The Study And Application Of Polysaccharides Immunostimulants And Probiotics For Sea Cucumber (apostichopus Japonicus Selenka)

Three feeding experiments were conducted to investigate the effects of three main polysaccharides immunostimulants includingβ-glucan, peptitoglycan and chitosan on growth, immunity and disease resistance in sea cucumber (Apostichopus japonicus Selenka). Nineteen potential probiotics were isolated from intestine of sea cucumber, culturing water and the mud of the pond. Five potential probiotics were selected for further study according to inhibitory ability against phathogen, colony morphologic, species and the source of isolation. Then, the effects of those five potential probiotics on growth, immunity and disease resisitance in juvenile sea cucumber (Initial weight 0.2-0.4 g) were studied. Three strains (T13, TC22 and EN25) of five potential probiotics were found to be beneficial to juvenile sea cucumber. Then three feeding experiments were conducted to investigate the effects of T13, TC22 and EN25 on growth, immunity, microflora and disease resisitance in young sea cucumber (Initial weight 3-5 g). The interaction between three probiotics and fructooligosaccharide respectively was also studied. At the end of the experiment, those three probiotics were identified and the better condition for growth of three probiotics was preliminarily studied. The results are summarized as follows:1. An 8 week feeding trial was conducted to determine the effects of dietary supplementation ofβ-glucan on the growth, immunity and resistance of sea cucumber against Vibrio splendidus infection. A basal diet was formulated to contain 20.6% crude protein and 4.8% crude lipid. Two levels (1.25 and 2.50 g/kg) ofβ-glucan were added to the basal diet to replace wheat. After the feeding trial, a V. splendidus injection challenge was executed to test the effects ofβ-glucan on disease resistance. Enhanced growth was observed in sea cucumber fed the diet supplemented with 1.25 g glucan/kg, but not in sea cucumber fed the diet supplemented with 2.50 g glucan/kg. The coelomocyte phagocytosis activity of sea cucumber fedβ-glucan supplemented diets was significantly (P<0.05) higher than those of sea cucumbers fed the basal diet. In addition, phenoloxidase activity of coelomocytes was significantly (P<0.05) enhanced by dietary supplementation ofβ-glucan. Sea cucumbers fed 1.25 g/kg glucan had a significant (P<0.05) increase in respiratory burst of coelomocytes, compared to sea cucumber fed diet containing 2.50 gβ-glucan/kg; however, coelomocyte respiratory burst of sea cucumber fed 2.50 gβ-glucan/kg diet was not significantly different from those of sea cucumber fed the basal diet. The challenge test showed that dietary supplementation ofβ-glucan at inclusion level of 1.25 g/kg conferred significant protection to sea cucumber against V. splendidus infection. However, protective effect ofβ-glucan supplementation at 2.50 g/kg was marginal. It is concluded that dietaryβ-glucan has potential for use in diet formulations of sea cucumber to limit the adverse effects of V. splendidus; however, dosage should be an important consideration in administration.2. An 8 week feeding experiment was conducted to determine the effects of dietary peptidoglycan on growth, immunity and resistance of sea cucumber against Vibrio splendidus infection. The basal diet was supplemented with 0, 200 and 500 mg/kg peptidoglycan to formulate three experimental diets. Results showed that growth of sea cucumber was improved with increasing dietary peptidoglycan. But no significant difference was observed between the control and peptidoglycan supplementation groups. The coelomocyte phagocytosis activity of sea cucumbers fed peptidoglycan supplemented diets was significantly (P<0.05) higher than those of sea cucumbers fed the basal diet. However, respiratory burst of sea cucumber coelomocytes was not significantly influenced by dietary peptidoglycan (P>0.05). Phenoloxidase activity of coelomocytes was significantly increased in animals fed the diet with 200 mg/kg peptidoglycan, but it was not affected by dietary peptidoglycan at 500 mg/kg compared to control (P>0.05). The acid phosphatase activity was decreased with increasing dietary peptidoglycan, and it was significantly lower in sea cucumbers fed the diet with 500 mg/kg peptidoglycan compared to animals fed other two experiment diets (P<0.05). The challenge test showed that dietary peptidoglycan at inclusion level of 200 mg/kg led to significant protection to sea cucumber against V. splendidus infection (P<0.05). It is concluded that dietary peptidoglycan has potential for use in diet formulations of sea cucumber to limit the adverse effects of V. splendidus.3. 8 week feeding experiment was conducted to determine the effects of dietary chitosan on growth, immunity and resistance of sea cucumber against Vibrio splendidus infection. The basal diet was supplemented with 0, 2.5, 5 and 10~ g/kg chitosan to formulate four experimental diets. Results showed that growth of sea cucumber was not significantly affected by dietary chitosan (P>0.05). The coelomocyte density and coelomocyte phagocytosis activity of sea cucumbers fed chitosan at 10 g/kg was significantly (P<0.05) higher than those of sea cucumbers fed the basal diet. However, respiratory burst of sea cucumber coelomocytes was not significantly influenced by dietary chitosan (P>0.05). There was no significant difference in phenoloxidase activity of coelomocytes (P>0.05). The acid phosphatase activity was significantly decreased in sea cucumbers fed diets with chitosan compared to animals fed the control diet (P<0.05). The challenge test showed that dietary chitosan conferred better protection to sea cucumber against V. splendidus infection, but no significant difference was observed compared to control group (P>0.05). Therefore, further studies on application of chitosan in sea cucumber are necessary.4. A total of 182 strains were isolated from the intestine of healthy sea cucumbers, intestine and skin of sea cucumbers affected by skin ulcer, mud and sea water of the pond cultured sea cucumbers by 2216E, TSB and MRS bacterial media. All selected isolates were tested for their inhibitory activity to two pathogens of sea cucumber, Vibrio splendidus and Pseudoalteromonas nigrifaciens by spot-on-lawn method. The 19 isolates of those strains were found to be inhibitory to both pathogens or one of them. Morphological analysis was conducted including colony morphologic and Gram coloration. The potential probiotics were identified by 16S rDNA preliminarily. The results showed that all 19 isolates were Gram-positive and Bacillus strains. All strains had amylase activity and 18 of them had proteinase activity. In the end, 5 isolates of 19 strains were selected for the safety test according to source of isolate, colony morphologic and inhibition against pathogens. The safety test of 5 selected potential probiotics showed that these 5 isolates could not induce disease and mortality of sea cucumber. Therefore, these 5 isolates (TMRC14, TC116, T13, TC22 and EN25) could be used for further study to investigate whether these 5 isolates can be applied for sea cucumber to increase the health of animals or not.5. The effects of potential probiotic TMRC14 and TC116 on growth, immune capacity, and disease resistance in juvenile sea cucumber Apostichopus japonicus were studied. Animals were fed with diet containing TMRC14 or TC116 at 0, 10~5, 10~7 and 10~9 CFU/g for 30 days. At the end of the feeding trial, fifteen sea cucumbers from each aquarium were sampled for immune indices measurement. Then twenty sea cucumbers of every replicate were challenged by immersion with Vibrio splendidus. The results revealed that administration of TMRC14 at all levels or TC116 at 10~5-10~7 CFU/g diet had no significant effect on the growth of sea cucumbers (P>0.05), but the growth of sea cucumber fed with diet containing TC116 at 10~9 CFU/g was significantly decreased (P<0.05). No statistical difference was found in the total coelomocytes counts in sea cucumbers fed the diet containing TMRC14 or TC116 (P>0.05). Phagocytosis of sea cucumber coelomocytes was significantly improved in animals fed with TMRC14 at 10~9 CFU/g diet or TC116 at 10~7 CFU/g diet (P<0.05). Dietary TMRC14 at 10~9 CFU/g increased the respiratory burst activity of sea cucumber coelomocytes, although there was no significant difference (P>0.05). Respiratory burst activity of sea cucumber fed the diet containing TC116 at 10~7 CFU/g was significantly higher than the control and other two groups. The cumulative mortality after V. splendidus challenge was decreased in the sea cucumbers fed with TMRC14 at 10~7-10~9 CFU/g or TC116 at 10~5-10~7 CFU/g, however no significant differences were observed between experimental and the control groups (P>0.05). Therefore, the effect of potential probiotic TMRC14 or TC116 on growth, immunity and protective effect against V. splendidus in sea cucumber were marginal.6. The effects of potential probiotic T13, TC22 and EN25 on growth, immune capacity, and disease resistance in juvenile sea cucumber Apostichopus japonicus were studied. Animals were fed the diet containing T13, TC22 and EN25 at 0, 10~5, 10~7 and 10~9 CFU/g for 30 days. At the end of the feeding trial, fifteen sea cucumbers from each aquarium were sampled for immune indices measurement. Then twenty sea cucumbers of every replicate were challenged by immersion with Vibrio splendidus. The results revealed that administration of T13 or TC22 at 10~9 CFU/g diet had significant effect on the growth of sea cucumbers (P<0.05), but dietary EN25 had no effect on the growth of animals (P>0.05). Phagocytosis, respiratory burst activity and total nitric oxide synthase (T-NOS) activity of sea cucumber coelomocytes were significantly improved in animals fed the diet with T13 or TC22 at 10~9 CFU/g diet or EN25 at 10~7 CFU/g diet (P<0.05). No statistical difference was found in the total coelomocytes counts and superoxide dismutase (SOD) activity in sea cucumbers fed with diet containing T13 or TC22 at 10~5-10~9 CFU/g or EN25 at 10~5-10~7 CFU/g (P>0.05), however, the SOD activity was significantly decreased in the group fed with diet containing EN25 at 10~9 CFU/g(P<0.05). Dietary supplementation of T13, TC22 or EN25 did not significantly influence acid phosphatase (ACP) activity of sea cucumber coelomocytes. The cumulative mortality after V. splendidus challenge was decreased significantly in the group fed the diet with T13 or TC22 at dose of 10~9 CFU/g feed or EN25 at 10~7 CFU/g feed (P<0.05). The results of present study confirmed the potential beneficial effects of T13, TC22 and EN25 as dietary probiotic in sea cucumber.7. The effects of probiotic T13 and the synergistic effects of T13 and fructooligosaccharide (FOS) on growth, immune response, microflora and disease resistance in sea cucumber Apostichopus japonicus (initial weight 4.893±0.028 g) were studied. Animals were fed on diet with T13 at doses of 0, 10~7, and 10~9 CFU/g feed with or without 0.5% FOS for 56d. At the end of the feeding trial, six sea cucumbers per tank were sampled for bacterial quantification and immune indices measurement. Then all the sea cucumbers left were challenged by injecting Vibrio splendidus. The results revealed that the SGR of sea cucumber fed the diet containing different doses of T13 with or without FOS was higher than control group, though no significant difference was observed (P>0.05). The phagocytosis activity, respiratory burst activity, phenoloxidase activity and total nitric oxide synthase of sea cucumber fed with diet containing T13 were improved, among these four indices, phenoloxidase activity was significantly higher than control (P<0.05). The total viable bacteria and Vibrio bacteria counts were enhanced significantly in sea cucumber fed with T13 at dose of 10~9 CFU/g feed (P<0.05). The cumulative mortality after V. splendidus challenge was decreased significantly in the group fed with T13 at dose of 10~9 CFU/g feed (P<0.05). The phagocytosis activity, respiratory burst activity and phenoloxidase activity of sea cucumbers fed with 0.5% FOS were increased significantly. And the sea cucumber fed with 0.5% FOS had a significant lower cumulative mortality than the control group (P<0.05). The total viable bacteria counts of sea cucumber were not influenced by dietary FOS with or without T13 (P>0.05). The total Vibrio bacteria counts was significantly decreased in sea cucumber fed with 0.5% FOS (P<0.05), however, it was not affected in sea cucumber fed diet containing FOS and T13 (P>0.05). The combination of 0.5% FOS with T13 increased the phagocytosis activity, respiratory burst activity and phenoloxidase activity of sea cucumbers without significant difference compared to control (P>0.05). The total nitric oxide synthase of sea cucumber fed with diet containing FOS and T13 at 10~9 CFU/g was significantly increased compared to control (P<0.05). The combination of 0.5% FOS with T13 improved disease resistance of sea cucumber compared with control (P<0.05).8. The effects of probiotic TC22 and the synergistic effects of TC22 and fructooligosaccharide (FOS) on growth, immune capacity, microflora and disease resistance in sea cucumber Apostichopus japonicus (initial weight 4.918±0.022 g) were studied. Animals were fed the diet with TC22 at doses of 0, 10~7, and 10~9 CFU/g feed with or without 0.5% FOS for 56d. At the end of the feeding trial, six sea cucumbers per tank were sampled for bacterial quantification and immune indices measurement. Then all the sea cucumbers left were challenged by injecting Vibrio splendidus. The results revealed that different doses of TC22 with or without FOS had no significant influence on sea cucumber growth (P>0.05). However, along with the increasing of TC22 dosage, the phagocytosis activity, respiratory burst activity and phenoloxidase activity of sea cucumber were improved significantly (P<0.05), while the total nitric oxide synthase acitivity of animals was not significantly influenced (P>0.05). The total viable bacteria counts was significantly enhanced in the sea cucmber fed with TC22 at dose of 10~9 CFU/g feed (P<0.05). But no significant difference was found on total Vibrio counts between trial treatment and the control (P>0.05). The cumulative mortality after V. splendidus challenge decreased significantly in the groups fed with TC22 at dose of 10~9 CFU/g feed (P<0.05). Animals fed with 0.5% FOS showed higher immune response and lower cumulative mortality than the control group (P<0.05). The combination of 0.5% FOS with TC22 at dose of 10~9 CFU/g feed produced significantly positive synergistic effects on sea cucumber immune responses and disease resistance compared with control group(P<0.05). Results of this experiment confirmed the potential of TC22 as dietary probiotic and the synergistic effects of TC22 and FOS in sea cucumber.9. The effects of probiotic EN25 and the synergistic effects of EN25 and fructooligosaccharide (FOS) on growth, immune response, microflora and disease resistance in sea cucumber Apostichopus japonicus (initial weight 3.127±0.012 g) were studied. Animals were fed the diet with EN25 at doses of 0, 10~7, and 10~9 CFU/g feed with or without 0.5% FOS for 56d. At the end of the feeding trial, six sea cucumbers per tank were sampled for bacterial quantification and immune indices measurement. Then all the sea cucumbers left were challenged by injecting Vibrio splendidus. The results showed that the SGR of sea cucumber was not significantly influenced by dietary EN25 with or without FOS (P>0.05). The phagocytosis activity and respiratory burst was significantly increased by dietary EN25 without FOS (P<0.05). Howerver, the phenoloxidase activity and total nitric oxide synthase of sea cucumber were not significantly influenced by dietary EN25 without FOS (P>0.05). Dietary EN25 without FOS did not produce significant influence on the counts of total viable bacteria of sea cucumber (P>0.05), but it significantly decreased the counts of Vibrio bacteria of animals (P<0.05). The cumulative mortality after V. splendidus challenge was decreased significantly in sea cucumber fed the diet containing EN25 without FOS (P<0.05). Sea cucumbers fed with 0.5% FOS had higher phagocytosis activity, respiratory burst activity, phenoloxidase activity and total nitric oxide synthase than control group, and had a significant lower cumulative mortality after challenged by V. splendidus (P<0.05). The combination of 0.5% FOS and EN25 increased the phagocytosis activity, respiratory burst activity, phenoloxidase activity and total nitric oxide synthase of sea cucumbers. Among these immune indices, the phagocytosis activity was significantly increased in sea cucumber fed FOS and EN25 (P<0.05) and respiratory burst activity was also significantly improved in sea cucumber fed on FOS combined with EN25 at 10~7 CFU/g (P<0.05). The total viable bacteria counts of sea cucumber intestine was not affected by dietary FOS with or without EN25 (P>0.05). The total Vibrio bacteria counts was significantly decreased in sea cucumber fed the diet with 0.5% FOS and EN25 at dose of 10~7 CFU/g (P<0.05). The disease resistance was significantly increased in sea cucumber fed on 0.5% FOS with EN25 at dose of 10~9 CFU/g.10~. The selected probiotics (T13, TC22 and EN25) were identified by Biolog microbes identification system and the cluster analysis of 16S rDNA. The optimum condition of culture for these three probiotics was preliminarily studied. The results showed that T13 was very similar to Bacillus subtilis and the optimum condition for growth was as follows: inoculation level 2%, liquid volumn 50ml/250ml, initial pH 7.0, temperature 32℃, rotating speed 180 r/min. TC22 was very similar to Bacillus licheniformis and the optimum condition for growth was as follows: inoculation level 4%, liquid volumn 75ml/250ml, initial pH 7.2, temperature 30℃, rotating speed 180 r/min. EN25 was very similar to Bacillus cereus and the optimum condition for growth was as follows: inoculation level 1%, liquid volumn 25ml/250ml, initial pH 8.0, temperature 30℃, rotating speed 180 r/min.