Int. J. Aquat. Biol. (2016) 4(1): 11-16 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology Original Article Effects of Pediococcus pentosaceus as a probiotic on intestinal microbiota and body composition of Siberian sturgeon, Acipenser baerii Brandt, 1869 Fateme Moslehi1, Masoud Sattari*1, Alireza Shenavar Masouleh2 1Department of Fisheries, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Iran. 2Department of Aquatic Animal Health, International sturgeon research institute, Rasht, Iran. Article history: Received 19 October 2015 Accepted 14 December 2015 Available online 2 5 February 2016 Keywords: Sturgeon Probiotic Intestine microflora Body composition Abstract: An eight-week experiment was carried out to determine the effects of dietary Pediococcus pentosaceus as probiotic on the body composition and gut microbiota of Siberian sturgeon, Acipenser baerii. A total of 180 fish with mean body weight of 143±0.01g were randomly distributed into 12 200L fiberglass tanks as four treatments with three replicates, including groups fed with diet containing 2×107, 2×108 and 2×109 CFU g-1 of P. pentosaceus and probiotic-free as control group. Body composition of treatment groups was not influenced by P. pentosaceus except for fat and moisture. The bacteria had a significant colonization in the intestine of fish fed with supplemented diet with P. pentosaceus. High level of acid lactic bacterial load was found in the treatment fed with highest amount of the probiotic i.e. 2×109 CFU g-1. The results showed that application of P. pentosaceus has positive effect on the body composition and intestinal microflora of the A. baerii. Introduction Probiotics are commonly defined as ‘Live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance’ (Fuller, 1989) and play a beneficial role on the health of the host if used in adequate amount (FAO/WHO, 2002). The application of the probiotics in aquaculture is based on the concept that the balance of intestinal microorganisms in healthy animals increases resistance to diseases, and efficient digestion and maximum absorption of nutrients (Fuller, 1992; Verschuere, 2000). Probiotics can also improve the immune response system, reduce mortality, increase growth parameters, improve water quality, enhance stress resistance, and increase reproduction efficiency (Bairagi et al., 2002; Wang and Xu, 2004; Gomez-Gil et al., 2000; Martınez Cruz et al., 2012). The selection of suitable strain of a microorganism is a primary requirement for the use of probiotics. Different types of microalgae, yeasts, gram-positive and gram-negative bacteria are * Corresponding author: Masoud Sattari DOI: http://dx.doi.org/10.7508/ijab.2016.01.002 E-mail address: msattari@guilan.ac.ir considered as probiotics. Several bacterial strains which are common member of the non-pathogenic microflora are capable of inhabiting fish pathogenic bacteria in in-vitro assay (Joborn et al., 1997; Austin and Zhang, 2006; Gibson et al., 1998). This has been demonstrated for lactic acid bacteria (Joborn et al., 1997), Vibrio sp. (Austin and Zhang, 2006), and Bacillus sp. (Gibson et al., 1998) etc. Pediococcus pentosaceus is a gram-positive, facultative anaerobic, non-motile, and non-spore forming bacterium. It is a member of the industrially important lactic acid bacteria that grows on lactobacilli MRS broth at 37°C. Ferguson et al. (2010) stated that dietary supplementation of Pediococcus acidilactici increases the intestinal microbial load of red tilapia, Oreochromis niloticus. Merrifield et al. (2011) also reported that this bacterium has a significant effect on intestinal microflora of rainbow trout, Oncorhynchus mykiss. They reported that this probiotic has a proper colonization in the gut of treated fish species. Acipenseridae are the most commercially 12 Moslehi et al./ Effects of P. pentosaceus on intestinal microbiota and body composition A. baerii important fish species in the Caspian Sea basin. They are endangered due to loss of habitat, overfishing and deterioration of water quality (Bahmani et al., 2001). Among sturgeon fishes, the Siberian sturgeon, Acipenser baerii, is a proper candidate for aquaculture because it can easily be adapted to hand feeding in captive condition. Hence, in the present study, the effect of P. pentosaceus was examined as a probiotic on the body composition and intestinal microflora of the Siberian sturgeon, A. baerii. Materials and Methods Probiotic: Pediococcus pentosaceus was isolated from the intestine of Persian sturgeon, A. Persicus identifying by 16S rRNA gene based on Merrifield et al. (2009). The probiotic powder was prepared in Guilan Science and Technology Park (Rasht, Guilan Province, Iran) with 1012 bacterial count. Experimental diet: A commercial diet composed of 36% crude protein, 14% lipid, 1% phosphorus, 11% moisture, 10% ash and 4% fiber was used as a control diet (Chineh commercial feed), and fish were acclimated to this diet for two weeks before beginning the experiment. The experimental diet was supplemented with P. pentosaceus. It was prepared by slowly spraying the mixture of 500 ml saline serum with 0.2, 2 and 20 g probiotic powder on 10 kg diet with 2×107, 2×108 and 2×109 CFU g-1 bacterial count, respectively, according to Merrifield et al. (2009). Experimental design: The specimens of the Siberian sturgeons were obtained from Beheshti Sturgeon Reproduction and Restoration Center (Rasht, Guilan Province, Iran). A total of 180 fish having average body weight of 143±0.01 g were randomly distributed into 12 fiberglass tanks (with 2000 L capacity) each containing15 fish. Four treatments each with 3 replicates, including TA, TB, TC and C containing 2×107, 2×108, 2×109 CFU g-1 probiotic and probiotic free diet, respectively, were designed for this study. The treatments were fed on supplemented diet for 8 weeks at the rate of 2-2.5% of body weight 3 times a day (08:00, 14:00 and 20:00). The unconsumed feed was siphoned out six hrs after feeding. About 75% of water from each tank was changed daily with minimum disturbances to the fish. Intestinal Microbiology: At the end of experiment, after 24 hrs starvation, three fish from each tank were anesthetized by 200 ppm clove powder solution. The intestine of fish was removed in a sterile condition then washed thoroughly three times with saline serum (Merrifield et al., 2009). Samples were diluted with saline serum and then the specimens were added to TSA (Tryptic Soy Agar) culture medium to count total number of intestinal bacteria (Rawling et al., 2009) and MRS (Man Rogosa Sharpe) culture medium to count slightly lactic acid bacteria (Merrifield et al., 2009). Chemical analysis: At the end of experiment, three fish per tank were taken to analyze carcass composition according to protocols of AOAC (1990) for measuring moisture, ash, lipid and protein. Amount of moisture was determined by drying the fish muscle in oven at 103-105°C for 18-24 hrs. Thereafter, the dried samples were used to determine ash content by combusting them in a furnace. Amount of lipid was estimated by ether extraction in Soxhlet system. Micro-Kjeldahl system was used to determine the nitrogen content followed by multiplying the amount of nitrogen by 6.25. Statistical Analysis: Data analysis were performed using SPSS program as installed by the University of Guilan. Differences between treatments were performed using One Way ANOVA and Duncan tests. The results were considered significant at the 95% (P<0.05) level. Results The results showed that P. pentosaceus has affected on fat and moisture of fish fed with probiotic- supplemented diets. The highest level of moisture was found in the control group and a significant differences were found between probiotic-fed and control fish (P<0.05). The maximum level of fat was found in TA (fish fed with 2×107 CFU g-1 probiotic in the diet) and a significant difference was found between TA and the other groups in term of fat level. 13 Int. J. Aquat. Biol. (2016) 4(1): 11-16 The lowest level of fat was observed in control group. There were no significant differences between probiotic-fed fish and control group in terms of protein and ash (Table 1). TSA (Tryptic Soy Agar) culture medium was used to calculate the Total Viable Count (TVC). The results showed that TVC ranged from log 6.37 CFU g-1 in TA (fed with 2×107 CFU g-1 of the probiotic) to log 6.15 CFU g-1 in C (control group). However, no significant differences were found between treatments (Fig. 1). The results of MRS (Man Rogosa Sharpe) culture medium showed that the amounts of P. pentosaceus ranged from log 4.46 CFU g-1 in TA (fed with 2×107 CFU g-1 of the probiotic) to log 5.65 CFU g-1 in TC (fed with 2×109 CFU g-1 ) and the differences between the probiotic-fed fish were significant (P<0.05) (Fig. 2). Discussion In the present study, the isolated P. pentosaceus from the intestine of Persian sturgeon was used as a probiotic in the diet of Siberian sturgeon. Based on the results, the supplementation of P. pentosaceus as probiotic affected the fat and moisture of body composition but it had no significant effect on protein and ash of treated fish. Merrifield et al. (2010) used Chlorogloeopsis cyanobacter as probiotic on the diet of O. niloticus and reported no significant effect on the body composition viz. crude protein, crude lipid and ash of the treated fish. Hoseinifar et al. (2011) and Merrifield et al. (2011) found similar results in H. huso and O. mykiss, by using Saccharomyces cerevisiae and P. acidilactici, respectively, as probiotics. In contrast to our findings, Bagheri et al. (2008) showed that the diet containing commercial Bacillus probiotic has a significant effect on body composition of O. mykiss, but they did not observe significant difference between the groups consumed the lowest level of probiotic and control group. Bagheri et al. (2008) also observed significant Parameter Treatment Moisture (%) Ash (%) Fat (%) Protein (%) C a76.383±0.12 a1.063±0.006 b6.53±0.33 a18.03±0.05 TA b74.52±0.28 a1.133±0.006 a8.59±0.26 a16.14±0.05 TB b75.073±0.12 a1.163±0.09 b7.28±0.21 a17.6±0.03 TC b74.99±0.25 a1.136±0.008 b7.01±0.31 a17.82±0.05 Different superscript letters denote significant (P<0.05) difference. Table 1. Siberian sturgeon’s body composition at the end of the experiment. Figure 1. Effect of P. pentosaceus on Siberian sturgeon intestinal total viable count of bacteria (log). Figure 2. Effect of P. pentosaceus on the number of Siberian sturgeon intestinal P. pentosaceus bacterium (log). 14 Moslehi et al./ Effects of P. pentosaceus on intestinal microbiota and body composition A. baerii differences in carcass moisture and lipid content between the groups fed with the probiotic. El- Rhman et al. (2009) also showed that the body composition in O. niloticus, were significantly affected by the Pseudomonas and Micrococcus lateus as a probiotic, except for moisture. The difference between above mentioned findings and our results may refers to different bacteria and host. The results of the present study showed no significant differences in TVC among all treatments, but the highest number of P. pentosaceus was observed in group fed with 2×109 CFU g-1 probiotic. This means that probiotic has been colonized in the intestine of treated fish. An important action of probiotic is competitive exclusion by competition for adhesion sites, nutrient, oxygen, and by-product inhibitory compounds (Irianto and Austin, 2002). Intestinal microbial flora change after application of probiotic and then pathogen bacteria can decrease in host animal. In addition, probiotic can reduce the pathogen by secretion of bacteriocins and then reduce intestinal pH (Irianto and Austin, 2002). Similar to our results, Ferguson et al. (2010) reported that the number of lactic acid bacteria (LAB) is significantly higher in the intestine of the probiotic-fed O. niloticus. However there was no significant difference between the population of heterotrophic anaerobic and aerobic bacteria (Ferguson et al., 2010). Merrifield et al. (2011) stated that O. mykiss had a successful colonization of P. acidlactici in its digestive tract and the TVC of mucosal bacteria was significantly higher in the probiotic-fed fish. In another study, the TVC of heterotrophic bacteria in Huso huso were not affected by Saccharomyces cerevisia, but the levels of autochthonous lactic acid bacteria significantly increased in the fish fed with 2% yeast (Hoseinifar et al., 2011). The effects of Lactobasillus curvatus and Leuconostoc mesenteroides on the gut microflora of H. huso and A. persicus were examined by Askarian et al. (2011). They reported that the A. persicus fed with L. mesenteoides had higher levels of LAB than fish fed with L. curvatus and LAB mixture, but the highest level of LAB in H. huso was observed when the fish were fed with L. curvatus. They reported that the levels of LAB in H. huso and A. persicus fed with LAB mixture, were similar to control group. As conclusion, the findings of this study showed that application of P. pentosaceus as a probiotic has positive effect on the body composition and intestinal microflora of the Siberian sturgeon. References Abd El-Rhman A.M., Khattab Y.A.E., Shalaby A.M.E. (2009). Micrococcus luteus and Pseudomonas species as probiotics for promoting the growth performance and health of Nile tilapia, Oreochromis niloticus. Fish and Shellfish Immunology, 27: 175-180. AOAC (1990). Official Methods of Analysis of Association of Official Analytical Chemists (AOAC), 15th ed, Washington, USA. Askarian F., Kousha A., Salma W., Ringo E. (2011). The effect of Lactic acid bacteria on growth, digestive enzyme activity and gut microbita in Persian sturgeon (Acipenser persicus) and beluga (Huso huso) fry. Aquaculture Nutrition, 17: 488- 495. Austin B., Zhang X.H. (2006). Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Letters in Applied Microbiology, 43:119-124. Bagheri T., Hedayati S.A.A., Yavari V., Alizade M., Farzanfar A. (2008). Growth, survival and gut microbial load of rainbow trout (Onchorhynchus mykiss) fry given diet supplemented with probiotic during the two months of first feeding. Turkish Journal of Fisheries and Aquatic Sciences, 8: 43-48. Bahmani M., Kazemi R., Donskaya P. (2001). A comparative study of some hematological features in young reared sturgeons (Acipenser persicus and Huso huso). Fish Physiology and Biochemistry, 24: 135- 140. Bairagi A., Ghosh K.S., Sen S.K., Ray A.K. (2002). Enzyme producing bacterial flora isolated from fish digestive tracts. Aquaculture International, 10: 109- 121. FAO/WHO.2002. Guidelines for the evaluation of probiotics in food. London Ontario Canada. 11 p. Ferguson R.M.W., Merrifield1 D.L., Harper G.M., Rawling M.D., Mustafa1 S., Picchietti S., Balcazar J. L., Davies S.J. (2010). The effect of Pediococcus 15 Int. J. Aquat. Biol. (2016) 4(1): 11-16 acidilactici on the gut microbiota and immune status of on-growing red tilapia (Oreochromis niloticus). Journal of Applied Microbiology 109(3): 51-62. Fuller R. (1989). A review, probiotics in man and animals. Journal of Application in Bacteriology, 66: 365-378. Fuller R. (1992). History and development of probiotics. In: R. Fuller (Ed.). Probiotics, the scientific basis. London, UK: Chapman and Hall. pp: 1-8. Gibson L.F., Woodworth J., George A.M. (1998). Probiotic activity of Aeromonas media on the Pacific oyster, Crassostrea gigus, when challenged with Vibrio tubiashii. Aquaculture, 169: 111-120. Gomez-Gil B., Roque A., Turnbull, J.F. (2000). The use and selection of probiotics bacteria for use in the culture of larval organism. Aquaculture, 191: 259-270. Hoseinifar S.H., Mirvaghefi A., Merrifield D.L. (2011). The effects of dietary inactive brewer's yeast Saccharomyces cerevisiae var. ellipsoideus on the growth, physiological responses and gut microbiota of juvenile beluga (Huso huso). Aquaculture, 318: 90- 94. Irianto A., Austin B. (2002). Probiotics in aquaculture. Journal of Fish Diseases, 25: 633-642. Joborn A., Olsson C., Westerdahl A., Conway P.L., Kjellberg S. (1997). Colonization in the fish intestinal tract and production of inhibitory substances in intestinal mucus and faecal extract by Carnobacterium sp. strain K. Journal of Fish Diseases, 20: 383-392. Martínez Cruz P., Ibáñez A.L., Monroy Hermosillo O.A., Ramírez Saad H.C. (2012). Use of Probiotics in Aquaculture. ISRN Microbiology, ID 916845: 1-13. Merrifield D.L., Dimitroglou A., Bradley G., Baker R.T.M., Davies S.J. (2009). Probiotic applications for rainbow trout (Oncorhynchus mykiss walbaum) I. Effects on growth performance, feed utilization, intestinal microbiota and related health criteria. Aquaculture Nutrition, 10: 1365-2095. Merrifield D.L., Güroy D., Güroy B., Emery M.J., Llewellyn C.A., Skill S., Davies C.J. (2010). Assessment of Chlorogloeopsis as a novel microbial dietary supplement for red tilapia (Oreochromis niloticus). Aquaculture, 299: 128-133. Merrifield D.L., Bradley G., Harper G.M., Baker R.T.M., Munn C.B., Davies S.J. (2011). Assessment of the effects of vegetative and lyophilized Pediococcuc acidlactici on growth, feed utilization, intestinal colonization and health parameters of rainbow trout (Oncorhynchus mykiss walbaum). Aquaculture Nutrition, 17: 73-79 Verschuere L., Rombaut G., Verstraete W. (2000). Probiotic bacteria as biological agents in aquaculture. Microbiology and Molecular Biology, 64(4): 655-671. Wang Y.B., Xu Z.R. (2004). Probiotic treatment as method of biocontrol in aquaculture. Feed Research, 12: 42-45. Int. J. Aquat. Biol. (2016) 4(1): 11-16 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology چکیده فارسی و ترکیب بدن تاسماهی سیبری، روده عنوان پروبیوتیک بر فلور میکروبیبه Pediococcus pentosaceusاثرات Acipenser baerii Brandt, 1869 2ماسوله ، علیرضا شناور1*، مسعود ستاری1فاطمه مصلحی سرا، ایران.صومعه گیالن، طبیعی، دانشگاه منابع دانشکده، شیالت گروه1 رشت، ایران.، المللی تحقیقات ماهیان خاویاریبهداشت آبزیان، انستیتو بین گروه2 چکیده: میکروبی فلوربر ترکیب بدن و عنوان پروبیوتیکبه Pediococcus pentosaceusای ای به منظور تعیین اثرات تغذیهیک آزمایش هشت هفته یتری ل 200تانک فایبرگالس 12صورت تصادفی بین گرم به 143±01/0قطعه ماهی با وزن متوسط 180اجرا گردید. تعداد روده تاسماهی سیبری P. pentosaceusباکتری 910x2 1-CFU gو 710x2 ،810x2حاوی هایهای تغذیه شده با خوراکوهرشامل گ عنوان چهار تیمار با سه تکراربه ه باکتریوسیلرطوبت بههای تیمار به جز چربی و ترکیب بدن گروهبراساس نتایج .تقسیم شدندعنوان تیمار شاهد هو خوراک بدون پروبیوتیک ب P. pentosaceus داری در روده ماهیان تغذیه شده با خوراک حاوی پروبیوتیک شد. سطح سازی معنیرار نگرفت. باکتری سبب کلنیقتحت تاثیر یافت شد. نتایج نشان 910x2 1-CFU gتیمار عبارت دیگر ار پروبیوتیک بهدهای اسیدالکتیک در تیمار تغذیه شده با بیشترین مقباکتری باالی لود میکروفلور روده تاسماهی سیبری دارد. ومثبتی بر ترکیب بدن اتاثر P. pentosaceusباکتری داد که کاربرد .میکروفلور روده، ترکیب بدن ،خاویاری، پروبیوتیکماهی :کلمات کلیدی