| In this study one high ectoenzyme-producing bacillus strain was screened fromintestinal tract of150±5.0g grass carp (Ctenopharyngodon idellus). Two inclusion levels ofthe Bacillus subtilis strain by fermented were added into the commercial feed to evaluatevariation of spores quantity of Bacillus subtilis in feed at different feed processingtechnology, processing sections, storage time and preservation methods. Moreover, theeffects of dietary supplement of Bacillus subtilis was evaluated on growth and immuneperformance, mucosal morphology of grass carp. Actual effects of dietary supplement ofBacillus subtilis on water quality had been studied as well. The results of the series studywere shown as follows:Experiment1: Three strains suspected bacillus strain (G1, G2, G3) were isolatedfrom intestinal tract of150±5.0g healthy grass carp, which were confirmed as Bacillussubtilis strains through analysis of colony characteristics, physiological and biochemicalcharacteristics and16S rDNAsequence. Moreover, the ability of the G1, G2and G3strainsproducing cellulase, amylase and protease were assessed by D/d(D: diameler of hydrolysistransparent circle; d: colony diameter), in which G1has higher zymogenicities of protease,amylase, cellulase(2.9，2.2and3.3, respectively) than G2, G3. Storage and usage of G1strain for following study.Experiment2: Two kinds of commercial feed processing technology(A and B) wereapplied to4experimental diets in which named A1(9.10×10~7cfu/g)、A2(1.05×10~7cfu/g)、B1(9.10×10~7cfu/g) and B2(1.05×10~7cfu/g) respectively, to evaluate the effects of differentprocessing technology, processing section and different supplement on spores quantity ofBacillus subtilis in feed. And the variation of spores quantity of Bacillus subtilis in diet B1and diet B2by two different preservation methods (closed or open) also had been studied.The results showed that, a decreasing trend of spores quantity appeared with the sequenceof processing section in both Aand B，especially granulating section. The survival rates ofspores in diet A1and A2were41.90%and37.47%respectively, while those of the diet B1and B2were28.35%and41.90%. In A and B processing technology, the relationshipbetween the amount of spores in the high dose group and low dose group showed a goodlinear in each section, The linear relationship of A was Y=0.0702X+2×10~6(r=0.9822), and the linear relationship of B was Y=0.1323X+972974(r=0.9999). In the same technologicalconditions, the survival rate of spores was consistent in the different concentration ofBacillus, which indicated the survival rate of dietary spores was not related toconcentration in feed. However, spore quantity in commercial feed were controlled bysupplementation. The spores quantity in diet B1and B2which were preserved by twodifferent preservation methods(closed and open, respectively) was detected on the1st,7th,14th,21st,28th,35th,100thday, the spores quantity were not significantly different betweenthe1stand the100thday(P＞0.05), suggesting that spore quantity was not affected by thetime of preservation.Experiment3: A10d-feeding trial was conducted to investigate the effects ofdifferent supplement levels of Bacillus subtilis spores in feed on water quality (NO2-N,NH3-N and COD), the spores quantity in the grass carp(Ctenopharyngodon idellus) faecesand cultured water. The basal diet was applied as control, in which S1(2.6×10~7cfu/g), S2(4.4×10~6cfu/g) were as tested diets, The grass carp with150g mean initial body weightwere distributed in0.48m~3closed tanks at a rate of25grass carp per tank, using acompletely randomized design with three treatments and three replicate groups pertreatment. The spores quantity in the grass carp (Ctenopharyngodon idellus) faeces wasdetected on the1st,4th,7th,10thday, the spores quantity, NO2-N, NH3-N and COD of thewater was detected on the1st,3rd,6th,9thday. During the experimental period，the group S1and S2spores quantity in water increased in the first6d. Since the6thday，where S1andS2group spore contents in water were stable,2.22×10~6cfu/g and3.98×10~5cfu/grespectively, and meanwhile the spores quantity in water as a control group was still0cfu/L. During the ten days, the spores quantity in dry matter of grass carp fecal was stable,in which S1group was8.05×10~6cfu/g, S2group was1.96×10~6cfu/g, control was0cfu/g.Compared with the spores quantity in feed, group S1and group S2fecal Bacillus subtilisspores were reduced by89.76%~90.71%and83.83~86.84%.Even though water quality ofin closed water group A and B could ameliorate to some extent by Bacillus subtilis sporesfrom faeces of Grass carp fed dietary Bacillus subtilis sproes,there were no significationdifference in the content of NO2-N, NH3-N or COD in closed water.Experiment4: Under the test3designs, A21d-growth trial was conducted toinvestigate the effects of different levels Bacillus subtilis spores supplement on growth,immune performance, intestinal mucosal morphology of grass carp. The results showedthat in S1, S2group, the weight gain of grass carp were increased by12.97%and29.08%,and the feed conversion ratio were decreased by0.90%and22.17%respectively, but all of those were no significantly different(P>0.05). The activity of ACP, AKP, AST, ALT and thecontent of TP in blood serum was a little higher than that in the control group, but nosignificantly different as well(P>0.05). The micro-sections of the grass carp showed thatthe foregut foldings were758.40μm and820.04μm, the foregut mucosal were813.51μmand872.79μm in S1, S2group, respectively which were significantly higher than thecontrol of677.02μm and720.17μm(P<0.05); moreover, the midgut foldings (761.11μmand813.13μm, respectively) and the midgut mucosal(793.37μm and848.80μm) in S1, S2group were very significantly higher than the control of610.91μm and657.53μm(P<0.01). The foregut muscular layer of S2group(238.88μm) were very significantlyhigher than the control of200.96μm, and the midgut muscular layer of S1, S2group were195.99μm and176.25μm, significantly higher than the control of152.95μm（P<0.05）.In conclusion, it was entirely feasible to improve the growth, immunity performanceand intestinal health of grass carp and ameliorate water quality to some extent by fedscreening of Bacillus subtilis from Grass carp intestine. |
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