.S.A. and the European Union (Tauxe, 2002;Greig and Ravel, 2009).
Due to the worldwide awareness on chemical preservatives, the food industry is now reflected by the consumer opinions for safer additives and thus is focusing on natural safe preservatives (Dillon and Board, 1994).Some plant materials are used as natural antimicrobials in food systems, as well as to prevent the growth of foodborne bacteria and molds resulting in shelf-life extension of processed foods.Fruits, vegetables, grains and food constituents can be contaminated by various microorganisms and their hazardous toxic metabolites.Enterotoxins produced by Escherichia coli, S. pyogenes, Salmonella, Yersinia and Clostridium species are responsible for toxicity in the intestinal tract causing vomiting, diarrhea, etc.Moreover, microorganisms are also associated with food spoilage causing economical loss (Harris, 1988;Rocourt et al., 2003).Although most healthy humans are not significantly affected by low doses of the bacteria, the pathogen can be more harmful for people with weak immune systems or during pregnancy.Among severe infections, even listeriosis by Listeria sp., has been associated with high mortality rate (Datta, 2003).
In this report, we focused on the studies for the investigation of anti-bacterial activities of Korean domestic algae against foodborne pathogens, which were highly issued recently in South Korea.It is anticipated that new food preservatives can be explored and developed on the basis of this study.

MATERIALS AND METHODS
Sea weeds materials: All sea weed was collected in sea near Jeju island Korea, authenticated by Jeju Biodiversity Research Institute (JBRI), Jeju, Korea and deposited as the voucher specimen (Table 1) to JBRI.
Each sea weed was washed, dried by freeze drier and crushed.Crushed weeds were extracted with 70% methanol (in water) at room temperature.Extracts were filtered through Whatman No. 1 filter paper and were concentrated by evaporator under reduced pressure.Sea weed-extract was redissolved in DMSO to 100 mg/mL, stored at -20°C and used it as a stock.

Agar-well diffusion assay:
The antimicrobial activities of sea weeds extracts were evaluated by agar-well diffusion assay.The assay was carried out according to the method (Owen and Palombo, 2007) of with some modifications.After 50 mL of each bacterial suspension (OD600 = 0.2-0.3,approximately 1×10 7 cfu/mL) were spread uniformly onto the surface of each agar plate (1.5%), the well was cut into the set agar using a sterile cork-borer (5 mm diameter).Sea weeds extracts (10 µg/well) were dispensed into the wells.Agar plates were left at room temperature for 30 min to allow the liquid to diffuse into the agar before overnight incubation at 37°C.All assays were carried out in triplicate.A clear zone of the inhibited microbial growth surrounded substances was considered positive exhibiting antibacterial properties.The solvent system used for the preparation of sea weeds extract samples was used for blank well.After incubation, the inhibition zones around wells were measured in mm using a caliper (Fazeli et al., 2007).Ampicillin and streptomycin (10 µg/well) were used as positive controls.
Statistical assay: All result was expressed as mean±Standard Deviation (S.D.) and were analyzed using one way Analysis of Variance (ANOVA) and Dunnett's multiple comparison test for individual comparisons.Results were considered statistically significant when p-values were p<0.05.

Anti-bacterial activity of marine algal extracts against food-borne pathogens:
The huge number of marine algal species inhabit in the coast of Jeju-island, Korea (Lee, 2008).Fifty one algae collected from seashore of Jeju-island were investigated for antibacterial activity against food-borne pathogens.The aim of this study is to explore algal species with antibacterial activity against six food-borne pathogens issued in Korea recently.Antibacterial activity of marine algae was determined by agar well diffusion assay.Control treatment (DMSO) showed no clear zone around agar-wells against any of pathogens (Fig. 1).
Among 51 marine algae, Laurencia okamurae Yamada and Dictyopteris undulata Holmes was elucidated to have a potent anti-bacterial activity against food-borne pathogens (Fig. 1 and Table 1).L. monocytogenes and were superior to streptomycin, the authentic antibiotics.However, streptomycin, which is a broad-spectrum antibiotics, showed the clear zone for all tested food-borne pathogens.
Great attention has been focusing on the marine seaweeds as potential and promising sources of bioactive compounds in recent years.Seaweeds are able to produce a great variety of secondary metabolites with versatile activities, such as antiviral, anthelmintic, antifungal and antibacterial activities (Lindequist and Schweder, 2001;Newman et al., 2003).Red algal genus Laurencia (Rhodomelaceae, Ceramiales) produces the numerous specialized secondary metabolites with diverse structural features.Its three major classes, which are sesquiterpenes, diterpenes and acetylenes, have not yet been encountered in other terrestrial organisms (Vairappan, 2003;Erikson, 1983;Fenical, 1975).Most of these metabolites are characterized by the presence of halogen atoms in their chemical formula.Bromine-containing compounds are more abundant than either chlorine-or iodinecontaining ones.Halogenated compounds and metabolites from Laurencia sp., have been investigated to be due to antimicrobial, insecticidal, cytotoxic and feeding-deterrent activities (Konig and Wright, 1997;Hay et al., 1987).
Due to increases in bacterial resistance against commercial antibiotics, there is a growing need, not only in human but also in veterinary medicine, for new antibacterial compounds that are active against pathogenic bacteria.It is reasonable to suggest some of these compounds as seaweed's defense chemicals to protect themselves against pathogens and survive in the marine ecosystem to potential anti-bacterial agents.It was reported that metabolites isolated from Malaysian Laurencia majuscula could inhibit its surface bacteria, suggesting the presence of an inherently available antibacterial defense mechanism (Vairappan et al., 2001).Some halogenated metabolites from Laurencia have been shown to have antibacterial activity against terrestrial bacteria (Vairappan et al., 2001), thus suggesting that halogenated compounds have the potential to function as chemical defense substances against pathogenic bacteria.Isolated metabolites including halogenated metabolites, such as 10acetoxyangasiol, aplysiadiol, cupalaurenol, 10acetoxyangasiol, aplysidiol, cupalaurenol and chamigrane epoxide, exhibited potent antibacterial activities against clinical bacteria, Staphylococcus aureus, Staphylococcus sp., Streptococcus pyogenes, Salmonella sp. and Vibrio cholerae.To date, there has been minimal research regarding the anti-bacterial activity against food-borne pathogens.
Brown algae, Phaeophyceae, are a class of almost exclusively marine organisms that have been explored for the bioactivity potential of its metabolic products, especially those of the representative family Dictyotaceae.Dictyotaceae natural products include a rich production of terpenoids of different origins and are therefore important in studies of metabolites of marine origin (Vallim et al., 2010).The most bioactive products have three or four isoprenoid units, corresponding to the sesquiterpenes and diterpenes, respectively.These two classes of terpenoids can have many different structures and variants, including the addition of halogen atoms or fragments from other biogenic pathways.Halogenated sesquiterpenes in which a phloroglucinol unit were reported in Dictyopteris divaricata (Ji et al., 2009) and D. undulata (Song et al., 2004a).The sesquiterpenes of these genera are known to exhibit activity as an inhibitor of herbivores and antifungal, cytotoxic, antibiotic and ichthyotoxic activities.In addition, the cytotoxic activities were evaluated in several human tumor cell lines for sesquiterpenes from Dictyopteris (Fenical et al., 1973) and also the antifungal activity was found only for sesquiterpenes from Dictyopteris.Therefore a polysaccharide-rich extracts from Dictyopteris sp., are expected to exhibit assorted biological activities, including anticoagulant, antiproliferative and antioxidant activities.

CONCLUSION
This result is able to provide a fundamental data of screening for marine algae with anti-bacterial activity against food-borne pathogens.Laurencia okamurae Yamada and Dictyopteris undulata Holmes was investigated to have a potent anti-bacterial activity against food-borne pathogens.And it is expected to be possible to develop a novel and natural food preservative using extracts of Laurencia okamurae Yamada and Dictyopteris undulata Holmes.

Fig. 1 :
Fig. 1: Anti-bacterial activities of Laurencia okamurae Yamada and Dictyopteris undulata Holmes against food-borne pathogens.After 50 µL of B. cereus, L. monocytogenes and S. aureus culture suspension (OD600 = 0.2-0.3,approximately 1×10 7 cfu/mL) were spread uniformly onto the surface of each agar plate (1.5%), the well was cut into the set agar using a sterile cork-borer (5 mm diameter).The Solvent system (DMSO) used for the preparation of extract samples was used for control.Extracts (10 µg/well) of Laurencia okamurae Yamada and Dictyopteris undulata Holmes were dispensed into the wells.After 24 h incubation, the inhibition zones around wells were measured Laurencia okamurae Yamada showed the inhibition zone of approximately 4.0±0.2mm around agar well on B. cereus-spreading agar plate.In addition, L. okamurae Yamada had antibacterial activities against S. aureus and L. monocytogenes with approximately 3.0±0.3and 2.6±0.5 mm, respectively.Another marine algae, Dictyopteris undulata Holmes had the antibacterial activity against S. chorelaesuis approximately 0.5±0.1 mm, B. cereus with 5.0±0.2mm, S. aureus with approximately 4.0±0.3mm and L. monocytogenes with approximately 9.0±1.0mm clear zones on bacterial spreading agar plates.Antibacterial activity of L. okamurae Yamada and D. undulata Holmes had specifically susceptibility for B. cereus, S. aureus andL.monocytogenes and were superior to streptomycin, the authentic antibiotics.However, streptomycin, which is a broad-spectrum antibiotics, showed the clear zone for all tested food-borne pathogens.Great attention has been focusing on the marine seaweeds as potential and promising sources of bioactive compounds in recent years.Seaweeds are able to produce a great variety of secondary metabolites with versatile activities, such as antiviral, anthelmintic, antifungal and antibacterial activities(Lindequist and Schweder, 2001;Newman et al., 2003).Red algal genus Laurencia (Rhodomelaceae, Ceramiales) produces the numerous specialized secondary metabolites with diverse structural features.Its three

Table 1 :
Agar diffusion susceptibility of foodborne pathogens to Korea domestic algae extract

:
Four grampositive (S. aureus, B. cereus, B. subtilis and Listeria monocytogenes) and two gram-negative foodborne pathogens (S. choleraesuis and S. enterica) were used in the experiments.S. aureus American Tissue Culture