Fish and shellfish are excellent protein sources for human consumption due to their high content in vitamins, minerals and polyunsaturated fatty acids [1
]. The organoleptic quality of marine products can deteriorate rapidly post mortem
as a consequence of various microbial and biochemical breakdown processes. In fact, the degree of alteration depends on the initial bacterial count in the fresh fish and shellfish, which is also affected by the microbiological quality of the water, its temperature and salinity [2
]. The bacterial flora in fish includes those naturally present in freshwater environments, and those associated with pollution of the aquatic environment. A third group includes bacteria introduced to fish and fish products during post-harvest handling and processing. In fact, procedures of handling, harvesting, processing (e.g., deheading, eviscerating, and cutting) and storage temperature are the main factors affecting the microbiological quality of fish and shellfish products post-mortem
]. Seafood may be a vehicle for most known bacterial pathogens, such as Salmonella
, Edwardsiella tarda
, Plesiomonas shigelloides
, motile Aeromonas
spp. strains [7
]. Filter feeders, such as mussels and oysters, concentrate all bacteria and viruses that live in the surrounding water in their filtration systems [8
]. Recently, the 16S rDNA sequencing revealed Shewanella baltica
as the main species isolated as hydrogen sulfide-producers from marine fish, followed by Serratia
spp. and other Shewanella
Essential oils are aromatic and volatile oily liquids containing a mixture of compounds resulting from plant’s secondary metabolism formed in special cells found in leaves and stems, and commonly concentrated in one particular region such as leaves, bark or fruit. Some essential oils have shown promise as potential food safety interventions when added to processed and raw foods. Extracts/essential oils from dietary herbal species have been used as sources of medicine and food preservatives for over 4000 years [12
]. Recently, some of the bioactive substances responsible for these medicinal and preservative activities have been identified as phenolic compounds like thymol and rosmarinic acid [13
]. Many aromatic/medicinal spices and herbs are generally used in the Mediterranean basin to flavor fish and shellfish seafood preparations. These plants and their derivatives are also used as preservation agents to avoid bacterial seafood contamination [12
]. Essential oils from oregano and mint are effective against the spoilage organism Photobacterium phosphoreum
, even in fatty fish like codfish, and spreading the essential oil on the surface of whole fish or in a coating film for shrimps appears effective in inhibiting the respective natural spoilage flora [12
]. The Oreochromis niloticus
(L.) fry diet supplemented with ginger, black cumin, thyme, clove, watercress, fennel or garlic essential oils improves their fingerling performance [15
]. The essential oils are recognized as safe according to the Food and Drug Administration [18
]. In the food processing industry, additives and other ingredients are used during the technological process of production. Today, the most important trends in the food industry are the application of natural flavors, spices, antioxidants and pigments. Herbal drug extracts and essential oils are added in different kinds of food products as natural preservatives. Their basic performance is to improve the taste, smell and organoleptic effect on food production, digestion of food products, increase the freshness of products and at least preserve of the products in a proper way [12
]. In Tunisia, zinger powder, dill aerial parts and dry laurel leaves are commonly added to fish and shellfish seafood dishes. Considering the great number of different chemical compounds groups present in the essential oils extracted from these plants, they are known to possess several biological activities including antibacterial, antifungal and antioxidant activities, and are used in various industries including medicine, food, and cosmetics [19
The leaves of L. nobilis
) are usually used to treat gastrointestinal disorders, an in the cosmetics and food industry as a fragrance component. Laurel essential oil is generally dominated by the monoterpene compound 1,8-cineole. This species is used as a food flavouring agent, and in the pharmaceutical industry for drug formulations. Laurel essential oils are recognized for their antimicrobial activity against a wide panel of tested foodborne spoilage and pathogenic bacteria and fungi, antiviral and antibiofilm activities [21
]. Commonly known as dill, Anethum graveolens
) seeds are widely used in food and pharmaceutical industries to treat gastrointestinal problems (carminative, stomachic) and rheumatism. Carvone is the predominant odorant of dill seed and α-phellandrene, limonene, dill ether, myristicin are the most important odorants of dill herb. The essential oil of this annual aromatic herb is recognized to exhibited a wide range of biological activities including antibacterial, antifungal, antioxidant, anti-inflammatory, anti-hyperlipidemic and anti-hypercholesterolemic effects [23
]. The plant Zingiber officinale
, belonging to the family Zingiberaceae
is indigenous to warm tropical climates, particularly southeastern Asia and is cultivated in India, China, Africa, Jamaica, Mexico and Hawaii. Both the essential oil and oleoresins extracted from the rhizomes (spice of commerce) are used in many food preparations, soft drinks and beverages [26
]. The analysis of volatile oils from dill rhizome showed camphene, p
-cineole, α-terpineol, zingiberene and pentadecanoic acid as major components. This oil possesses different biological properties including antibacterial, antifungal, ant-oxidative, anticancer, larvicidal, antidiabetic, anti-inflammatory and nephro/hepato-protective properties [27
The main objectives of the present work were to study the antibacterial activities of Laurus nobilis (laurel), Anethum graveolens (dill) and Zingiber officinale (ginger) essential oils against bacteria isolated from fish and shellfish frequently consumed in Tunisia. The chemical composition and the antioxidant activities of the three essential oils were also studied.
2.1. Identification and Characterization of Isolates
Seventy-threes trains were identified from all fish samples, mollusks and crustaceans analyzed based on biochemical characters obtained on the API galleries (20E, 20NE, Staph) and interpretation of results was performed by using the API software (bio Mérieux SA, Marcy l’Etoile, France). Ten species belonging to the genus Staphylococcus were identified in all samples tested: S. lentus, S. xylosus, S. sciuri, S. lugdunensis, S. saprophyticus, S. epidermidis, S. aureus, S. hominis and S. cohnii spp. cohnii, with dominance of the two species S. lugdunensis and S. sciuri. Only one strain isolated from the skin of sea bream was identified as Micrococcus spp.
Using the API20E galleries, the colonies growing on CHROMagar™ E. coli
agar plates were identified as Enterobacter cloacae
, Klebsiella ornithinolytica
, K. oxytoca
and Serratia odorifera
. In addition, the 44 strains isolated from CHROMagar™ Vibrio
agar plates and identified by using API20NE strips belonged to the genera Aeromonas
. Only one strain isolated from the skin of sea bream (V36
) has not been identified by the API 20NE system. Aeromonas hydrophila
is the dominant species in all fish samples, mollusks and crustaceans analyzed. The strains selected in this study produced several hydrolytic enzymes such as amylase (100%), caseinase (100%), lecithinase (12/34: 35.29%) and DNase 24/34: 70.58%). The majority of strains were beta-haemolytic (24/34: 70.58%). All these data are summarized in Table 1
. Using the Cristal Violet technique, all strains were interpreted as non-biofilm forming bacteria on polystyrene 96-well microtiter plate U-bottom as the optical densities estimated at 595 nm were less than 1. All tested strains grew on Congo red agar medium (Figure 1
) giving five morphotypes with different color (red, pinkish-red, bordeaux, pink colonies with a darkening at the center, black). Among the isolated strains, three A. hydrophila
and one S. lentus
(11.76%) were slime producers characterized by black colonies, and the remaining 31 strains were non-slime producers.
2.2. Chemical Composition of the Essential Oils
The GC/MS analysis permitted the identification of 29, 24 and 60 components in the essential oils of laurel, dill, and ginger, respectively. Laurel essential oil is rich in monoterpene hydrocarbons (26.9%) and oxygenated monoterpenes (64.8%). The main constituents is this oil were 1,8-cineole (56%), α-terpinyl acetate (9.0%), 4-terpineol (5.2%), methyleugenol (3.6%), sabinene (3.5%) and α-pinene (3.2%). Carvone (27%), piperitone (25.7%), limonene (20.6%), dill apiol (8%), trans
-dihydrocarvone (4.9%) and camphor (4.4%) were the key components in the essential oil of dill. Hydrocarbon monoterpenes (32%), oxygenated monoterpenes (31%) and hydrocarbon sesquiterpenes (22.2%) were the main chemical classes in ginger essential oil. This oil is rather rich in camphene (11.5%), β-phellandrene (10.7%), 1,8-cineole (10.4%), α-zingiberene (6.9%), borneol (6.4%), ar
-curcumene (4.6%) and sesquiphellandrene (4.1%). The quali-quantitative compositions of the three oils are given in Table 2
2.3. Antioxidant Activities
The assessment of antioxidant activity by a single method underestimates the antioxidant potential of an extract or molecule and it reflects only the ability to inhibit a precise class of oxidants present. The combination of different complementary assays can give a clearer idea of the antioxidant activity. For this, we evaluated in this study the antioxidant activity of the three essential oils using four antioxidant tests (DPPH assay, Ferrous Reducing power, capacities to inhibit the bleaching of β-carotene and to neutralize the superoxide anion).The results are reported in Table 3
The DPPH assay is considered a valid and easy assay to evaluate the radical scavenging activity of antioxidants, since the radical compound is stable and does not have to be generated as in other radical scavenging assays.The scavenging effects on the DPPH radical expressed as IC50 values was the highest for L. nobilis essential oil (135 µg/mL) followed by Z. officinalis (470 µg/mL) and A. graveolens essential oils (3000 µg/mL), showing a radical scavenging activity clearly less important than that the positive control BHT (IC50 11.5 µg/mL).
The inhibition of β-carotene bleaching is another type of antioxidant assay associated with lipid peroxidation. In oil–water emulsion-based system, linoleic acid undergoes thermally induced oxidation, thereby producing free radicals that attack the β-carotene chromophore resulting in a bleaching effect. The IC50 value registered in the β-carotene bleaching assay was lower for Z. officinalis (1900 mg/mL) followed by L. nobilis (3600 mg/mL) and A. graveolens (3000 mg/mL) essential oils. With regards to the superoxide radical anion assay, our results indicated a high scavenging ability for the ginger essential oil (IC50 320 µg/mL) as compared to the laurel and dill essential oils, although lower than the standard, BHT (IC50 1.5 µg/mL).
As shown in Table 3
, the reducing power of the three essential oils, expressed as EC50
, was clearly less important than that of positive control, ascorbic acid (37.3 µg/mL), and the highest activity was registered for L. nobilis
essential oil (1850 mg/mL), followed by Z. officinalis
essential oil (1900 mg/mL) and A. graveolens
2.4. Antibacterial Activities
The antimicrobial activities of the three essential oils against the microorganisms isolated from seafoods products were qualitatively and quantitatively evaluated by the presence or the absence of inhibition zone diameter, MICs and MBCs values. The results were given in Table 4
Based on the observed means compared using the Duncan’s multiple range tests for means, three groups were observed and L. nobilis essential oil showed the highest activity, regardless of the bacteria tested (mean = 14.25 mm) followed by A. graveolens (mean = 12.31 mm) and Z. officinale essential oils (mean = 11.36 mm). The results showed that the three essential oils had substantial antimicrobial activity against the 34 bacteria tested as compared to the results obtained with the standard compounds erythromycin and kanamycin. In fact, the data obtained in terms of zones of growth inhibition (mm) scored in Mueller-Hinton agar demonstrated that Z. officinale essential oil produced the highest diameters of growth inhibition (between 6–22.33 mm). This oil was active against all Gram negative bacteria, especially A. hydrophila with a diameter of inhibition ranging from 10.33 to 22.33 mm and the Gram negative Staphylococcus spp. with a diameter of inhibition ranging from 6 to 15.33 mm. The highest diameter of inhibition (about 25.33) mm was obtained when A. graveolens essential oil was tested against the A. hydrophila (R5) isolated from the blue mussels Mytilus galloprovincialis.
The MIC and MBC values indicate that the tested essential oils were active against A. hydrophila, Enterococcus spp., Klebsiella spp., Staphylococcus spp., S. odorifera and V. alginolyticus strains, with low MICs values ranging from 0.05 to 0.39 mg/mL, while high concentrations of essential oils were needed to inhibit growth of almost all isolated bacteria. The lowest MBCs values were obtained with the laurel oil and the concentrations needed to inhibit the growth of A. hydrophila strains ranged from 6.25 to 50 mg/mL.
The three essential oils tested in the present study possess different degrees of antibacterial activity against several Gram positive and Gram negative bacterial species isolated from fish and shellfish products. It seems that laurel essential oil possesses the highest antibacterial activity against all tested bacteria. Hence, they could potentially be used as a natural preservatives against food-borne pathogens, in order to inhibit lipid oxidation and delay microbial growth in fish and shellfish. These essential oils can also be mixed with fish diets to prevent the multiplication of natural spoilage bacteria. In rearing tanks of Sparus aurata or Dicentrarchus labrax fishes, or their live prey (rotifer cultures), these essential oils can reduce the count of the pathogenic bacteria like Vibrio spp. strains.
Additionally, the antioxidant-antiradical activity registered can be important to preserve marine products known to contain a number of components prone to chemical degradation. It seems that laurel essential oil was the most effective as compared to the dill and zinger oils. Therefore this oil can be used to prolong seafood shelf life because of its protective effects against both microbiological and chemical deterioration thus preventing off flavors and formation of all the above toxic agents.
Further experiments must be done to confirm the viability of the commercial use of these essential oils, and especially laurel volatile oil, if used to prevent the multiplication of natural spoilage fish and shellfish flora in the food industry, or in fish hatcheries to control the multiplication of pathogenic bacteria like Vibrionaceae and Aeromonadaceae families members in live prey cultures (Artemia salina and Brachionus plicatilis) and fish rearing tanks.