| In aquaculture, the abuse of antimicrobial drugs could lead to outbreaks of resistant bacteria, environmental hazards, drug residues and other issues. As a consequence, the development of alternatives to antibiotics is receiving considerable attention. Currently, a lot of reports about probiotics have indicated that they are not only the substitutes for antibiotics in aquaculture but also have lots of probiotic effects, such as improved fish growth, higher feed efficiency, modified gut microbiota, better immune response, against pathogens, and refined water quality. Therefore, screening and application of safe and effective probiotic strains are essential for the sustainable development of aquaculture. In this study, by in vitro experiments, exoenzymatic profiles, antagonistic profiles and gastrointestinal tolerance of the isolates (from environment and gastrointestinal tract) were evaluated. On the other hand, according to the outcomes of in vitro tests, strains of Bacillus G024 (isolated from water environment) and Bacillus M001 (isolated from flounder Paralichthys olivaceus digestive tract) were selected to be used in the in vivo test to study their potential probiotic effects on growth performance, immunity, disease resistence and gut microbiota of turbotS. maximus.1. Isolation and identification of marine extrocellular enzymatic bacteriaExtracellular enzyme producing strains isolated from the digestive tract of fish samples (P. olivaceus, S. maximus, E. coioides, Konosiruspunctatus) and open breeding pond of sea cucumber were screened by respectively using skimmed milk 2216E agar plate (contain skimmed milk 1%, w/v), potato starch 2216E agar plate (contain potato starch 0.3%, w/v), carboxymethylcellulose 2216E agar plate (contain carboxymethylcellulose 1%, w/v), TW-802216E agar plate (contain TW-800.8%, v/v). A total of 98 extracellular enzymatic producing bacteria were screened. In these strains,44 isolates of them were gram-positive bacteria and the other 54 isolates were gram-negative bacteria. Then, the colonies showing different morphological appearance, more broaden enzymatic pattern and higher enzymatic acitivities were taken out to be sequenced and their 16S rRNA gene amplicons were analysed. The BLAST comparison results showed that the 16 sequencing isolates respectively belong to Lactococcus (A005), Bacillus (B005, G012, G024, K003, M001, N004, P008, Y005), Kocuria (C005), Pseudoalteromonas (G002, W003), Vibrio (H002, P010), Listonella (H012), Mucus bacterium (J003). Besides that, we studied their resistence to the common aquatic drugs (furazolidone, tetracycline, ampicillin, rifampicin, ciprofloxacin, chloramphenicol). The outcomes of resistence showed that G002,H012, P010 have varying degrees of resistence to ampicillin.2. Evaluation of antagonistic effect on aquatc pathogens of the extracellular enzymatic Bacillus spp.By the disc diffusion method, the diameter of inhibition zone formed by cell free supernatent from the extracellular enzymatic Bacillus B005, G012, G024, K003, M001, N004, P008, Y005 against indicator bacteria (Edwardsiella tarda LSE40, Streptococcus sp. CF, Staphyloccocus aureus, B. cereus, V. anguillarum M3, V. campbellii 1.1596, V. fluvialis 1.1608, V. harveyi 1.1593, V. vulnificus L32, V. parahamolyticus 1.1587, V. alginolyticus 1.1607, V. splendidus 1.1604) were measured. The results showed that strains of Bacillus G012, Bacillus Y005 can not inhibit any indicator bacteria growth, but the other 6 strains could antagonize the indicator bacteria in different degrees. Then, by scanning electron microscope (SEM), strains of Bacillus G024, Bacillus N004 were selected to characterize the effect of their cell free supernatent on the indicator bacteria. The outcomes showed that the cell supernatent of the enzyme producing bacteria destructed the surface structure of the indicator bacteria.3. Studies on the growth characteristics, tolerance to the environment in digestive tract and animal safty of potential probiotic Bacillus spp.The test Bacillus B005、G012、G024、K003、M001、N004、P008、Y005 could grow in a wide range of temperature (10-40℃), pH (6-8) and NaCl (0.5-12.5%) and the logarithmic growth phase of most of the strains is 6 h. The strains of B005, G012, G024, K003, Y005 could grow under relatively higher oxgall salt concentration (0.015%, w/v). The test bacteria of G012, G024, K003, M001 showed relatively stronger tolerance to the artificial gastric juice (0.1% pepsin,0.9% NaCl, pH 4) (within 3 h, the amount of such strains reduced by 2-3 orders of maginitude). In addition, the biofilm formation capacity of strains of B005, G024 were more stronger (P< 0.05). Besides that, all the 8 test Bacillus showed hemolytic activity to haemocyte of Scophthalmus maximus, no lecithinase activity. Additionally, the plasmid of the these isolates could not be detected. The outcome of the animal safty assay showed that all the test bacteria were not pathogenic to turbot (S. maximus).4. Evaluation of potential probiotic Bacillus G024, M001 on growth performance, digestive enzyme activity, innate immunity, gut microflora and disease resistence of turbot Scophthalmus maximusThe fresh bacterial culuture (strains of G024, M001) was mixed with commercial fish diet, and make the concentration of bactria in fish diet to get 5.0×108 CFU/g diet for a 42-day breeding trial of S. maximus. The determination of BW (body weight), WG (weitht gain), SGR (specific growth ratio), FR (feed ratio) of the test fish were performed. The outcomes did not showed any difference between the treatment groups and the control{P > 0.05). The activities of the digestive enzymes of gastrointestinal tract and hepatopancreas were determined. The outcomes showed that, compared with the control group, the activities of hepatopancreas lipase, stomach protease, stomach lipase, stomach amylase of G024 treatment group were significantly rised (P< 0.05); the activities of hepatopancreas protease, hepatopancreas lipase of M001 treatment group have better performance than the control group (P< 0.05). Then, the serum innate immunological indexes were determined. The results showed that the activity of serum lysozyme of G024 treatment group were significantly higher than control group (P< 0.05); the activity of superoxide dismutase and the level of serum total protine content were higher than control group (P< 0.05). But compared with control goup, the activities of serum nitric oxide synthase, serum acidphosphatase, serum alkaline phosphatase of potential protiotic treatment groups did not change significantly. The control group and the potential probiotic treatment groups were challenged with the pathogenic V. anguillarum M3 at 4.8 x 108 CFU/mL by intramuscular injection. The challenge test showed that the accumulative mortalities of G024 and M001 treatment groups totaled 10.67±2.31%and 33.33±12.86%, respectively. Additionally, the accumulative mortalities of the both two treatment groups were all significantly lower than that of control (89±8.33%) (P< 0.05). The effects of such two potential probiotics on the gastrointestinal mirobiota were analyzed by PCR-DGGE method. The outcomes showed that, compared with the control group, there are 4 dominant bands, such as 14 (Rubritalea spongiae strain YM21-132),15 (Janibacter anophelis strain CCUG 49715),16 (Uncultured bacterium),17 (Uncultured bacterium), disappeared in different degrees in treatment groups. Additionally, compared with control, the number of DGGE bands and shannon index in treatment groups were lower, but without significant defference (P> 0.05). Above outcomes demonstrate that the strains of G024, M001 could play a significant role in the probiotic effects on nutrition and immunity of the aquatic animal turbot S. maximus and could be used as potential probiotics. |
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