Int. J. Aquat. Biol. (2016) 4(2): 96-101; DOI: ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology Original Article The study of enrichment capability of adult Artemia franciscana with singular or combined administration of Pediococcus acidilactici and fructooligosaccharide Mahmood Azimirad*1, Saeid Meshkini2, Nasrollah Ahmadifard1, Seyed hossein Hoseinifar3 1Fisheries Department, Faculty of Natural Resources, Urmia University, Iran. 2Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Iran. 3Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran. Article history: Received 9 February 2016 Accepted 12 April 2016 Available online 2 5 April 2016 Keywords: Probiotic Synbiotic P. acidilactici Enrichment Artemia franciscana Abstract: The present study investigates the possibility of enriching adult Artemia franciscana with singular or combined administration of Pediococcus acidilactici and fructooligosaccharide (FOS). The experiment was conducted in a completely randomized design with four treatments, including synbiotic, P. acidilactici +FOS (T1), probiotic, P. acidilactici (T2), prebiotic, FOS (T3) and control (T4). To evaluate the enrichment of adult Artemia with each treatment, sampling was performed at 2, 4 and 6 hrs post enrichment. The bacterial counts was measured using the microbial culture and expressed as log CFU per g Artemia. A pre-experiment has been designed and probiotic was used in three levels (107, 108 and 109 CFU per litter of suspension) and prebiotic was used in three levels of 1, 2 and 5 g per litter of suspension. Based on pre experiment results, 108 CFU per litter of probiotic and 5 g per litter of prebiotic was selected. The results of this experiment showed that over time, consumed bacteria increased by adult Artemia and there was a significant difference between sampling in terms of ingested bacteria. The highest bacterial count (6.78±0.03 log CFU g-1) was observed 6 hrs after the start of enrichment. Based on microbial culture, the number of bacteria P. acidilactici in T1 and T2 was significantly higher than those in T4 (control) and T3 (prebiotic). There was no significance difference between T2 (probiotic) and T1 (synbiotic). In conclusion, the results of this study showed that adult Artemia in a short time (about 4 hrs) can retain a large amount of probiotic bacteria. Introduction During the past decade, the use of probiotics in aquaculture is become prevalent and can overcome many of the problems associated with bacterial diseases. The use of probiotics as a food supplement for farmed animals goes back to the 1970s (Fuller, 1989). Various types of microalgae (Tetraselmis), yeasts (Phaffia and Saccharomyces), Gram-positive bacteria (Bacillus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Strepto- coccus and Weissella) and Gram-negative bacteria (Aeromonas, Alteromonas, Photorhodobacterium, Pseudomonas and Vibrio) have been studied as probiotics (Gatesoupe et al., 2010). The doubts in the use of probiotics such as the non-guaranteed viability * Corresponding author: Mahmood Azimirad E-mail address: mahmoodazimirad@gmail.com of the probiotics in the gastrointestinal tract, necessity of competition autochthonous microbiota, the colonization ability and the long-term sustainability of the colony, caused the researchers to suggest the idea of prebiotic (Gibson, 2004; Mahious and Ollevier, 2005). The prebiotics increase numbers and dominance of beneficial bacteria due to selectively fermentation (Roberfroid, 2007). Researches in this field have shown that non-digestible oligosaccharides such as inulin and oligofructose are the most important materials that have prebiotic function (Flickinger et al., 2003). Because of the inability of probiotic species to form stable masses and maintain dominance in the aquatic microbiota, simultaneous 97 Int. J. Aquat. Biol. (2016) 4(2): 96-101 use of probiotics species with appropriate prebiotics (synbiotic) as a substrate to increase dominance and sustainable growth of probiotics bacteria has been suggested (Hoseinifar et al., 2015). Regarding the use of synbiotics in aquaculture, few studies have been performed and their positive effects on physiology and immunity have been reported (Rodriguez-Estrada et al., 2009; Merrifield et al., 2010; Montajami et al., 2012; Abid et al., 2013; Hosseinifar et al., 2015). However, the use of synbiotics during the early life stages of fish through the enrichment of live food and the effects on growth, physiology and immunity has not been considered. The use of synbiotic in Artemia could be considered as a food for Artemia, and also could affect the intestinal microbiota, immune system and increase resistance to pathogenic bacteria, enhance health and reduce the risk of disease outbreaks. Artemia is among the live foods that widely used in the culture of ornamental fishes due to the high nutritional value, the proper size and the enrichment capability (Sorgeloos et al., 2001). Artemia can be used as the carrier of particles used in aquaculture such as nutrients (fatty acids, vitamins, etc.), antimicrobial substances, vaccines and probiotics (Ziaei-Nejad et al., 2006) Application of live, useful and non-pathogenic bacteria to culture medium or Artemia culture can positively affects cultured fish species by improving the intestinal microbial microbiota, eliminating harmful bacteria and improving the nutritional value of Artemia (Havennar et al., 1992; Ringo et al., 1992). The number of bacteria in the Artemia exponentially increases at the time of Artemia hatching and enrichment processes by nutrients (Ritar et al., 2004). It also has been observed that during the early stages of fish development, the increase in the number of bacteria in the intestinal microbiota of fish, is mainly associated with the bacteria in live food (Makridis et al., 2000). It can be concluded that mortality increases in the intensive culture of early life stages of fish along with elevation of the number of opportunistic bacteria in the fish intestine. Therefore, control of bacterial population in the live feed may lead to higher survival rates of fish larvae and profitability in hatcheries (Olafsen, 2001). Therefore, this study was conducted to study enrichment capability of adult Artemia franciscana with singular or combined administration of Pediococcus acidilactici and fructooligosaccharide (FOS) as probiotic and prebiotic, respectively. Materials and Methods Artemia culture conditions and Bacterial strain: Artemia cysts (A. franciscana) was obtained from Great Salt Company, USA. Chorionic layer of cysts were separated using sodium hypochlorite during decapsulation. Hatching of the decapsulated cysts was performed by a cone-shaped container with a volume of 120 litters and sea water (with salinity of 30 g per litter). Cysts were incubated with a density of 5 g per litter at 30°C with 2000 lux lighting conditions and vigorous aeration (Sorgeloos et al., 1986). Artemia naupliis were transferred to culture environment after hatching. The culture environment was a 150L cone-shaped plastic containers that were aerated by aeration pipes connected to the central pump. Nauplii were fed during the first few days by spirulina algae (Spirulina platensis), and then by a mixture of rice bran, baker's yeast and spirulina. Feeding was performed three times a day with an interval of 4 hrs. Stocking density was three nauplii per ml and culture period was 20 days to reach sexual maturity (Teresita et al., 2005). During culture period, all physical and chemical parameters were measured and recorded daily. Physical and chemical factors, including water temperature, salinity, dissolved oxygen, light and pH were 28.69°C, 32 g L-1, 7.75 mg L-1, 1500 lux and 7.88, respectively. The used commercial probiotic used in this experiment was obtained from Tak Gene Company with Pedi-guard brand name contains bacteria P. acidilactici to amount of 1×1010 CFU g-1. Prebiotic, Oligofructose (Raftilose P95) was supplied from Orafti Company, Belgium. Enrichment of synbiotic to adult Artemia: For 98 Azimirad et al./ Enrichment capability of Artemia with of P. acidilactici and Fructooligosaccharide enrichment of the adult Artemia by synbiotic, combinations of the probiotics and prebiotics were used along with singular administration of the probiotic and prebiotic as described in Table 1. For preparation of the synbiotic suspension, first a ratio of 0.1:10 lecithin and water at 40°C were poured into a clean and dry beaker and mixed using an electric mixer. Then, the rapeseed oil was added to the solution and mixed very well by mixer. The ratio of lecithin, oil and water in suspension was 0.1, 1 and 10, respectively. To evaluate the diameter of oil particle, some samples were poured on slide and observed under light microscope. 150 ml was separated from the prepared suspension, 700 mg probiotic, P. acidilactici and 100 mg of prebiotic, FOS were transferred to the beaker and were uniformed with an electric mixer, then mixed in 2 litters of seawater. The adult Artemia with the number of 4000 was placed inside the culture container (Agh and Sorgeloos, 2005; Daniels et al., 2013) (Table 1). To determine the best level of the probiotic, P. acidilactici and prebiotic FOS in enrichment suspension of Artemia, a pre-experiment has been designed and probiotic was used in two levels with an amount of 107 CFU and 108 CFU per litter of suspension, and prebiotic was used in two levels of 2 and 5 g per litter of suspension. The results of this pre-experiment has been used as level of probiotic, prebiotic and synbiotic in this experiment. To examine the process of enrichment, sampling was performed from the all treatment at 2, 4 and 6 hrs (Dhont and Lavens, 1996). In each sampling time, 100 ml (containing 0.5 g of adult Artemia) were collected using a sterile pipette and were transferred to a filter with a mesh size of 300 micrometer, then to elimination of the bacteria in the external surface of Artemia body, were washed for 60 seconds in a salt solution, Benzalkolium chloride (0.1%) and again were washed with sterile water and after that, water of samples was taken after a while (Makridis et al., 2000). The sterile samples were weighted and transferred to sterile porcelain mortar. After the homogenization of samples using a sterile saline solution (0.87% w/v), dilutions of 10-1 to 10-7 were prepared. From prepared dilutions, under sterile conditions, 0.1 mm was removed and spread on surface of the MRS agar plates (for determine the number of lactic acid bacteria). The incubation of plates was conducted for 3-5 days in an incubator at a temperature of 30°C and under aerobic conditions. After the incubation period, the bacteria were counted, and recorded according to the logarithm of the colony unit (the number of bacterial colonies grown on culture medium × dilution coefficient -1) per g of Artemia (Rengpipat et al., 1998). Pediococcus acidilactici was identified based on apparent characteristics, gram staining and also standard biochemical tests such as phenol red, citrate, indole, motion and methyl red (Peter and Sneath, 1986). Statistical analysis: Statistical analysis was performed using the SPSS software package (version 18). One-way ANOVA was used for comparison between treatments and Duncan's multiple range test was used for the comparison of means at confidence level of 0.05% (P<0.05). Results The effects of different treatments and sampling time on the amount of bacteria in the Artemia is shown in Table 2. The results indicated that probiotic bacteria in each sampling time, were successfully enriched in Artemia. The enrichment trend of A. franciscana was Treatments Rapeseed oil suspension (ml L-1) Probiotic, P. acidilactici (mg L-1) Prebiotic, FOS (mg L-1) Synbiotic (T1) 150 700 100 Probiotic (T2) 150 700 0 prebiotic (T3) 150 0 100 Control (T4) 150 0 0 Table 1. The enrichment condition adult Artemia in different treatments. 99 Int. J. Aquat. Biol. (2016) 4(2): 96-101 different at different sampling times. In terms of the enrichment time, the results showed significant difference in the capability of Artemia enrichment (P<0.05). Regarding the synbiotic and probiotic treatments at 4 and 6 hrs enrichment, there was no significant difference in the number bacteria per g of Artemia (P>0.05). The results of bacterial count in prebiotic and control treatments showed that the concentration of lactic acid bacteria in these treatments were lower than 20 CFU g-1 and no significant difference were observed between sampling times (P>0.05). The bacterial counts in treatments enriched by probiotic and synbiotic were almost at the same level, but increasing trend was observed along with enrichment period (Fig. 1). However, no statistically significant differences was observed between bacterial level in adult Artemia at 4 and 6 hrs of enrichment (P>0.05). Discussion In this experiment, bacterial levels used in the enrichment solutions at all sampling times were at a level of 1010 CFU g-1. Gomez-Gil et al. (1998) were applied the concentrations of 107 CFU g-1 and 108 CFU g-1 of Vibrio parahaemolyticus and V. alginolyticus, respectively, during enrichment experiment of A. franciscana and reported the same pattern in their changes at different sampling times. Similar study were not observed regarding to enrichment of adult Artemia with probiotic and synbiotic. Based on the results, the concentration of bacteria in adult Artemia showed a positive correlation with the duration of enrichment, similar to the results of Parta et al. (2003) during the enrichment of A. franciscana nauplii with yeast (Saccharamyces baulardii) which revealed accumulation of yeast in nauplii at a level of 3.5×103 CFU g-1. However, enrichment of A. franciscana Hours Treatments Synbiotic (T1) Probiotic (T2) Prebiotic (T3) Control (T4) 2 5.50±0.07b 5.58±0.04b 1.15±0.02a 1.09±0.05a 4 6.61±0.07b 6.67±0.03b 1.04±0.02a 1.23±0.04a 6 6.71±0.04b 6.78±0.03b 0.83±0.07a 1.15±0.04a Table 2. The cultivable lactic acid bacteria levels (log CFU g-1 Artemia) in A. fransiscana enriched in pre-, pro- and symbiotic. Figure 1: The process of enrichment adult Artemia enrichment at different times in different treatments. 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 2 hour 4 hour 6 hour L O G C F U /G synbiotic Probiotic Prebiotic Control 100 Azimirad et al./ Enrichment capability of Artemia with of P. acidilactici and Fructooligosaccharide nauplii with two strains of Vibrio sp. showed different patterns, so that, attached bacteria to Artemia nauplii began to increase at first 30 minutes of enrichment, then suddenly declined at 8 hrs after enrichment and again a sharp rise occurred at 24 hrs at the levels of bacteria in nauplii which all naupliis died at the end of this time (Gomez-Gil et al., 1998). The A. urmiana showed a gradual trend in enrichment with mentioned probiotic increased over time. Furthermore, Campbell et al. (1993) enriched A. franciscana with the formalin-killed V. angualiurum and showed that when the concentration of bacteria in enrichment solution is 1.5×107 CFU g-1, the maximum accumulation of Vibrio sp. in the Artemia nauplii is happened at 60 min. Moreover, in concentrations lower than 5×106 CFU g-1, the maximum accumulation is occurred at 120 min after the start of enrichment. Changes in the number of bacteria in the A. franciscana is not limited by the number of bacteria in enrichment suspension and the same results reported by Makridis et al. (2000) in the enrichment of A. franciscana nauplii with the probiotic bacteria. In conclusion, the results of this experiment indicated that adult Artemia has high ability to be enriched with the probiotic bacteria, P. acidilactici and bacterial levels in Artemia that is increased along with enrichment time. Acknowledgments The authors express their thanks to A. Esteghlalian for his assistance during experiment. 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(2016) 4(2): 96-101 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology چکیده فارسی Pediococcus acidilacticiبا کاربرد منفرد یا همزمان Artemia franciscanaسازی آرتمیا بالغ، مطالعه امکان غنی فروکتوالیگوساکارید و 3فر حسینی حسین سید ،1فرد احمدی نصراهلل ،2مشکینی سعید ،، 1*راد عظیمی محمود .ایران ،ارومیه ارومیه، دانشگاه طبیعی منابع دانشکده شیالت، گروه1 .ایران ،ارومیه ارومیه، دانشگاه طبیعی منابع دانشکده ،دامپزشکی دانشکده غذایی، مواد بهداشت گروه2 .ایران ،گرگان گرگان، طبیعی منابع و کشاورزی علوم دانشگاه ،زیست محیط و شیالت دانشکده آبزیان، پرورش و تکثیر گروه3 چکیده: Pediococcus acidilactici از ترکیبی بیوتیکسین با بالغ (Artemia franciscana) فرانسیسکانا آرتمیا سازی غنی امکان مطالعه این در P. acidilactici بیوتیک سین شامل تیمار چهار قالب در تصادفی کامالً طرح صورت به آزمایش این. گرفت قرار بررسی مورد فروکتوالیگوساکارید و جهت. گردید اجرا (T4) شاهد تیمار و (T3) ساکارید فروکتوالیگو بیوتیک پری ،P. acidilactici (T2) پروبیوتیک ،(T1) فروکتوالیگوساکارید و هایباکتری تعداد و انجام بردارینمونه سازی،غنی شروع از پس ساعت 6 و 4 ،2 هایزمان در تیمارها، از یک هر با بالغ آرتمیای سازیغنی ارزیابی که داد نشان نتایج. گردید شمارش آرتمیا گرم هر در CFU لگاریتم حسب بر هاباکتری تعداد میکروبی، کشت از پس آرتمیا بدن داخل در موجود نسبت الغب آرتمیای به شده الحاق باکتری تعداد و سازیغنی زمان بین و بوده بیشتر بالغ آرتمیای توسط شده مصرف هایباکتری زمان، گذشت با زمان با که گردید الحاق بالغ آرتمیای به( 2/0 × 615 ± 56/5) باکتری تعداد بیشترین سازی،غنی ساعت 6 از بعد(. P<50/5) دارد وجود یدارمعنی باکتری تعدادکه داد نشانهمچنین نتایج . نداد نشان داری معنی اختالف سازیغنی ساعت 4 زمان مدت با ولینشان داد دارمعنی اختالف ساعت 2 P. acidilactici باشدمی بیوتیک پری فقط واجد تیمار و شاهد تیمار از بیشتر داریمعنی میزان به 2 و 1 تیمارهای در (50/5P>.) اختالف اما ( ساعت 4 حدود) کوتاهی زمان مدت در بالغ آرتمیایداد که نشان همچنین نتایج. نگردید مشاهده بیوتیکسین و پروبیوتیک تیمار بین داریمعنی .نماید ذخیره خود در را پروبیوتیک باکتری از باالیی میزان تواندمی .فرانسیسکانا آرتمیا سازی، غنی فروکتوالیگوساکارید، ،Pediococcus acidilactici بیوتیک،سین :کلمات کلیدی