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Determination of Lactic Acid Bacteria Viability In the Small Intestine of Catfish (Pangasius djambal) by using a Radioisotope of 32P Irawan Sugoro1, Dimar Fairuz2, Ania Citraresmini1, and Adria Prilianti Murni1 1 Center for Application of Isotope and Radiation, National Nuclear Energy Agency Jl. Lebak Bulus Raya No. 49, Jakarta, Indonesia 2 Departement of Biology – Faculty of Science and Technology UIN Syarif Hidayatullah Jakarta, Indonesia ARTICLE INFO AIJ use only: Received date Revised date Accepted date Keywords: Catfish Viability Probiotic Lactic acid bacteria Radioisotope of 32P ABSTRACT The viability is important to be determined, as its probiotic potency in ther small instestine of fish. The result can be used as a basis to determine the feeding frequency of probiotic when applied to fish. The aim of this study is to gain information about Lactic Acid Bacteria (LAB) viability’s in the fish small intestine, by using 32P isotope technique. Catfish (Pangasius djambal) was used as a tested fish, and LAB with code of P2.1 PTB as the subject of the experiment. Before the viability tested, LAB has been labelled with radioisotope 32P, then mixed into catfish feed. The viability could be determined through the activity of 32P counted. The result showed that percentage of LAB viabilty in the saml intestine of catfish was declined until the day of 7. Percentage of LAB viability was decreased at an amount of 30% at the days of 3. Based on this result, the frequency feeding of LAB P2.1 PTB is could be done for every 3 days. © 2015 Atom Indonesia. All rights reserved INTRODUCTION The role of probiotic and its mechanism of action in the aquaculture is continuously being researched and developed. This is to consider with its potency to improve the quantity and quality of aquaculture production. Probiotics are living microorganisms which provide health benefit to the host, when taken in sufficient quantities [1]. The benefits of probiotics for aquaculture are improving the immune system [2], improve feed efficiency and protein retention [3] and enhance the growth rate [4]. One of the terms for microorganisms to be used as probiotics is the ability to colonize the intestinal epithelial cells, thus preventing or reducing the colonization of pathogenic microorganisms [5]. Research on the application of probiotics to fish in general is still centered on its ability to increase the growth rate of fish and the antimicrobial effects. However, research about probiotic viability in the digestive tract of its host is still rarely found. The ability of microorganisms to colonize the host’s digestive tract can be determined by the viability test using a radioisotope tracer compound. Radioisotope used in this study is radioisotope phosporus-32 (32P). The 32P had a unique trait, as a label and radiation source, therefore these compounds can be used as tracer in the experiments of molecular biology for animals, plants and bacteria [6]. Phosphorus-32 is a beta-emitter (1.71 MeV) with a half-life of 14.3 days, which 1 is able to replace the phosphorus compound where the cellular components made up, such as cell membranes, RNA, DNA, and ATP as energy cell [7] [8]. Thus it makes radioisotopes of 32P compounds easier to binds with microorganism cell in the viability test. This study aims to determine the viability of Lactic Acid Bacteria (LAB) in the small intestine of catfish to gain the empirical data about its potency as probiotics. The result of viability test can also be used as a basis to determine the feeding frequency of probiotic when applied to fish. A lactic acid bacterium (LAB) with code of P2.1 PTB was used in this experiment. This bacterium was isolated from cat fish intestine [9]. EXPERIMENTAL METHODS Growth Curve Analysis A lactic acid bacterium (LAB) was inoculated into NB media in the Erlenmeyer. The cultures were incubated with a shaker incubator for 0, 2, 4, 8, 12, 18, 24 to 30 hours for measure the number of cells by using of spectrophotometer (λ600 nm) and pH of media. Measurement of % incorporation of 32P Measurement of % incorporation was performed at the same time with measurement of growth curve. A 100 µL of 32P (KH232PO4; 10 mCi) was mixed into each of Erlenmeyer flask which also contained NB and LAB. Each of Erlenmeyer was coded and was incubated using shaker incubator for 0, 2, 4, 8, 12, 18, 24 and 30 hours. Each isolate in NB media was taken 5 ml and transferred into a tube. Each of sample tubes was centrifuged at 10,000 rpm, and then rinsed with distilled water 2 times. The supernatant was separated to measure its activity with Liquid Scintillation Counter (LSC). Percentage of incorporation was determined by using the following formula : % incorporation = (A-B)/A x (100%) Notes: A : activity of 32P in media on day of 0 (cpm) B : activity of 32P in media on day of t (cpm) Determination of Viability using 32P Lactic Acid Bacteria culture with the highest % incorporation in NB medium was inoculated into 100 ml of NB medium. The 100 µL of 32P was added and incubated subsequently at room temperature with 120 rpm agitation until the peak time of incorporation. After that, the supernatant and pellet of LAB culture was separated (precipitation) by centrifuging at 10,000 rpm and temperature of 40C, then rinsed with distilled water for 2 times. The pellet was diluted again with distilled water and mixed with the feed. This mixture was left for 2 hours. The pellets were inserted into an aquarium that already contains 20 cat fishes with length of 16-19 cm. The proportion of feed given was 3% of fish’s body weight/fish. Observations were conducted on days 0, 1, 3, 7 and 14. Bacterial viability was known by detecting the presence of LAB-labelled 32P in small intestine. It was cut from the end of the ventriculous to the base of the large intestine and the sample was soaked in 5 ml of HCl 35%. The mixture was homogenized with a vortex and centrifuged at a speed of 5000 rpm. The activity of supernatant was measured with LSC. Viability of the bacteria was determined by using the following formula : % viability = A/B x (100%) Notes: A : activity on day of t (cpm) B : activity on day of 0 (cpm) RESULTS AND DISCUSSION Growth Curve The growth curve of LAB showed there’s no adaptation phase in its phase (Figure 1). The bacteria was directly come into exponential phase. It could be due to the same media in inoculum and growth culture, hence the adaptation could be in shorter time. After 2 12 hours, the bacteria were entered stationary phase until the end of incubation (30 hours) but the decline or death phase was not occured. continue to divide. When the cells die, the phosphorous will be released back into the media and absorbed again by the living cells. Fig. 1. Comparison of cell growth with a pH value. Fig. 2. % incorporation of 32 P on bacteria cells. During the bacteria growth, pH of media was decreased, for until 4 hours incubation. After that, the pH was increased until 18 hours. It’s indicating that the bacteria utilized nutrition in media. Acid condition could be occured because of the degradation of organic compound that producing volatile fatty acid such as acetic, lactic, and propionic acid. Degradation of protein such as peptone, meat extract and yeast extract, which was contained in NB, producing ammonia and have become the causing of base condition in media [10]. The pH value is very effecting for the metabolism of bacteria, especially for enzyme rate [11]. Fig. 3. Mechanism of bacteria labelled by radioisotope of % Incorporation of 32P % incorporation of 32P in bacteria cell was increasing during incubation (Figure 2). The highest of % incorporation was occured at the hour of 12th at the observation. This time can be used as a reference for further studies to measure cell viability in the catfish intestine. The curve pattern showed similarity with the pattern of growth curve (Figure 1). It was suspected that during the division, incorporated radioisotope of 32P has become the part of cell such as DNA, membrane and ATP (Figure 3). This isotope only could be absorbed when the cells are still alive and still 32 P. Determination of LAB Viability The percentage of LAB viability in the small intestine of catfish was declined until the day of 7th (Figure 4). Percentage of LAB viability was decreased less than 30% at the day of 3rd. If the number of LAB cells decreased, it will effecting the performance rate of probiotic for the fish growth. We are expecting the LAB’s probiotic function will not be exhausted in the gut. The occurence of this condition could be due to the bacterial release with the excretion of feces, therefore only a small 3 amount could be colonized. It can be shown that until the day of 7th, the viability of LAB was 5%. Other possibility is the bacteria were died because it lost the ability to compete with indigenous bacteria [12]. It showed that these isolates were able to survived and colonized the small intestine of catfish. Description on how the bacteria could be viable can be seen in the figure 5. The results of this study showed that isolates of LAB with code P2.1 PTB had potentially become a candidate probiotic. In addition, the decrease of LAB viability on the day of 3rd could become the reference for the feeding frequency. It could be done for every 3 days. The application of LAB based on the viability data in the small intestine is useful for the reduction in production costs due to administration that probiotics must be given every day. CONCLUSION The viability of Lactic Acid Bacteria (LAB) could be determined by using a radioisotope of 32P. The frequency feeding of LAB with code P2.1 PTB was done for every 3 days. Fig. 4. Viability of LAB in the small intestine of catfish by using a radioisotope of 32P. Catfish REFERENCES 1. FAO ⁄WHO. Report of A Joint FAO ⁄WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria (2001). 2. Septiarini, E. Harpeni, dan Wardiyanto, EJournal of Rekayasa dan Teknologi dan Budidaya Perairan 1 (2012) 39. 3. Setiawati, J.E., Tarsim, Y.T. Adiputra dan S. Hudaidah, E-Jurnal Rekayasa dan Teknologi dan Budidaya Perairan 1 (2013) 151. Labelled bacteria 4. Jusadi, D., E. Gandara dan I. Mokoginta, Jurnal Akuakultur Indonesia 3 (2004) 15. Digestive tract Fig. 5. Viability of cell labelled- 32P in fish. Research by Dyvia et al. [13] revealed that the probiotic was able to live and reduced the amount of pathogenic bacteria in the intestine of fish larvae Puntius conchonius significantly. Vine et al. [14] examined the competition between 5 candidates of probiotic bacteria candidates and 2 pathogenic bacteria to attach to the fish's intestinal mucus in vitro. 5. Balcazar, J.L., I.D. Blas, I. Ruiz-Zarzuela, D. Cunningham, D. Vendrell and J.L. Muzquiz, Veterinary Microbiology 114 (2006) 173. 6. Creager, A.N.H., Studies in History and Philosophy of Biological and Biomedical Sciences 40 (2009) 29. 7. Wyttenbach, A and A.M. Tolkovsky, Molecular & Cellular Proteomics 5 (2006) 553. 8. Purnami, S., M. Syaifudin dan Giyatmi, JFN 3 (2009) 11. 4 9. Hanifa, D.. Thesis. Hidayatullah (2014). UIN Syarif 10. Pelczar, M. J. & Chan, E. C. 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