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1 Data are representative of mean contents of triplicate samples.
2 (#) not enough microalga sample for biochemical analysis.
Microalgae growth rates and their biochemical composition are two important characteristics, which will determine the potential of certain species to be used for aquaculture purposes (De Castro Araujo and Garcia, 2005). Results showed that, the growth parameters measured for C. muelleri were significantly higher at harvest time in cultures with SWE, particularly extracts of U. lactuca, E. intestinalis and G. corticata as a supplement compared to just using the f/2 medium. The growth-enhancing potential of these seaweed extracts might be attributed to the fact that these SWE provided sufficient nutrients for microalgae growth as long as the SWE were replenished as needed. The increased growth of microalgae obtained with these SWE might be attributed to either the presence of macro and micronutrients (Rathore et al, 2009) or the presence of growth substances such as cytokinins or auxins, which are known to occur at relatively high concentration in various seaweeds (Blunden, 1991; Crouch and Staden, 1993). Moreover, use of seaweed extracts as a supplement significantly enhance nutrient uptake by microalgae (Rathore et al., 2009). The increases in microalgae
chlorophyll a content could be attributed to high microalgal cell density that produces shadows and thus stimulates increased chlorophyll production in microalgal cells (Saoudi-Helis et al., 1994). Lipids, proteins and carbohydrates are the most important biochemical components in microalgal biomass (Boechat and Giani, 2000). When SWE were used as a supplement or as an alternative, the protein, lipid and carbohydrate content had no notable changes. Rohani et al. (2012), Cho et al. (1999) and Alvarado et al. (2008) reported that the proximate biochemical composition of I. galbana cultured with and without SWE were similar. Since culture of C. muelleri with these SWE produced, the same protein and lipid content compared to the f/2 medium, these SWE can therefore be used as a substitute to f/2 medium. In conclusion, the results clearly showed that the addition of SWE produced the equal amount of biomass and proximate biochemical composition when they were used as an alternative media or even greater biomass of microalga as a supplement media during the same period than using the f/2 media. Therefore, we believe that the use of locally sourced SWE could constitute a viable alternative to conventional f/2 medium as a food source in aquaculture operations.
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Effects of replacing fish meal/oil with plant sources in diet of Beluga sturgeon (Huso huso) and rainbow trout (Oncorhynchus mykiss) on the distal gut microbial counts
Maryam Roohi1*, Naser Agh2, Reza Jalili3
1M.S.Student in microbiology, Department of microbiology, Islamic Azad university, Urmia, Iran
2 Department of Fisheries, Artemia and Aquatic Animals Research Institute, Urmia University, Iran
3 Department of Fisheries, Faculty of Natural Resources, Urmia University, Iran
* Corresponding author. Tel.: +98 441 3467097. E-mail address: firstname.lastname@example.org
The present study was performed to examine the effect of replacing fish meal and fish oil with plant sources on microbial counts of rainbow trout and Beluga sturgeon. The control diet contained only fish meal and fish oil as the primary sources of protein and lipid, while the 6 remaining diets either contained 20% fish oil and 80% the canola:linseed:safflower oil blend (40:30:30, respectively) as the primary lipid source and 40, 60, 80 and 100 percent replacement of fish meal with plant protein sources. Results showed no significant difference was observed among the mean weights of the groups. However, no significant differences were detected in microbial counts of fish fed control diet and those fed on diet containing vegetable oil diet in both fish. But, replacing fish meal with 60, 80 and 100% plant protein resulted in significantly decreased microbial counts in rainbow trout and beluga sturgeon compared to control group.
Keywords: Plant sources, Microbial counts, Beluga sturgeon, Rainbow trout.
A major obstacle to this work is that these plant based diets induce a variety of histological and functional changes in the gastrointestinal tracts of fish. Observed effects include change in the intestinal structure, inflammation, reduced growth as well as nutrient digestion and absorption, and an increased susceptibility to disease (Krogdahl et al., 2010). These changes are thought to be due to direct effects of anti-nutritional factors in plant ingredients and/or the indirect result of diet-induced changes in the structure and function of the intestinal microbiota (Ring0 et al., 2006). Plant ingredients such as soybean, canola and peas have been used previously in the manufacturing of aquaculture diets (Gatlin et al., 2007). These ingredients are known to contain anti-nutritional factors such as saponins, glucosinolates, phytate and trypsin inhibitors, although processing the ingredients can reduce their levels (Francis et al., 2001). Previous studies have shown that soy protein concentrate (SPC) and canola protein concentrate (CPC) can be used at maximum inclusion rates of 50% and pea protein concentrate (PPC) at 27% without having a negative impact on fish growth (Krogdahl et al., 2010). However, the effects of these ingredients on intestinal physiology and microbiota of salmonid fish have not been investigated. However, there are few reports on the effect of diet on the intestinal microbiota of fish (Dimitroglou et al., 2009). Therefore, the present study evaluated the effect of replacing fish meal/oil with plant sources in diet of Beluga sturgeon (Huso huso) and rainbow trout (Oncorhynchus mykiss) on the distal gut microbial counts.
Materials and methods
Animals and experimental design
Beluga sturgeon (Huso huso) were reared at 18-19°C (18 fibreglass tanks, 300 l capacity; 30 individuals per tank) and The rainbow trout (Oncorhynchus mykiss) were reared in running fresh water at 14-15°C (18 fibreglass tanks, 300 l capacity; 30 individuals per tank). Six experimental diets with similar protein, lipid and energy content were
formulated to contain graded levels of plant protein and blend vegetable oil sources to replace fish meal and fish oil (Table 1). Fish were fed two times per day at 1-2% body weight for 6 weeks. No mortality was observed during the experiment. The Beluga sturgeon grew from 133±5 g to 256±10 g, and the rainbow trout grew from 65±2 g to 138±2 g. No significant difference was observed among the mean weights of the groups.
Table 1. Percentages of fish meal and fish oil replacement._
Protein source Oil source
Dietary Treatments 1 2
Fish meal Plant sources Fish oil Vegetable oil
1plant protein source included: wheat gluten-based, corn gluten and soybean meal. 2vegetable oil: was formulated using canola oil (30%), linseed oil (30%), sunflower (30%) and safflower oil (10%).
At the end of the experiments, in both Beluga sturgeon and rainbow trout, six individuals were sampled in each tank. After euthanasia using excess anaesthetic, intestine of both fish were aseptically removed by dissection, and the contents were squeezed out and collected. Then, tenfold dilutions from faeces, intestine contents were prepared to obtain 10-1-10-2-10-3-10-4-10-5 dilutions, respectively. One millilitre of the dilutions (faeces) was spread on plate. Aliquots of 100 ul of the dilutions from faeces contents were spread onto tryptic soy agar (TSA, Scharlau Chemie, Spain), to counting microbial. The plates were incubated at 20°C and inspected for up to five days.
The intestinal rnicrobiota plays critical roles in intestinal development, pathogen and digestion in fish (Merrifield et al., 2010). Ideally, any manipulation of diets to increase plant ingredient content would cause minimal disruption in the structure of the microbiome or movement away from the ideal microbiome associated with a gold standard, FM diet. However, there are few reports on the effect of diet on the intestinal microbiota of fish (Dimitroglou et al., 2009).
In the present study, no significant differences were detected in microbial counts of fish fed control diet (100FM/FO) and those fed on diet containing vegetable oil diet (100FM/VO) in both fish.
Table 2. Comparison of bacterial counts in intestinal contents sampled in rainbow trout and beluga sturgeon fed the experimental diets.
Beluga sturgeon (10-5)
For each index, the means were compared by ANOVA, and those without common superscript were significantly different after Tukey test. Data are presented as mean (standard deviations).
In rainbow trout replacement of fish meal with 40% (60FM/80VO), 60% (40FM/80VO), 80% (20FM/80VO) and 100% (0FM/80VO) plant protein in combination with both fish meal and 80% replacement fish oil resulted in decreased microbial counts compared to the control fish. But in beluga sturgeon microbial counts of fishes fed 40% fish meal replaced (60FM/80VO) diet showed no significant differences compared to the control fish, whereas microbial counts was significantly lower in
40FM/80VO, 20FM/80VO and 0FM/80VO treatments.
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Effect of Cuminum cyminum Essential oil and Nisin on the growth inhibition of Streptococcus iniae, the cause of zoonotic disease in farmed fish (Aquaculture)
*Laleh Roomiani1, Mansoreh Ghaeni2 and Nasrin Choobkar3
1 Department of Fisheries, Abadan Branch, Islamic Azad University, Abadan, Iran.
2Department of Fisheries, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran.
3 Department of Fisheries, Kermanshah Branch, Islamic Azad University, Kermanshah,
*Corresponding author: Laleh Roomiani(L.roomiani @ yahoo.com). 09166436971.
The aquatic zoonotic pathogen Streptococcus iniae represents a major health and economic problem in fish species worldwide. In this study, the effect of Cuminum cyminum L. essential oil (EO) and Nisin on growth of Streptococcus iniae in fillets of Oncorhynchus mykiss was evaluated. The experiment included different levels of EO (0, 0.045, 0.135, 0.405 and 0.81%) and Nisin (0, 0.25 and 0.75 ug/mL) to assess their effects on S. iniae count for 21 days in 4c°. The significant (P < 0.05) inhibitory effects of EO (even at its lowest concentration) and Nisin on this organism were observed alone and in combination together. The best inhibitory effect was obtained at combination of EO = 0.81 % and Nisin = 0.75%.
Key Words: Streptococcus iniae, Cuminum cyminum, Nisin, Oncorhynchus mykiss.
Streptococcus iniae is a major fish pathogen causing streptococcosis. Streptococcosis of cultured fish has become a major problem in many countries, for example Iran (Soltani et al., 2008). This disease caused by
Streptococcus iniae accounts for significant economical losses in the aquaculture industry worldwide (Shoemaker et al., 2001). It leads to mortality, reduced growth and unmarketable appearance. Streptococci can cause acute infections in fish, resulting in a greater than 50% mortality rate over a period of 3-7 days (Ghasemi Pirbalouti et al., 2011). Rainbow trout (Oncorhynchus mykiss) is one of the most cultured fish species in Iran and is gaining popularity in other parts of the world. Soltani et al., 2009 found that Streptococcus iniae is the most commonly isolated bacterium from diseased rainbow trout in aquaculture farms in Iran. Bacterial diseases in aquaculture are mainly controlled by antibiotics. However, continuous intensive use of antibiotics is undesirable as this leads to the development of drug resistance and thereby to a reduced efficacy of the drugs (Ozyurt et al., 2011). In the public health context, antibiotic resistance can be transferred to environmental and human pathogenic bacteria. In addition, antibiotics accumulate in the environment and fish, posing a potential risk to consumers and to the environment in general. Antibiotics (such as oxytetracycline, erythromycin, tetracycline and etc.) are widely used to prevent bacterial disease in fish. However, the indiscriminate use of antibiotics by fish farmers can lead to the emergence of drug resistant strains and can create serious public health problems because some streptococci are zoonotic agents (Can, 2012). The rapidly expanding aquaculture industry in Iran has suffered heavy economic losses due to bacterial pathogens, particularly Streptococcus iniae and Lactococcus garvieae, which are the major agents of streptococcosis in rainbow trout (Akhlaghi and Mahjoor, 2000). Increased public awareness of the negative effects caused by overexposure to synthetic chemicals has led to the search for ''green solutions'', such as organic and synthetic chemical-free food products (Abutbul et al., 2004). To enable organic fish production it is essential to develop antibacterial treatments that are based on materials from natural sources. Nisin, an antibacterial substance produced by Lactococcuslactis (formerly Streptococcus lactis, Lancefield group N), discovered more than 80 years ago, is widely used as a food preservative. It is regarded as a bacteriocin, but is atypical in having a wide spectrum of activity against Gram-positive bacteria (Sanlibaba et al., 2009). This paper
describes the use of EO and Nisin as treatment against streptococcal disease caused by Streptococcus iniae in rainbow trout.
Materials and Methods
The air-dried seed of C. cyminum was collected in Kerman province of Iran and identified by Iranian Institute of Medicinal Plants, Tehran, Iran. The obtained EO was dried over anhydrous sodium sulfate until the last traces of water were removed and then stored in dark glass bottles at 4C. The EO was analyzed by gas chromatography (GC) (Agilent 6890, Wilmington, PA). Lyophilized culture of S.iniae GQ850377obtained from Department of Aquatic Animals Health, Faculty of Veterinary Medicine, University of Tehran, Iran, was used in this study. Nisin containing 2.5% active Nisin was purchased from SIGMA-ALDRICH Inc. (United Kingdom, EC 215-807-5). Nisin stock solution was prepared with 0.02 mol-1 HCl (pH 1.6), and was filter sterilized through a 0.45 um sterile, disposal and non-pyrogenic syringe filter (NALGENE, USA). Oncorhynchus mykiss were caught from a fish farm in Province of Alborz. The fish were further skinned, filleted and cut into square pieces (8 cm x 3 cm about 30 g). The fillets were packed in plastic bags and immediately transferred to Atomic Energy Organization of Iran, Nuclear Research Center for Agriculture and Medicine, Amirabad, Tehran, Iran. The fillets were exposed to Gamma irradiation (5 KGy, using cobalt-60 irradiator) to eliminate any likely background of S.iniae . At the same day, the irradiated fillets were transported in refrigeration condition to the laboratory.
GC-MS analysis resulted in the identification of 11 components representing 95.69% of the oil. The major compounds were cuminaldehyde (29.02%), alphaterpinene- 7-al (20.70%) and gamma-terpinene (12.94%).
Table1. Growth response (viable count) of S.iniae (at inoculation level of 105 cfu g-1) in fish fillets as affected by different concentrations of essential oil (EO), Nisin (N) and their combinations during 21 days of storage at 4°C.
logio cfu g"1 ± SD
on sampling days
6 9 b
12 15 18 21
The significant inhibitory (P < 0.05) effect of Nisin was observed during the storage of the treated filets and the number of organism reached 2.66 log10 cfu g-1 (after 3 d) and 2.10 log10 cfu g-1 (after 2 d) for Nisin concenterations of 0.25 and 0.75 ug ml-1, respectively. The inhibitory effect of EO was also significantly (P < 0.05) increased by adding the N even at its minimum concentration level (0.25 ug ml-1) used in this study e.g. at the combination of EO = 0.045 % and N = 0.25 ug ml-1, the log10 cfu g-1of the organism reached 3.41and <2 at the days 1 and 3 of the study, respectively.
The synergistic effect between essential oils (and their constituents) and other antimicrobial substances such as Nisin has been conclusively demonstrated (Yamazaki et al., 2004; Rajkovic et al., 2005). Basti(et al ., 2007) found significant inhibitory effect of Z. multiflora Boiss. essential oil on gram negative and gram positive bacteria (S. typhimurium and S. aureus) in BHI broth. Moosavy (et al., 2008) also obtained significant inhibitory effect of Z. multiflora Boiss. essential oil on both S. typhimurium and S. aureus in a liquid food (barley soup) model system which was increased by adding Nisin at 25 and 8 °C for S. aureus and only at 8 °C for S. typhimurium. In this study, we obtained similar results on S.iniae in fish fillets. Our results (Tables 1) showed significant inhibitory effect of EO on the studied micro-organism which was significantly increased by adding Nisin. At lower temperatures the proportion of unsaturated fatty acid chains of the lipids is increased resulting in higher membrane fluidity (Pol and Smid, 1999) which could facilitate the incorporation of lipophilic compounds as Nisin or cuminaldehyde. Therefore, this strong synergistic inhibitory effect of EO by Nisin reduces the total quantity of the preservatives incorporated in food products for adequate inhibitory effect on the bacteria and prevents any likely undesirable sensory effect of the essential oil.
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