| The Apostichopus japonicus (sea cucumber) is the most profitable aquaculture animal with the highest output as single variety in our country, which is of high nutritional value and economic value. In recent years, as the growth of the market demand and decrease of natural resources, Apostichopus japonicus?s artificial breeding scale expands rapidly. With the high density and intensive development model of Apostichopus japonicus breeding, feed problem which account for more than 50% of the total production cost becomes the most important point which restricts the healthy development of the Apostichopus japonicus culture. The lack of bait with high quality, low feed utilization rate and the water quality issues caused by remnant corruption is the most crucial factor. In this study, we focus on Laminaria japonica, which has high output and low cost. By degrading the algin in the Laminaria japonica with screened beneficial microorganism fermentation, an algin-degraded Laminaria japonica feed with value-added function was obtained. Algin composition in the Laminaria japonica which was hard assimilated can be effectively degraded by microorganism, which improves the digestion and absorption of feed, and significantly improves the growth of Apostichopus japonicus. Besides, Degradation products alginate oligosaccharides are a kind of immunostimulant, which convert the large number of seaweed alginate into potential natural immune enhancer for Apostichopus japonicus. In addition, the highly active beneficial microbes in the feed play an effective role of strengthening the digestive enzyme activity in the intestinal of Apostichopus japonicus, promoting nutrient uptake, restraining the growth of potential pathogens, promoting nonspecific immune response of Apostichopus japonicus, and improving the resistance to the infection of pathogenic vibrio.Main work in this thesis is as follows:(1) An algin-degrading bacteria WB1 was isolated and purified from sea mud utilizing the medium with the sodium alginate as the sole carbon source. Based on the results of 16S rDNA homology analysis, and together with the morphological, physiological, and biochemical characteristics, the algin-degrading strain was identified as a Bacillus sp. and designated as Bacillus amyloliquefaciens WB1. The risk of disease associated with the isolated algin-degrading bacterium was tested by injecting the sea cucumber with a high dose of the bacterial suspension at the site of the coelom, which showed that strain WB1 has no pathogenicity to the sea cucumber; Bacteriostatic test in vitro showed that B. amyloliquifaciens WB1 has certain inhibition effect to three common pathogenic vibrio of Apostichopus japonicus(Vibrio splendidus, Vibrio tasmaniensis, Vibrio cyclitrophicus); Final product of alginate lyase digestion of algin by B. amyloliquifaciens WB1 was analyzed by thin-layer chromatography and molecular exclusion chromatography. It was found that its major ingredient are disaccharide and trisaccharide, and the relative contents were 74.1% and 25.9% respectively; The average molecular weight of oligose in the enzymolysis end-products of brown algae is 560 Da which is determined by high gel permeation chromatography.(2) Eight factors affecting growth and algin-degrading capacity of WB1 were investigated. The results of Plackett-Burman design indicated that fermentation time, content of beef extract in the culture, and solvent to solid ratio were the significant parameters. Furthermore, the mutual interaction between the solvent to solid ratio and beef extract concentration was more significant than the other pairs of parameters on algin degradation. Optimal values obtained from Central-Composite Design were 113.94 h for fermentation time, 0.3%(w/v) beef extract concentration and 44.87 (v/w) ratio of solvent to feedstuff. Under optimal conditions,56.88% of the algin was degraded when a 50-fold scale-up fermentation was carried out, using a 5-L fermenter.(3) The non-specific immunostimulatory effect of alginate oligosaccharides (AOS) with three types of molecular weights, as well as their two types of corresponding purified uronic acid oligomers (mannuronate and guluronate oligomers) were studied on sea cucumber (Apostichopus japonicus) coelomocyte in vitro. Coelomocytes cultured in L-15 medium containing different AOS concentrations were sampled, and the results showed that non-specific immune responses, such as phagocytic capacity, reactive oxygen species, lysozyme activity, peroxidase activity, total nitric oxide synthase and acid phosphatase activity were improved by treating with the three types of AOS, however, the significant difference appeared at different concentrations and incubation times depending on the AOS molecular weights. Furthermore, among the significant differences of various molecular weights of the AOS groups relative to the control sample, these immune parameters in mannuronate or guluronate oligomer groups were all significantly higher than that in the control sample (P<0.05), in addition, the A1 mannuronate group had a higher increase in phagocytic activity than that of guluronate, and no significant difference was observed in other immune parameters between corresponding mannuronate and guluronate oligomer groups.(4) The feeding trials were conducted to study the improving effect of algin-degraded Laminaria japonica-based diet on growth and immunity of Apostichopus japonicus through degrading which by microorganism. The results showed that the growth performance of sea cucumber was significantly increased by a diet consisted of B. amyloliquefaciens WB1-treated L.japonica, compared to those fed with a diet containing untreated L. japonica, which even provided the same level of energy and nutrients as the commercial diet made of S. thunbergii; the activities of many immune-related enzymes of sea cucumber were significantly increased and the disease-resistant capacity of sea cucumbers could be improved when these individuals were fed with a diet containing L. japonica that had been treated with B. amyloliquefaciens WB1; the total number of aerobic bacteria and algin-degrading bacteria in the intestinal tract of sea cucumbers fed with a diet consisted of B. amyloliquefaciens WB1-treated L.japonica was increased significantly, and the total number of Vibrio sp. in the intestinal tracts of sea cucumbers fed with a diet consisted of B. amyloliquefaciens WB1-treated L. japonica was significantly lower, compared to those of sea cucumbers fed with the other diets. |
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