Frontiers in Marine Science (Jun 2014)
Survival of the probiotic bacteria <i>Lactobacillus rhamnosus</i> in seawater and its bioencapsulation in the brine shrimp <i>Artemia</i>
Abstract
The probiotic Lactobacillus rhamnosus IMC 501® (Synbiotec Srl, Camerino, Italy) has demonstrated a positive effect in growth and early development of some fish species as clownfish (Amphiprion ocellaris, Cuvier 1830) and zebrafish (Danio rerio, Hamilton-Buchanan, 1822). The probiotic effects in A. ocellaris were an increase in body weight, a metamorphosis and skeletal head development acceleration and a reduction in the response to general stress (Avella et al. 2010). Besides this, an acceleration of the backbone calcification and gonadal differentiation were described in D. rerio (Avella et al. 2012). Despite those investigations, there is a lack of knowledge on the survival of L. rhamnosus in seawater and the bioencapsulation efficiency in Artemia. In the present study, the survival of L. rhamnosus in seawater and the efficiency of the probiotic bioencapsulation in 24h Artemia metanauplii were assessed. In a first experiment, 1 gr/l of probiotic bacteria was resuspended in 50 ml seawater and incubated in the conditions used for Artemia culture (26°C and gentle aeration). Samples were taken at 0, 15, 30, 60 min and at 3, 6, 24 and 30h. Serial dilutions were plated on selective MRS-Agar for probiotic bacteria isolation and on MRSA-B Agar (MRS-Agar supplemented by aniline blue for total CFU staining)(Fig.1). Plates were incubated for 7 days at 30°C in obscurity and CFU counted. In the bioencapsulation experiment, two conditions were tested: AS, Artemia standard (negative control, Fig.2) and AP, Artemia supplemented by probiotic (positive control, Fig.3). For AP bioencapsulation 30,000 Artemia 24h-metanauplii were suspended in 300 ml SSW and 1gr/l of probiotic was added. Samples were taken at 0 (after 15 min of bioencapsulation), 0.5, 1, 3, 6 and 24h. For that, the samples were filtered in a 125 µm mesh, washed and homogenized for microbiological analysis. Serial dilutions were plated on Marine Agar, TCBS Agar and MRSA-B Agar for quantification of total bacteria, total Vibrionaceae and lactic-acid bacteria, respectively. All experiments were performed in duplicate. The results obtained in the first test showed that the L. rhamnosus was able to survive in seawater during the whole experiment (30h), maintaining densities of 10e7 CFU/ml during the first 6h although decreasing progressively afterwards (10e3 CFU/ml at 30h). This allows adequate levels at sufficient time for Artemia to incorporate the probiotic. In fact, bioencapsulation test demonstrated that Artemia metanauplii were able to bioencapsulate the probiotic, reaching the highest concentration in Artemia after 30 min of bioencapsulation (10e4 CFU/Artemia). A slight further decrease (10e3 CFU/Artemia) was observed after 24h. Interestingly, L. rhamnosus reduced in 1Log total Vibrionaceae bacteria in Artemia during the 3 first hours. Therefore, 3 hours was the time established for the bioencapsulation protocol and further studies are in progress to determine the ability of Artemia metanauplii to maintain bioencapsulated L. rhamnosus once transferred to rearing tanks. Also, the capability of the probiotic to inhibit potential pathogenic or opportunistic bacteria will be assessed.
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