多粘芽孢杆菌分解饲料蛋白和促进鱼体生长的研究
摘要
本研究采集了四川境内多个养殖池塘和湖泊鲜活鲤鱼的肠道内容物,经初筛获得55株芽孢杆菌菌株,其中具有产蛋白酶能力的菌株有19株,最终筛选出一株产蛋白酶能力强而且效果稳定的菌株,经鉴定为多粘芽孢杆菌(Bacillus polymyxa strain),编号为A7。A7菌株能耐受pH 2.5的酸性环境和0.5%胆盐浓度,是耐酸性和耐胆盐较强的菌株,并且该菌株对嗜水气单胞菌具有很强的抑制作用。菌株的安全性试验表明,A7菌株在水产养殖中应用是安全的。
通过A7菌株产酶条件的单因素优化试验发现,其最佳产酶条件是:pH值7.0,温度37℃,培养时间48h。A7菌株在28℃,110r/min水域振荡培养72h,产酶量达到最高,其酶活力为2998U。
以玉米粉300g、麦麸400g、豆粕300g和pH6.8磷酸盐缓冲液500mL为固体发酵基础培养基,对固体发酵条件进行单因素优化试验,测定出A7菌株的最佳固体发酵条件是:在温度28℃,发酵时间48h和料水比1:1.0时,接种1.2×10~9cfu/mL的种子液5%于装量40g的250mL容量瓶中,发酵后固体活菌数可达2.85×10~(12)cfu/g。
采用体外消化法研究了A7菌株对鱼粉、豆粕、菜籽粕、棉籽粕4种饲料蛋白质的体外消化能力。试验结果显示,添加A7菌株的所有试验组均对鱼粉干物质和粗蛋白的体外消化率没有明显促进作用(P＞0.05),而添加含10~8cfu/mLA7菌株试验组对豆粕、菜籽粕、棉籽粕干物质和粗蛋白的体外消化率均有显著的促进作用(P＜0.05),其消化率分别提高了44.85%、10.66%和53.05%;8.45%、、28.78%和63.9%。
从鲤鱼幼鱼饲喂试验可以看出,在饲喂鲤鱼幼鱼50d后,饲料中添加A7菌株固体发酵物试验组幼鱼的平均增重率和特定生长率分别高于对照组的23%和11.8%,干物质表观消化率和蛋白质表观消化率的平均值分别为65.85%和85%,均显著高于添加酵母组、酵母+A7组和对照组(P＜0.05)。
This study gathered the intestinal contents of fresh common carp from the breeding ponds and lakes in Sichuan. Carries on selection have certain proteinase-producing ability bacillus strain after the gathering sample, altogether separates and purifies to 55 strains, then chosen 19 strains which all have proteinase-producing ability, to carry on the advance screening. Finally, we screen one to have the high proteinase-producing ability and the effect stable strain to the milk culture medium, after the appraisal, we sure that this strain is Bacillus polymyxa strain, and named A7. The strain has tolerance to bile salt concentration of 0.5% and pH2.5 ability. The experiments show that the strain of A7 has significantly inhibitory effect on the aquaculture-conditional pathogennic bacteria Aeromonas hydrophila and then thought hemolytic test, small white mouse safety test and common carp safety test finally we determination A7 safety on aquaculture.
Adopted solid fermentation medium is corn meal 300g、wheat bran 400g、soybean meal 300g and phosphate butter 500mL. The optimization experiments of solid fermentation results shows that culture conditions included: temperature 28℃, capacity ratio 16%, material and water ratio 1:1.0, inoculation quantity 5%, fermentation time 48h,drying time 48h. The strain revival ration is above 60% on the solid fermentation broth storage at normal temperature of one mouth..
Using A7 solid fermentation broth mixture of diets fed common juvenile carp fish, the trial was carried out 50d. The results showed that the addition of A7 in foundation diets experiment group average of growth rate has the control group 1.23 times and specific growth rates higher than the control group, though the addition of yeast in foundation diets experiment group average of growth rate has lower than the control group 2.1% and specific growth rates lower than the control growth. The apparent digestibility of dry-matter and crude protein feed ingredients which addition of A7 in foundation diets experiment group has been 65.85% and 85% respectively, significantly higher than other test groups(P<0.05).
To the A7 strain degeneration protease, produces the enzyme condition and the enzyme activity research discovery that the optimally produces the enzyme temperature of the strain is 37℃, pH value is 7. The strain in 28℃,110Or/min water shake cultivation 72h,produces the enzyme quantity to achieve the highest, its enzyme activity is 2998U.
The in vitro digestibility of 4 feed protein ingredients i.e. fish meal, soybean meal, rapeseed meal and cottonseed meal for common carp was studied by means of the in vitro digestibility method in the experiment. The results shows that there was no significant in all different test in vitro dry-matter digestibility and crude protein coefficient for fish meal, though there was significant in all different test in vitro dry-matter digestibility and crude protein coefficient for soybean meal, rapeseed meal and cottonseed meal.
通过A7菌株产酶条件的单因素优化试验发现,其最佳产酶条件是:pH值7.0,温度37℃,培养时间48h。A7菌株在28℃,110r/min水域振荡培养72h,产酶量达到最高,其酶活力为2998U。
以玉米粉300g、麦麸400g、豆粕300g和pH6.8磷酸盐缓冲液500mL为固体发酵基础培养基,对固体发酵条件进行单因素优化试验,测定出A7菌株的最佳固体发酵条件是:在温度28℃,发酵时间48h和料水比1:1.0时,接种1.2×10~9cfu/mL的种子液5%于装量40g的250mL容量瓶中,发酵后固体活菌数可达2.85×10~(12)cfu/g。
采用体外消化法研究了A7菌株对鱼粉、豆粕、菜籽粕、棉籽粕4种饲料蛋白质的体外消化能力。试验结果显示,添加A7菌株的所有试验组均对鱼粉干物质和粗蛋白的体外消化率没有明显促进作用(P＞0.05),而添加含10~8cfu/mLA7菌株试验组对豆粕、菜籽粕、棉籽粕干物质和粗蛋白的体外消化率均有显著的促进作用(P＜0.05),其消化率分别提高了44.85%、10.66%和53.05%;8.45%、、28.78%和63.9%。
从鲤鱼幼鱼饲喂试验可以看出,在饲喂鲤鱼幼鱼50d后,饲料中添加A7菌株固体发酵物试验组幼鱼的平均增重率和特定生长率分别高于对照组的23%和11.8%,干物质表观消化率和蛋白质表观消化率的平均值分别为65.85%和85%,均显著高于添加酵母组、酵母+A7组和对照组(P＜0.05)。
This study gathered the intestinal contents of fresh common carp from the breeding ponds and lakes in Sichuan. Carries on selection have certain proteinase-producing ability bacillus strain after the gathering sample, altogether separates and purifies to 55 strains, then chosen 19 strains which all have proteinase-producing ability, to carry on the advance screening. Finally, we screen one to have the high proteinase-producing ability and the effect stable strain to the milk culture medium, after the appraisal, we sure that this strain is Bacillus polymyxa strain, and named A7. The strain has tolerance to bile salt concentration of 0.5% and pH2.5 ability. The experiments show that the strain of A7 has significantly inhibitory effect on the aquaculture-conditional pathogennic bacteria Aeromonas hydrophila and then thought hemolytic test, small white mouse safety test and common carp safety test finally we determination A7 safety on aquaculture.
Adopted solid fermentation medium is corn meal 300g、wheat bran 400g、soybean meal 300g and phosphate butter 500mL. The optimization experiments of solid fermentation results shows that culture conditions included: temperature 28℃, capacity ratio 16%, material and water ratio 1:1.0, inoculation quantity 5%, fermentation time 48h,drying time 48h. The strain revival ration is above 60% on the solid fermentation broth storage at normal temperature of one mouth..
Using A7 solid fermentation broth mixture of diets fed common juvenile carp fish, the trial was carried out 50d. The results showed that the addition of A7 in foundation diets experiment group average of growth rate has the control group 1.23 times and specific growth rates higher than the control group, though the addition of yeast in foundation diets experiment group average of growth rate has lower than the control group 2.1% and specific growth rates lower than the control growth. The apparent digestibility of dry-matter and crude protein feed ingredients which addition of A7 in foundation diets experiment group has been 65.85% and 85% respectively, significantly higher than other test groups(P<0.05).
To the A7 strain degeneration protease, produces the enzyme condition and the enzyme activity research discovery that the optimally produces the enzyme temperature of the strain is 37℃, pH value is 7. The strain in 28℃,110Or/min water shake cultivation 72h,produces the enzyme quantity to achieve the highest, its enzyme activity is 2998U.
The in vitro digestibility of 4 feed protein ingredients i.e. fish meal, soybean meal, rapeseed meal and cottonseed meal for common carp was studied by means of the in vitro digestibility method in the experiment. The results shows that there was no significant in all different test in vitro dry-matter digestibility and crude protein coefficient for fish meal, though there was significant in all different test in vitro dry-matter digestibility and crude protein coefficient for soybean meal, rapeseed meal and cottonseed meal.
引文
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[2]丁彦文,艾红.微生物在水产养殖中的应用[J].湛江海洋大学学报,2000.20(1):68-73;
[3]Elisabetta Chelossia,Luigi Vezzullib,Anna Milanoc et al,Antibiotic resistance of benthic bacteria in fish-farm and control sediments of the Western Mediterranean[J].Aquaculture,2002,219:83-97;
[4]Eleonor A Tendencia,et al,Level and percentage recovery of resistance to oxytetracycline and oxolinic acid of bacteria from shrimp ponds[J].Aquaculture,2002,213:1-13
[5]Fuller R.Probiotics in man and animals[J].J Appl Bacteriol,1989,66(5):365-378
[6]Adami A.Cavazzoni V.Occurrence of selected bacterial groups in the faeces of piglets fed with Bacillus coagulans as probiotic[J].J Basic Microbiol,1999,39:3-9;
[7]Alexopoulos C,Georgoulakis I E,Tzivara A,et al.Field evaluation of the efficacy of a probiotic containing bacillus licheniformis and Bacillus subtilis spores,on the health status and performance of sows and their litters[J].J Anim Physiol Anim Nutr,2004,88:381-392;
[8]Kritas S K.Morrison R B.Evaluation of probiotics as a substitute for antibiotics in a large pig nursery[J].Vet Rec,2005,156:447-448;
[9]Zani J L,da Cruz F W,dos Santos A F,et al.Effect of probiotic CenBiot on the control of diarrhoea and feed efficiency in pigs[J].JAppl Microbiol,1998,84:68-71
[10]何明清.我国动物微生态制剂的起源、发展战略及应用前景[J].中国微生态学杂志,2001,13(3):166-167,176
[11]Kozasa M1986.Toyocerin(Bacillus toyoi) as growth promoter for animal feeding[J].Microbial Aliment Nutr.164:351-358
[12]Ring E,gatesoupe F J.Lactic acid bacteria in fish:a view[J].Aquaculture,1998,160:177-203
[13]Rengpipat S,Phianphak W,Piyatiratitivorakul S,etal.Effects of a probiotic bacterium on black tiger shrimp(Penaeus monodon) survival and growth[J].Aquaculture,1998,167(3):301-313
[14]华雪铭,周洪琪,邱小琮等.饲料中添加芽孢杆菌和硒酵母对异育银鲫的生长及抗病力的影响[J].水产学:报,2001,25(5):448-453
[15]陈勇等.益康菌对鲤鱼肠道菌群生长的影响[J].北华大学学报(自然科学版)[J].2001,2(5): 441-444
[16]华雪铭,周洪琪,张宇峰周辉.饲料中添加壳聚糖和益生菌对暗纹东方鲀幼鱼生长及部分消化酶活性的影响[J].水生生物学报,2005,29(3):299-305
[17]王福强等.产蛋白酶益生菌Y2—2和Y1—13对牙鲆幼鱼生长促进作用的研究[J].海洋科学,2005,29(1):29-33
[18]刘波,刘文斌,王恬.地衣芽孢杆菌对异育银鲫消化机能和生长的影响[J].南京农业大学学报,2005,28(4):80-84
[19]倪学勤等.微生物饲料添加剂对鲤鱼生长性能影响的研究[J].中国饲料.2005,6,7-9
[20]易力、汪洋.不同益生菌剂对鲤鱼促生长作用研究[J].水利渔业.2006,26(5):86-87
[21]刘文斌等.3种益生素配伍对异育银鲫(Carassius auratus gibelio)生长、消化及肠道菌群组成的影响[J].海洋与湖沼.2007,27(1):29-35
[22]孙舰军,丁美丽.改善虾池环境增强中国对虾抗病力的研究[J].海洋科学.1999,1:3-5
[23]Kerovuo J,Lappalainen I,Reinikainen T.The Metal Dependence of Bacillus subtilis Phytase[J].Biochemical and Biophysical Research Communications,2000,(2):365-369
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