Effects Of Two Phytase Producing Bacteria Isolated From Bullfogs On Growth,Nutrient Absorption And Intestinal Health Of Bullfrog (Rana (Lithobates) Catesbeiana)

Feeding trials were conducted to investigate the effects of two phytase-producing bacteria(E.cloacae and B.subtilis)on growth,nutrient absorption and intestinal health of bullfrogs(Rana(Lithobates)catesbeiana).The results are presented as follows:1.Study on heat tolerance and enzyme production ability of phytase-producing(E.cloacae and B.subtilis)Two phytase-producing bacteria(E.cloacae and B.subtilis)were screened from the bullfrog's intestinal.Experiment 1,study the effects of different temperature(25 ?,28 ?,31 ?,34 ?,37 ?)and different culture cycles(24 h,48 h,72 h,96 h)on the sporulation ability of B.subtilis,and to study the optimum sporulation conditions.Experiment 2,to study the heat resistance of two enzyme-producing bacteria by heating five different temperatures(65 ??75 ??85 ??95 ??10~5 ?)for 10 min.Experiment 3,cultured 72 h under the condition of 30 ? to determine the enzyme production capacity of E.cloacae and B.subtilis.In conclusion: in Experiment 1,the optimum conditions for B.subtilis were(31 ?,96 h)and the spore rate>95%.In Experiment 2,B.subtilis has a high heat resistance,10~5 ? survival rate of up to 93.56%,but E.cloacae is not heat resistant.In Experiment 3,the enzyme production capacity of E.cloacae was 33.72 U/mL,and the activity of B.subtilis was 26.31 U/mL.2.Effects of different levels of phytase-producing B.subtilis and E.cloacae on growth performance,body composition and serum antioxidant capacity of bullfrogs.252 bullfrog with initial average weight of(42.5�0.2)g were randomly divided into seven groups with three replicates in each group and 12 bullfrog in each replicate.Two strains of phytaseproducing bacteria(E.cloacae and B.subtilis)were selected,each bacteria set up three addition gradients(10~5,10~7 and 10~9 cfu/g)and control group(sterile).A total of 7 feeds were prepared.B.subtilis 10~9 cfu/g dose group(KG)had the highest FE(1.21),which had no significant difference with B.subtilis 10~7 cfu/g dose group(KZ)(P>0.05),but significantly higher than other groups(P<0.05).Feed efficiency(FE)and protein efficiency rate(PER)increased with the increase of E.cloacae in E.cloacae group,reached the maximum at E.cloacae 10~9 cfu/g dose group(YG)and higher than the control group(P<0.05).but T-AOC increased first and then decreased with the increase of B.subtilis,and reached the maximum at B.subtilis 10~7 cfu/g dose group(KZ).KZ and KG group were significantly higher than E.cloacae group(P<0.05).With the increase of two phytase-producing bacteria in the feed,the level of serum CAT increased first and then decreased,reached the highest in YZ and KZ group;The levels of SOD increased first and then decreased,and reached the highest level in YZ and KZ groups respectively.In conclusion,the addition of E.cloacae(1�10~9)and B.subtilis(1�10~7 and 1�10~9)did not affect the growth performance of bullfrogs,but could improve FE and PER of bullfrogs and the antioxidant capacity of serum.3.Effects of phytase-producing bacteria on calcium and phosphorus deposition and apparent digestibility of nutrients.After 8 weeks culture experiment,feces were collected of the remaining bullfrog for each cylinders and assay nutrient digestibility.In conclusion,with the increase of the level of the two bacteria,the whole body calcium and phosphorus levels of bullfrog had an upward trend,and reached the maximum value at YG and KZ,which was significantly higher than control group(P<0.05).However,with the increase of B.subtilis level,serum phosphorus increased significantly(P<0.05),KG reached the maximum,and was significantly higher than control group(P<0.05).With the increase of two phytase-producing bacteria,alkaline phosphatase(AKP)had decrease trend.ADC of dry matter reached the maximum value at YG and KZ with the addition of two bacteria,which was significantly higher than control group(P<0.05).With the increase of B.subtilis level,ADC of crude protein increased first and then decreased,reached the maximum at KZ and was significantly higher than the control group(P<0.05).With the increase of two enzymeproducing bacteria,the ADC of calcium and phosphorus increased first and then decreased.The ADC of calcium reached the maximum at YZ and KZ,respectively,and was significantly higher than the control group(P<0.05).In conclusion,the results showed that the addition of two phytaseproducing bacteria could promote the deposition of calcium and phosphorus and the ADC of feed.4.Effects of phytase-producing B.subtilis and E.cloacae on intestinal tissue structure of bullfrogsUnivariate analysis of variance showed that the addition of two phytase-producing bacteria had significant effect on intestinal villus length,villus width,muscle thickness and intestinal epithelial cell height(P<0.05).Anterior intestine: with the increase of the two bacteria,the length of villi,the width of villi and the thickness of muscle layer showed an increasing trend.The height of intestinal epithelial cells was not affected by the addition of two bacteria(P>0.05).Midintestine: the trend is similar to the anterior intestine.Posterior intestine:the length of villus and the width of villi increased first and then decreased with the increase of the two enzyme producing bacteria,and reached the maximum at YZ and KZ,respectively.The intestinal epithelial cell height increased with the addition of two phytase-producing bacteria,and the YG and KG reached the maximum value,the YG was significantly higher than the control group(P<0.05).Observation of bullfrog intestinal tissue slice showed that two phytase-producing bacteria can improve the integrity of intestinal wall,villus integrity and the number of foregut,midgut goblet cells,reduce intestinal epithelial cell autolysis.In conclusion,feed add 1�10~7-1 �10~9 cfu/g of E.cloacae and 1 x 10~7 cfu/g B.subtilis conducive to maintaining intestinal health of bullfrog.5.Effects of phytase-producing B.subtilis and E.cloacae on intestinal microflora structure of bullfrogsThe addition of two phytase producing strains had significant effect on the intestinal OTUs of bullfrogs(P<0.05).With the increase of the level of two bacteria,OTUs showed an increasing trend,which reached the maximum at YG and KZ,respectively,and significantly higher than the control group(P<0.05).The addition of two bacteria resulted in a significant increase in the Alpha diversity index(P<0.05)of the intestinal flora of bullfrogs,which reached the maximum at YG and KZ,respectively,and was significantly higher than the control group(P<0.05).At the phylum level,the addition of the two bacteria changed the proportion of the dominant microflora.With the addition of two phytase-producing bacteria,the proportion of Proteobacteria in the intestine was also higher than the control group.However,with the addition of two bacteria,Firmicutes increased first and then decreased,and reached the maximum at YZ and KZ,respectively.At the genus level,the diversity of the flora was abundant among all treatment groups.With the increase of two phytase-producing bacteria,reduce the number of mycoplasma.With the addition of E.cloacae,the level of Klebsiella decreased and lower than the control group.Lactococcus had increased trend in the B.subtilis group and was higher than the control group.Bacillus had increased trend with the addition of B.subtilis,and it was higher than that the control group and E.cloacae group.In conclusion,Addition of E.cloacae and B.subtilis could increase the abundance and diversity of intestinal microorganisms.To a certain extent,it can increase the proportion of beneficial bacteria,and inhibit the proliferation of pathogenic bacteria.