| A Bacillus subtilis B1 and a Brevibacillus formosus Y5 with phytase producing ability were isolated from the intestinal mucosa of healthy bullfrogs in the previous experiment of the laboratory were used as the test bacteria.On this basis,we explored the fermentation characteristics of two strains and the best conditions for two strains to ferment soybean meal.In order to explore the effects of the fermented soybean meal on the growth performance,digestion and intestinal health of the bullfrog,based on the optimal fermentation conditions,we used the fermented soybean meal to replace different proportion of fish meal to prepare experimental diets,then the diets were used to the feeding trial for 9 weeks.The main results were as follows:(1)Study on fermentation characteristics of phytase producing bacteria isolated from the bullfrog.The effects of different fermentation time,temperature and initial medium pH on the growth of the two strains were studied.In conclusion,after 12 hours of culture,B1 and Y5 reached end exponential growth phase,the optimum growth temperature of B1 and Y5 were 34℃ and 37℃respectively,and the optimal initial medium pH of the two strains was 7.0.In addition,when the two strains were cultured at 33℃ and the initial pH of the medium was 7.0 for 10 hours,the phytase activity of B1 was 0.47μmol/min/ml,and that of Y5 was 0.38μmol/min/ml.And by intraperitoneal injection,it was found that the bacterial solution of two strains with concentration of 0.5mL of 1 X 109 cfu/mL(0.85%NaCl)had no negative effect on bullfrog’s health respectively.(2)Study on fermentation conditions of soybean meal by the two strains.To exploring the content of phytate phosphorus of soybean meal after fermentation under different conditions,based on the previous studies of the laboratory,the fermentation experiment was designed as 10 factors/level,the fixed factors were fermentation time(48h),the ratio of material to water(1:0.8)and temperature(33℃),the variables were inoculated strains(B1 and Y5),inoculated proportion(single and mixed fermentation(inoculated proportion of mixed fermentation were 2:1 and 1:1))and the inoculation amount(1×107 CFU/mL and 1×108 CFU/mL).The results showed that the best fermentation combination of mixed fermentation was Y5 and B1(the proportion of inoculation was 1:2),the amount of inoculation was 1×108 CFU/mL,the ratio of material to water was 1:0.8 based on fermentation at 33℃ for 48 hours.The optimal fermentation combination of single strain fermentation was inoculated with B1,the inoculation amount was 1×108 CFU/mL,the ratio of material to water was 1:0.8 based on fermentation at 33℃ for 48 hours.Under the best fermentation conditions,the 23.37%phytate phosphorus of soybean meal could be reduced by two fermentation combinations,respectively.Compared with soybean meal,after fermentation by mixed bacteria the crude protein and crude lipid of FMSM increased 7.01%and 20.13%,while the content of total amino acid remained unchanged.And the crude protein,crude lipid and total amino acid of FBSM increased 6.49%,18.24%and 3.63%after fermentation by single bacteria.(3)Effects of two fermented soybean meals on growth,digestion and intestinal health in bullfrog.Seven isonitrogenous and isolipid diets,included control group(including 20%fish meal),3 groups replacing 60%fish meal(60%SM group,60%FBSM group and 60%FMSM group)and 3 groups completely replacing fish meal(100%SM group,100%FBSM group and 100%FMSM group).In addition,we carried out a 9-week feeding trial on bullfrogs(about 43g)to explore the effect of two kinds of fermented soybean meals replacing fish meal on growth,digestion and intestinal health in bullfrogs.The results showed that the level of replacing fish meal and alternative protein sources had significant effect on the final body weight(FBW)and weight gain rate(WGR)of bullfrog(P<0.05),and with the increase of the level of soybean meal or fermented soybean meal instead of fish meal,those decreased.The feed coefficient rate(FCR)and feeding rate(FR)were significantly affected by the level of replacing fish meal(P<0.05),and which decreased with the increase of the level of replacing fish meal.The contents of crude lipid and protein of bullfrog gradually decreased with the increase of replacing level.Besides,at the same replacement level,the hepatosomatic index(HSI)in the replacement group of fermented soybean meal was lower than that in the replacement group of soybean meal,while the hind leg index(HLI)was higher than that in the replacement group of soybean meal.However,with the increase of the level of soybean meal or fermented soybean meal instead of fish meal,the HSI increased while the HLI decreased.The serum biochemical index and liver antioxidant index of bullfrog were significantly affected by the level of replacing fish meal and alternative protein sources(P<0.05),and there was significant interaction between the level of replacing fish meal and alternative protein sources on serum biochemical indexes and liver antioxidant indexes(P<0.05).With the increase of the level of soybean meal or fermented soybean meal instead of fish meal,the level of blood ammonia,blood urea nitrogen and MDA increased(P<0.05),while the other serum biochemical indexes and liver antioxidant indexes were decreased(P<0.05).The activity of intestinal protease,lipase and amylase were significantly affected by the level of replacing fish meal and alternative protein sources(P<0.05),and there was significant interaction between the level of replacing fish meal and alternative protein sources on intestinal lipase,amylase and protease(P<0.05).At the same level of replacement,there was no significant difference in the activity of intestinal lipase and protease between the two fermented soybean meal groups(P>0.05),and which were significantly higher than that of the soybean meal replacement group(P<0.05).But the activity of intestinal protease and lipase decreased with the increase of the level of soybean meal or fermented soybean meal instead of fish meal.The feces of bullfrog were collected after 9 weeks of feeding trial.Then the contents of dry matter,total calcium,total phosphorus,crude protein and crude lipid in the diets,feces and body of bullfrog were determined.The results showed that the apparent digestibility coefficients(ADC)of dry matter,crude protein,total calcium,total phosphorus and phytate phosphorus and the retention rate(RRi)of calcium and phosphorus were significantly affected by the level of replacing fish meal and alternative protein sources(P<0.05),and there was significant interaction between the level of replacing fish meal and alternative protein sources on the above ADC and RRi(P<0.05).In addition,the ADC of those in the fermented soybean meal replacement groups was higher than those in soybean meal replacement groups,but with the increase of the level of soybean meal or fermented soybean meal instead of fish meal,the ADC of those decreased(P<0.05).In addition,with the increase of the level of replacing fish meal,the deposition of nutrients in bullfrog feed first increased and then decreased.The results showed that the structure of jejunum of bullfrogs in control group,60%FBSM group and 60%FMSM group were complete,and the three-group owned thick muscularis,while the structure of jejunum of bullfrogs in the other groups showed different degrees of damage.The height and weight of mucosal folds of jejunum of fermented soybean meal replacement groups was significantly higher than those of soybean meal replacement group at the same level of replacement(P<0.05),but there was no significant difference between the two fermented soybean meal groups(P>0.05).Besides,the height and weight of mucosal folds and the thickness of muscularis decreased with the increase of the level of soybean meal or fermented soybean meal instead of fish meal.There was no significant difference in the thickness of lamina propria among the groups(P>0.05).The expression of IL-1β gene,IL-17 gene,IL-4 gene,IL-10 gene and TGF-β gene were significantly affected by the level of replacing fish meal and alternative protein sources(P<0.05),and there was significant interaction between the level of replacing fish meal and alternative protein sources on IL-1β gene,IL-4 gene,IL-10 gene and TGF-β gene(P<0.05).In addition,the expression of IL-4 gene,IL-10 gene and TGF-β gene in fermented soybean meal replacement groups were significantly higher than those in soybean meal replacement groups(P<0.05).But with the increase of the level of soybean meal or fermented soybean meal instead of fish meal,the expression of IL-1β gene,IL-17 gene and TNF-α gene of the jejunum of bullfrog increased,while the expression of IL-4 gene,IL-10 gene and TGF-β gene decreased.Compared with control group,the number of OTU in the other groups increased except for the 100%FBSM group.With the increase of the level of soybean meal or fermented soybean meal instead of fish meal,ACE index and Chao1 index decreased,while Simpson index increased first and then decreased.Compared with control group,the relative abundance of Proteus decreased except 60%FBSM group.In addition to 100%FBSM group,the relative abundances of Firmicutes increased in each alternative group.And the relative abundances of Bacteroides in 60%FBSM,100%FMSM and 60%SM groups increased.The total abundance of Mycoplasma and Aureobacteria of the four fermented soybean meal replacement groups was lower than that of soybean meal replacement groups.It explained that fermented soybean meal could inhibit the growth of harmful bacteria to a certain extent. |
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