Antibacterial Activity of Essential Oils and their Effects on Nile Tilapia Fingerlings Performance
M. S. Mohamed
S. Abd El-Shafi
This study investigated the antibacterial activity of five essential oils in-vitro.
Also, the strongest antibacterial oil (thyme oil) was tested on performance
of Nile tilapia (Oreochromis niloticus) fingerlings fish. Two experiments
were carried out, in the first one antimicrobial activity of essential oils
(EOs) of 5 medicinal plants, ginger, black cumin, thyme, clove and watercress
were evaluated (in-vitro). The screening of antibacterial activity was
against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli,
Listeria monocytogenes, Lactococcus lactis and Bacillus cereus.
In the second experiment (in-vivo) 120 fish were divided in 4 groups.
There were 3 replicate glass aquaria (per group) of 10 fish per aquarium. The
1st group fed basal diet (control) and the other groups (2-4) were fed basal
diet supplemented with 0.1, 0.25 and 0.5% thyme oil. First experiment showed
that thyme oil had the strongest antibacterial activity against all tested bacteria
except Staph. aureus and E. coli. The more sensitive bacteria
to thyme oil was Pseud. aeruginosa while. E. coli was least susceptible
to it. Second experiment, showed that thyme oil significantly increase growth
rate, feed intake and feed utilization of fish. The best level was 0.25%. Total
protein and globulin concentrations in plasma were significantly increased by
0.1 and 0.25% thyme oil. The best economical efficiency was obtained by 0.25%
Received: January 26, 2013;
Accepted: April 08, 2013;
Published: June 14, 2013
Improving growth performance and feed efficiency by synthetic and natural feed
additives have been widely used (Collington et al.,
1990; Lee et al., 2001). The residue of antibiotics
increase bacterial resistance and has bad effect on animal and human health.
Therefore, recent researches tended to use alternative natural product like
Essential Oil (EO). Therefore, demand for green aquaculture or organic aquaculture
has recently been increasing. Natural products like plant extracts contain secondary
metabolites. Generally, they can be structured into 3 groups: saponins, tannins
and Essential Oils (EO) (Calsamiglia et al., 2007).
It have been investigated for their therapeutic and prophylactic effects of
several fish diseases, inhibited the Lactococcus garviea and Aeromona
hydrophila, fish pathogenic bacteria (Rattanachaikusopon
and Phumkhachorn, 2009; Seden et al., 2009).
Thymol oil derived from thyme (Thymus vulgaris), has demonstrated biological
properties such as antimicrobial, antioxidant and antiseptic activities (Lee
and Ahn, 1998). It has high activity on inhibition of respiratory tract
pathogenic bacteria (Inouye et al., 2001). Also,
diet supplemented with Origanum vulgare (kind of thyme) improve performance
of Nile tilapia fingerlings (Seden et al., 2009).
These effects may change according to the differences in thyme species, cultivation,
origin, vegetative stage and growing season of the plants (Milos
et al., 2000; Martinez et al., 2006).
The EO recognized as safe admitted by the Food and Drug Administration (FDA,
However, there are limited studies conducted to investigate the effect of EO
on performance of fish. The present study was designed to evaluate the antimicrobial
activity of ginger, black cumin, thyme, clove and watercress EO on some pathogenic
bacteria (in-vitro). From in vitro results we choose the best
oil (thyme oil) to study its effect on performance of Nile tilapia (Oreochromis
MATERIALS AND METHODS
The experimental work was carried out in the Aquaculture Research Lab., Abbassa,
Abo-Hamad; Department of Animal Production, Faculty of Agriculture and Department
of Botany, Faculty of Science, Zagazig Univ., Egypt.
In vitro study
Essential oils: Essential oils of 5 herbs, ginger (Zingiber officinale),
black cumin (Nigella sativa), thyme (Thymus vulgaris), clove (Syzygium
aromaticum) and watercress (Eruca sativa) were purchased from El-Hawag
Factory, Badr City, Egypt.
Bacterial species: Six bacterial species belonging to Gram-negative
and Gram-positive were tested (Table 1). Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli were obtained from Faculty
of Medicine, Zagazig University, while Listeria monocytogenes, Lactococcus
lactis and Bacillus cereus were obtained from Faculty of Science,
Screening of antibacterial activity: Screening of antibacterial activity
was performed by standard disc diffusion method (Saeed
et al., 2007). Fifty sterilized discs of filter paper (6 mm diameter)
were soaked in 1 mL of oil, separately for 2 min and then used for screening.
The potency of each disc was 10 μL (each 50 discs of filter paper absorbed
0.5 mL). Nutrient agar was used as base medium and Nutrient broth was used for
the preparation of inoculums. A sterile cotton swab was dipped into the bacterial
test suspension to inoculate entire surface of a nutrient agar plate. Discs
of oil were placed on the surface of inoculated plates with the help of sterile
forceps. The inoculated plates were incubated at 37°C for 24 h. After incubation
inhibition zone diameters of 4 discs for each oil were measured to the nearest
Experimental design: A total number of 120 Nile tilapia fish (average body
weight 13 g) were used in 4 experimental groups.
|| Antibacterial activities of essential oils (in-vitro)
|a, b, c, dMeans in the same row bearing different
letters differ significantly (p<0.05)
|| Chemical composition (%) of the commercial basal diet
|DM: Dry matter, OM: Organic matter, CP: Crude protein, CF:
Crude fiber, EE: Ether extract, NFE: Nitrogen free extract
There were 3 replicate glass aquaria (per group) of 10 Nile tilapia fish (Oreochromis
niloticus) per aquarium. The 1st group fed basal diet (control) and the
other groups (2-4) were fed basal diet supplemented with 0.1, 0.25 and 0.5%
thyme oil, respectively.
Preparation of fish diets: The thyme oil was added to a ground commercials
diet which was pelleted again. Commercial diet composed of fish meal, soybean
meal meat meal, yellow corn, bone meal and a mixture of vitamins and minerals.
The chemical composition of diet (Table 2) was adopted according
to AOAC (1984).
Aquarium design and fish rearing: The dimensions of aquarium were 150x150x50
cm, these aquaria were supplied with dechlorinated tap water up to 80% of its
highest and continuous aeration was adapted by using an air pump and air stones.
Fish wastes were filtered by siphon method each day and the rearing water was
completely changed every 3 days. Mean water temperature was 27±2°C.
The fish were fed 2 times a day (09:00 and 16:00 h) at a rate of 4% of the total
body weight (at two equal meals). The fish were weighted monthly and the feed
quantities were readjusted according to the change in live body weight. The
period of experiment was 3 months.
Blood samples: At the end of the experiment, blood samples were taken
from the caudal vein of 12 fish for each treatment (4 fish/replicate). Blood
plasma was separated and stored at -20°C to analysis. Plasma total protein,
albumin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
were analyzed by using commercial kits from Diamond Diagnostics Company, Egypt.
Statistical analysis: Data of the experiment were statistically analyzed
using the General Linear Model Program of SAS (1996). Significant
differences between treatment means were tested by Duncans Multiple Range
Test (Duncan, 1955).
RESULTS AND DISCUSSION
In-vitro study: Essential oils used in this study exhibited antibacterial
activity against some tested bacteria with different degree of inhibition (Table
1). Based on the diameter of inhibition zone, thyme oil had the strongest
(p<0.05) antibacterial activity against all tested bacteria except Staphylococcus
aeures and E. coli. These results are similar to those obtained by Viuda-Martos
et al. (2011) who reported that thyme oil was better than lavender,
fennel, parsley and black oil in inhibition of Listeria innocua,
Serratia marcescens and Pseudomonas fluorescens. Also, Seden
et al. (2009) reported that Origanum vulgare (kind of thyme)
in diet of Nile tilapia fingerlings inhibited fish pathogenic bacteria (Aeromona
hydrophila). The more sensitive bacteria to thyme oil was Pseudomonas
aeruginosa while E. coli was least susceptible to it. These results
agree with those obtained by Inouye et al. (2001)
who reported that E. coli was least susceptible to 14 essential oils.
Also, Saeed and Tariq (2008) reported that E. coli
was the least sensitive to clove oil. Generally, The antimicrobial activity
of essential oils against bacteria varies depending on the source of an essential
oil and strain of bacteria. The composition of EO can vary among different parts
of the same plant (Dorman and Deans, 2000). Also, Martinez
et al. (2006) observed that the concentration of active components
(carvacrol, thymol, p-cymene and Y-terpinene) in thyme EO varied widely depending
on the species of the thyme plant. The inhibitory effect of thyme EO against
bacteria are due to interacting with bacterial cell membrane (Dorman
and Deans, 2000).
1- Body weight gain, feed intake and feed utilization: The chemical composition
(%) of the commercial basal diet was recorded in Table 2 according
to AOAC (1984). All levels of thyme oil (0.1, 0.25 and 0.5%)
significantly (p<0.05) increase live body weight, body weight gain, specific
and relative growth rate (Table 3), feed intake and feed utilization
(Table 4) of Nile tilapia fingerlings in all experimental
period. However, using 0.25% thyme oil was the best level. These results agree
with the findings of Seden et al. (2009) who
reported that 1% Origanum vulgare herb significantly improved growth
performance, feed intake, feed conversion, protein and energy utilization of
Nile tilapia fingerlings. Also, agree with Al-Kassie (2009)
who reported that 200 mg thyme oil kg-1 diet significantly improved
|| Effect of thyme oil on growth performance of Nile tilapia
|a, b, cMeans in the same row bearing different
letters differ significantly (p<0.05)
||Effect of thyme oil on feed intake and feed efficiency of
Nile tilapia fingerlings
|a, b, cMeans in the same row bearing different
letters differ significantly (p<0.05)
On the other hand Jang et al. (2007) reported
that growth performance, feed intake and feed efficiency were similar among
birds fed basal diet and the diet supplemented with 25 or 50 mg commercial blend
of EO/kg diet. These differences between researches may be due to the kind,
composition of EO which affected by cultivation, origin, vegetative stage and
growing season of the plants (Milos et al., 2000),
nutritional status of animals, infection, diet composition and environment (Giannenas
et al., 2003). The beneficial effect of EO on fingerlings body weight
gain and feed efficiency may be due to: firstly, its antimicrobial and antioxidant
effect (Milos et al., 2000). Thyme oil improve
intestinal microflora (decrease growth of pathogenic bacteria Aeromonas hydrophila
(Seden et al., 2009) and increase growth of
beneficial bacteria (Lactobacilli) (Jang et al.,
2007). The pathogenic bacteria increase rate of passage and thickness of
intestinal mucosa which reduce nutrient digestibility and absorption (Hu
et al., 2002; Xia et al., 2005). Secondly,
enhancing digestibility and absorption of nutrients. EO increase the digestive
enzyme activities of the pancreas (trypsin and α-amylase) and intestine
(maltase, alkaline phosphatase and leucine aminopeptidase) (Lee
et al., 2003; Jang et al., 2007).
Also, EO increase the intestinal microvilli enzymes such as disaccharide, alkaline
phosphatase and leucine aminopeptidase.
||Effect of thyme oil on plasma constituent and survival rate
of Nile tilapia fingerlings
|a, b, cMeans in the same row bearing different
letters differ significantly (p<0.05), AST: Aspartate amino transferase,
ALT: Alanine amino transferase enzymes
These enzymes are important constituents of the microvillus membrane in the
intestinal absorptive cells, where they associated with the degradation and
absorption of nutrients from the gut (Ferraris et al.,
1992). Thirdly, improve immunity and body health including positive effects
on cardiovascular diseases, some tumors, inflammatory processes and in general
diseases in which the uncontrolled proliferation of free radical is very damaging
(Trouillas et al., 2003).
Blood parameters: Some fish plasma constituents are shown in Table
5. Total protein and globulin concentrations were significantly increased
(p<0.05) by 0.1 and 0.25% thyme oil, respectively. These results agree with
the findings of Al-Kassie (2009) who reported that broiler
diet supplemented with 100 and 200 ppm thyme oil significantly increased plasma
total protein. The activity of aspartate amino transferase (AST) and alanine
amino transferase (ALT) enzymes significantly (p<0.05) increased by addition
of 0.5% thyme oil. These results indicate that 0.1 and 0.25% thyme oil improve
liver and kidney functions and body health.
Survival rate: The survival rate (Table 5) did not
significantly affected by all levels of thyme oil.
|| Effect of thyme oil on economical efficiency
|*Total feed intake x price, The price of 1 kg control, 0.1%
thyme oil, 0.5% thyme oil, 1% thyme oil were 250, 266.67, 291.68, 333.35
piasters respectively (price 2010), One mL thyme oil was 16.67 piasters,
respectively, **Total gainx20 (one kg 2000 piasters), ***Gain price-feed
cost, ****Relative profit for treatment/net revenue of controlx100
Economical efficiency: As shown in Table 6, the economical
efficiency results indicated that the best level was 0.25% thyme oil (119.39%).
Generally, the relative profit of control, 0.1, 0.25 and 0.5% thyme oil were
100, 115.19, 119.39 and 100.58%, respectively.
This study has shown that all tested essential oils had antibacterial effects
on Gram positive and Gram negative bacteria. The most potent oils was thyme
oil. Addition of thyme oil specially 0.25% to Nile tilapia fingerlings fish
diet improve body weight gain, feed efficiency, blood parameters and economical
efficiency. Therefore, thyme oil can be used as an alternative to antibiotics,
cheap and safe feed additive.
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