ANSB01G菌对玉米赤霉烯酮的降解机制及其动物试验效果研究
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摘要
试验一目的是抽样调查北京地区饲料及原料饲料中的玉米赤霉烯酮(zearalenone, ZEA)污染状况。调查结果显示,北京地区15个猪场的131份样品(玉米、豆粕、糠麸类、DDGS和猪全价饲料)普遍存在ZEA污染；且玉米、豆粕、麸皮、DDGS口猪全价饲料中ZEA的检出率分别达到100%、54.45%、100%、100%和100%,超标率分别为0.00%、0.00%、0.00%、41.18%和0.00%。其中,ZEA平均含量分别为109.08、9.19、14.92、882.68和58.88μg/kg。同时,抽检结果显示,DDGS中ZEA污染最为严重,超标率达41.18%,其中中度污染率达23.53%,严重污染率达17.65%。
试验二目的是降解ZEA的益生菌的筛选及其降解特性研究。本试验从79个各种动物肠道食糜或粪便、发霉的饲料和食品以及不同环境和地区的土壤样品中筛选得到一株能够高效降解ZEA的细菌ANSBO1G。其对液体培养基中ZEA的降解率为88.65%,对玉米、DDGS和猪全价饲料中ZEA的降解率分别为84.58、66.34和83.04%。经细菌形态学、生理生化特性以及16S rDNA鉴定,ANSB01G是一株枯草芽孢杆菌。并且,枯草芽孢杆菌ANSB01G对大肠杆菌、沙门氏菌和金黄色葡萄球菌具有很好的抑菌活性,同时在高温、胃酸和胆盐条件下具有较高的存活率和耐受性。研究还发现,该菌株的降解活性物质主要存在于发酵上清液中,且上清液对蛋白酶K和加热敏感,从而推断其作用机制是降解,而不是吸附。
试验三目的是降解ZEA活性物质的分离纯化及作用机理分析。本研究首先采用硫酸铵对活性物质进行沉淀,然后使用离子交换层析、疏水层析以及分子筛层析方法对硫酸铵沉淀得到的活性物质进行进一步分离纯化。结果显示,其活性组分分子量小于44.3kDa。用Q-TOF对降解产物进行分析,根据降解产物的质谱结果推测其作用机理,ZEA的降解首先是ZEA酚羟基与谷氨酸的Y-羧基结合,然后是ZEA的内酯环水解、脱羧、还原羰基和脱水。
试验四目的是研究枯草芽孢杆菌ANSB01G工业发酵干燥产品霉立解(MLJ),对采食ZEA污染日粮青年母猪的影响。试验选取体重为36.64±1.52kg青年母猪18头,随机分为三个处理,每个处理6个重复,试验期为24d。对照组(C)饲喂正常玉米型日粮(不含ZEA),处理组1(T1)饲喂霉变玉米型日粮(ZEA,238.57μg/kg),处理组2(T2)在T1的基础上添加2kg/T的MLJ(活菌数为1×109CFU/g)。研究结果表明,与对照组相比,被238.57μg/kg ZEA亏染的T1组日粮可导致青年母猪阴户红肿,阴户面积显著增加,繁殖器官指数显著增加(P<0.05),而T2组在霉变日粮中添加MLJ能缓解青年母猪阴户红肿和繁殖器官指数增加；T1组中青年母猪血清FSH、LH和E2水平没有显著影响,但是血清中PRL的水平显著升高(P<0.05)；T1组中子宫体细胞出现明显的细胞肿大和脂肪变性,肝脏出现细胞肿胀、炎症反应和淋巴细胞浸润现象,卵巢出现卵泡萎缩退化和内部空化,而在霉变日粮中添加霉立解(T2组)使这些症状明显减轻。
试验五目的是研究霉立解对采食ZEA污染日粮肉母鸡的影响。试验选取180只1日龄Ross308肉母鸡,随机分为三个处理,每个处理6个重复,每个重复10只鸡。试验期42天。对照组(C)饲喂正常玉米型日粮,处理组1(T1)饲喂霉变玉米型日粮(ZEA,248.76μg/kg),处理组2(T2)在T1的基础上添加1kg/T的霉立解(活菌数为1×109CFU/g)。试验结果显示,不同处理组之间,肉母鸡的生产性能、器官指数、肉品质以及血清和肝脏抗氧化能力均没有显著差异。本试验结果表明,含有248.76μg/kg ZEA的日粮对肉母鸡生产性能、器官指数、肉品质以及血清和肝脏抗氧化能力均没有显著影响。
Experiment1:The aim of this research was to analyze and evaluate the contamination situation of zearalenone (ZEA) in ingredients and complete feeds in Beijing region. Results showed that the detection rate of ZEA in corn, soybean meal, bran, DDGS, swine complete feeds were100%,54.45%,100%,100%and100%, respectively; the over standard rate were0.00%,0.00%,0.00%,41.18%and0.00%, respectively; and the average content were109.08,9.19,14.92,882.68and58.88μg/kg, respectively. These results indicated that the average content of ZEA in DDGS was the highest. Moderate contamination rate and severe contamination rate in DDGS were23.53%and17.65%. Therefore, ZEA contamination was widely found in both feeds and feedstuffs.
Experiment2:The aim of this research was to screen for probiotics caplable of degrading zearalenone and to study on its degradation characteristics. A bacteria ANSB01G strain which could degrade ZEA was isolated from79kinds of animal intestinal chyme, moldy feed and food, or soil samples. The results showed that ANSB01G strain was able to degrade88.65%of ZEA in liquid mediun. About84.58,66.34and83.04%of ZEA were degraded by ANSB01G in naturally contaminated maize, DDGS and swine complete feed, respectively. According to bacterial morphology, physiological and biochemical characteristics and16S rDNA identification results, ANSB01G is a Bacillus subtilis strain. The Bacillus subtilis ANSB01G has a good antibacterial activity on Escherichia coli, Salmonella typhimurium and Staphylococcus aureus, and has a high survival rate and tolerance in high temperature, acid or bile salt conditions. This experiment studied the ZEA degradation by ANSB01G strain in different pH, different reaction time and different reaction temperature. Both heating and treatment with proteinase K significantly reduced the ZEA degradation activity of the culture supernatant, indicating that ZEA degradation might be an enzymatic reaction.
Experiment3:The aim of the research was to study on the purification and separation of enzyme and products of ZEA degraded by ANSB01G. Extracellular proteins with ZEA transformation activity was precipitated with ammonium sulfate, and then purified by the ion-exchange chromatography, hydrophobic chromatography and Sephadex-Molecular sieve chromatography method. The result showed that the apparent molecular masses are less than44.3kDa. Mass spectrum (Q-TOF) of the degradation products indicated that the first step of ZEA degradation is to bind the phenolic hydroxyl of ZEA and the γ-carboxyl of glutamate, and the following reactions were the lactone ring of ZEA hydrolysis, decarboxylation, reduction of carbonyl and dehydration.
Experiment4:The aim of this research was to investigate the effect of MLJ(Bacillus subtilis ANSB01G) on prepubertal female gilts when exposed to ZEA. In this experiment, a total of18prepubertal female gilts were randomly allocated to three treatments with6replicate per treatment (one pig per replicate). The gilts in each treatment were randomly with one of the three diets:control (basal diet with normal corn; T1(Diet was prepared by substituting corn naturally contaminated by ZEA for all normal corn in basic diets, The concentrations of ZEA in diet is238.57μg/kg); T2(Diet was prepared by mixing the diets of T1with2kg/T of MLJ). The total viable count of MLJ was1×109CFU/g. This experiment lasted for24days. The results showed that ZEA in diets significantly increased the vulva size and reproductive organs weight of gilts in T1group compared with the control group (P<0.05), and the addition of MLJ to diets naturally contaminated by ZEA can ameliorate these symptoms as observed in the T2group. The presence of low doses of ZEA (238.57μg/kg) in the diet (T1group) had no significant effect (P>0.05) on the level of FSH, LH, and E2in serum of gilts, but the level of PRL in group T1was increased significantly (P<0.05). The gilts of T1group showed conspicuous cell enlargement and fatty degeneration of the corpus uteri, atrophy and cavitation of mature follicular tissue and swelling, inflammation, and lymphocyte infiltration of liver cells as compared to that in Control group. The addition of MLJ in the diet (T2group) can alleviate these hyperestrogenic effects caused by ZEA, maintaining the body of gilt in a healthy status. The results indicated that the short-term (24days) feeding diet concentrated with ZEA (238.57μg/kg of diet) can cause deleterious effects on prepubertal female gilts, and addition of2kg MLJ into a ton diet can effectively ameliorate these detrimental effects and alleviate ZEA toxicosis in gilts.
Experiment5:The aim of this research was to investigate the effect of MLJ (Bacillus subtilis ANSB01G) on female broilers when exposed to ZEA. A total of180female Ross308broilers were randomly assigned to3treatments with6replicate of10birds each. Control group (C) comprised basal diet with normal corn; Treatment1(T1) was prepared by substituting corn naturally contaminated by ZEA for all normal corn in basic diets (The concentrations of ZEA in diet is248.76μg/kg); Treatment2(T2) was prepared by mixing the diets of T1with1kg/T of MLJ. The total viable count of MLJ was1×109CFU/g. This experiment lasted for42days. The result showed that there were no significant effects on growing performance, relative weight of organ, meat quality and antioxidant capacity of serum and liver from female broilers in C, T1and T2groups (P>0.05). The results indicated that the adverse effect of low level (248.76μg/kg of diet) of ZEA in diet of female broilers was slight.
试验二目的是降解ZEA的益生菌的筛选及其降解特性研究。本试验从79个各种动物肠道食糜或粪便、发霉的饲料和食品以及不同环境和地区的土壤样品中筛选得到一株能够高效降解ZEA的细菌ANSBO1G。其对液体培养基中ZEA的降解率为88.65%,对玉米、DDGS和猪全价饲料中ZEA的降解率分别为84.58、66.34和83.04%。经细菌形态学、生理生化特性以及16S rDNA鉴定,ANSB01G是一株枯草芽孢杆菌。并且,枯草芽孢杆菌ANSB01G对大肠杆菌、沙门氏菌和金黄色葡萄球菌具有很好的抑菌活性,同时在高温、胃酸和胆盐条件下具有较高的存活率和耐受性。研究还发现,该菌株的降解活性物质主要存在于发酵上清液中,且上清液对蛋白酶K和加热敏感,从而推断其作用机制是降解,而不是吸附。
试验三目的是降解ZEA活性物质的分离纯化及作用机理分析。本研究首先采用硫酸铵对活性物质进行沉淀,然后使用离子交换层析、疏水层析以及分子筛层析方法对硫酸铵沉淀得到的活性物质进行进一步分离纯化。结果显示,其活性组分分子量小于44.3kDa。用Q-TOF对降解产物进行分析,根据降解产物的质谱结果推测其作用机理,ZEA的降解首先是ZEA酚羟基与谷氨酸的Y-羧基结合,然后是ZEA的内酯环水解、脱羧、还原羰基和脱水。
试验四目的是研究枯草芽孢杆菌ANSB01G工业发酵干燥产品霉立解(MLJ),对采食ZEA污染日粮青年母猪的影响。试验选取体重为36.64±1.52kg青年母猪18头,随机分为三个处理,每个处理6个重复,试验期为24d。对照组(C)饲喂正常玉米型日粮(不含ZEA),处理组1(T1)饲喂霉变玉米型日粮(ZEA,238.57μg/kg),处理组2(T2)在T1的基础上添加2kg/T的MLJ(活菌数为1×109CFU/g)。研究结果表明,与对照组相比,被238.57μg/kg ZEA亏染的T1组日粮可导致青年母猪阴户红肿,阴户面积显著增加,繁殖器官指数显著增加(P<0.05),而T2组在霉变日粮中添加MLJ能缓解青年母猪阴户红肿和繁殖器官指数增加；T1组中青年母猪血清FSH、LH和E2水平没有显著影响,但是血清中PRL的水平显著升高(P<0.05)；T1组中子宫体细胞出现明显的细胞肿大和脂肪变性,肝脏出现细胞肿胀、炎症反应和淋巴细胞浸润现象,卵巢出现卵泡萎缩退化和内部空化,而在霉变日粮中添加霉立解(T2组)使这些症状明显减轻。
试验五目的是研究霉立解对采食ZEA污染日粮肉母鸡的影响。试验选取180只1日龄Ross308肉母鸡,随机分为三个处理,每个处理6个重复,每个重复10只鸡。试验期42天。对照组(C)饲喂正常玉米型日粮,处理组1(T1)饲喂霉变玉米型日粮(ZEA,248.76μg/kg),处理组2(T2)在T1的基础上添加1kg/T的霉立解(活菌数为1×109CFU/g)。试验结果显示,不同处理组之间,肉母鸡的生产性能、器官指数、肉品质以及血清和肝脏抗氧化能力均没有显著差异。本试验结果表明,含有248.76μg/kg ZEA的日粮对肉母鸡生产性能、器官指数、肉品质以及血清和肝脏抗氧化能力均没有显著影响。
Experiment1:The aim of this research was to analyze and evaluate the contamination situation of zearalenone (ZEA) in ingredients and complete feeds in Beijing region. Results showed that the detection rate of ZEA in corn, soybean meal, bran, DDGS, swine complete feeds were100%,54.45%,100%,100%and100%, respectively; the over standard rate were0.00%,0.00%,0.00%,41.18%and0.00%, respectively; and the average content were109.08,9.19,14.92,882.68and58.88μg/kg, respectively. These results indicated that the average content of ZEA in DDGS was the highest. Moderate contamination rate and severe contamination rate in DDGS were23.53%and17.65%. Therefore, ZEA contamination was widely found in both feeds and feedstuffs.
Experiment2:The aim of this research was to screen for probiotics caplable of degrading zearalenone and to study on its degradation characteristics. A bacteria ANSB01G strain which could degrade ZEA was isolated from79kinds of animal intestinal chyme, moldy feed and food, or soil samples. The results showed that ANSB01G strain was able to degrade88.65%of ZEA in liquid mediun. About84.58,66.34and83.04%of ZEA were degraded by ANSB01G in naturally contaminated maize, DDGS and swine complete feed, respectively. According to bacterial morphology, physiological and biochemical characteristics and16S rDNA identification results, ANSB01G is a Bacillus subtilis strain. The Bacillus subtilis ANSB01G has a good antibacterial activity on Escherichia coli, Salmonella typhimurium and Staphylococcus aureus, and has a high survival rate and tolerance in high temperature, acid or bile salt conditions. This experiment studied the ZEA degradation by ANSB01G strain in different pH, different reaction time and different reaction temperature. Both heating and treatment with proteinase K significantly reduced the ZEA degradation activity of the culture supernatant, indicating that ZEA degradation might be an enzymatic reaction.
Experiment3:The aim of the research was to study on the purification and separation of enzyme and products of ZEA degraded by ANSB01G. Extracellular proteins with ZEA transformation activity was precipitated with ammonium sulfate, and then purified by the ion-exchange chromatography, hydrophobic chromatography and Sephadex-Molecular sieve chromatography method. The result showed that the apparent molecular masses are less than44.3kDa. Mass spectrum (Q-TOF) of the degradation products indicated that the first step of ZEA degradation is to bind the phenolic hydroxyl of ZEA and the γ-carboxyl of glutamate, and the following reactions were the lactone ring of ZEA hydrolysis, decarboxylation, reduction of carbonyl and dehydration.
Experiment4:The aim of this research was to investigate the effect of MLJ(Bacillus subtilis ANSB01G) on prepubertal female gilts when exposed to ZEA. In this experiment, a total of18prepubertal female gilts were randomly allocated to three treatments with6replicate per treatment (one pig per replicate). The gilts in each treatment were randomly with one of the three diets:control (basal diet with normal corn; T1(Diet was prepared by substituting corn naturally contaminated by ZEA for all normal corn in basic diets, The concentrations of ZEA in diet is238.57μg/kg); T2(Diet was prepared by mixing the diets of T1with2kg/T of MLJ). The total viable count of MLJ was1×109CFU/g. This experiment lasted for24days. The results showed that ZEA in diets significantly increased the vulva size and reproductive organs weight of gilts in T1group compared with the control group (P<0.05), and the addition of MLJ to diets naturally contaminated by ZEA can ameliorate these symptoms as observed in the T2group. The presence of low doses of ZEA (238.57μg/kg) in the diet (T1group) had no significant effect (P>0.05) on the level of FSH, LH, and E2in serum of gilts, but the level of PRL in group T1was increased significantly (P<0.05). The gilts of T1group showed conspicuous cell enlargement and fatty degeneration of the corpus uteri, atrophy and cavitation of mature follicular tissue and swelling, inflammation, and lymphocyte infiltration of liver cells as compared to that in Control group. The addition of MLJ in the diet (T2group) can alleviate these hyperestrogenic effects caused by ZEA, maintaining the body of gilt in a healthy status. The results indicated that the short-term (24days) feeding diet concentrated with ZEA (238.57μg/kg of diet) can cause deleterious effects on prepubertal female gilts, and addition of2kg MLJ into a ton diet can effectively ameliorate these detrimental effects and alleviate ZEA toxicosis in gilts.
Experiment5:The aim of this research was to investigate the effect of MLJ (Bacillus subtilis ANSB01G) on female broilers when exposed to ZEA. A total of180female Ross308broilers were randomly assigned to3treatments with6replicate of10birds each. Control group (C) comprised basal diet with normal corn; Treatment1(T1) was prepared by substituting corn naturally contaminated by ZEA for all normal corn in basic diets (The concentrations of ZEA in diet is248.76μg/kg); Treatment2(T2) was prepared by mixing the diets of T1with1kg/T of MLJ. The total viable count of MLJ was1×109CFU/g. This experiment lasted for42days. The result showed that there were no significant effects on growing performance, relative weight of organ, meat quality and antioxidant capacity of serum and liver from female broilers in C, T1and T2groups (P>0.05). The results indicated that the adverse effect of low level (248.76μg/kg of diet) of ZEA in diet of female broilers was slight.
引文
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