豆类丝核菌发酵液中苦马豆素的提取及其体外抑菌活性试验和对新城疫疫苗免疫效果的影响
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摘要
目的:改进从豆类丝核菌发酵液中提取分离苦马豆素的方法;对发酵液不同极性溶剂的萃取物进行体外抑菌试验的研究;对3.2871A豆类丝核菌菌株发酵液在雏鸡免疫功能方面的影响进行研究。
方法:1.豆类丝核菌保藏与移接,用马铃薯葡萄糖琼脂(PDA)培养基,发酵培养用改良的查氏(Czapek’s)培养基,温度25℃,温箱培养14d,收集发酵液并对其进行预处理。采用减压浓缩、分段萃取、D101大孔树脂吸附、硅胶柱层析、冷冻干燥、减压升华等方法从发酵液中提取分离SW,薄层色谱适时监测,薄层色谱、气相色谱对分离产物进行鉴定分析。
2.采用纸片扩散法与试管稀释法,选用金黄色葡萄球菌(Staphyloccocus aureus)、大肠埃希菌(Escherichia coli)、无乳链球菌(S.agalactiae)、李斯特菌(L.monocytogenes)、蜡状芽孢杆菌(Bacillus cereus)对发酵液各段萃取物进行定性体外抑菌试验,然后根据定性体外抑菌试验结果再进行定量体外抑菌试验,测定各段萃取物的最低抑菌浓度(MIC)。
3.研究豆类丝核菌发酵液对雏鸡免疫功能的影响时,在雏鸡免疫新城疫疫苗前、后3天对其注射SW含量为(1、2、4 mg·kg-1)3种剂量的豆类丝核菌发酵液,通过测定雏鸡的抗体效价、免疫器官指数、外周血白细胞数及脾脏淋巴细胞增殖情况来分析免疫效果,确定其使用剂量和使用时间。
结果:1.定期保藏与移接的各豆类丝核菌菌株生长状态良好;从3.2871A豆类丝核菌菌株发酵液中提取分离到白色粉末,经薄层色谱、气相色谱鉴定分析确定其中含有苦马豆素。
2.发酵液4个部分提取物均呈现不同程度的抑菌效果。豆类丝核菌发酵液正丁醇萃取部分抑菌作用明显,雷氏盐提取物也具有较好的抑菌效果,由于SW标准液浓度太小没能表现出明显的抑菌效果。经进一步试验发现正丁醇萃取部分对大肠埃希菌MIC为50mg/mL,对金黄色葡萄球菌、李斯特杆菌MIC均25mg/mL,对其他2种菌MIC均为12.5mg/mL;雷氏盐提取物对无乳链球菌、蜡状芽孢杆菌MIC均为50mg/mL,对其他3种菌MIC均为25mg/mL。
3.雏鸡免疫新城疫疫苗前后3天,注射中剂量豆类丝核菌发酵液,作佐剂,可使雏鸡血清特异性抗体效价极显著增高(P<0.01),外周血白细胞数增加(P<0.01),雏鸡免疫器官指数差异极显著(P<0.01);中剂量豆类丝核菌发酵液单独或协同刺激淋巴细胞时,淋巴细胞增殖极显著(P<0.01)。
结论:改进从3.2871A豆类丝核菌菌株发酵液中提取分离苦马豆素的方法,经薄层色谱、气相色谱鉴定分析确定其产物中含有苦马豆素,但未得到纯品SW,试验方法仍需进一步的研究与改进;发酵液4个部分提取物均呈现不同程度的抑菌效果,豆类丝核菌发酵液正丁醇萃取部分抑菌作用最明显,说明SW具有一定的抑菌作用;一定剂量的豆类丝核菌发酵液能增强雏鸡的免疫功能。
Objective: Improve the method of isolate SW from Rhizoctonia leguminicola fermentation broth;Study the bacteriostasis in vitro of fermentation broth extract from different polar solvent, widen the use scope of Rhizoctonia leguminicola fermentation broth,improve its utilization value;Study the effect to chicken's immunologic function of 3.2871A Rhizoctonia leguminicola fermentation broth。
Method:1.Use the potato dextrose agar(PDA) to preserve regularly and graft the Rhizoctonia leguminicola ,fermented culture use the improved Czapek's medium,temperature 25℃,Thermostate culture 14d,collect the fermentation broth and pretreat.Use the method of Reduced pressure concentration、stage extraction、D101macroporous resin adsorption、Silica gel column chromatography、freeze drying、Decompression sublimation etc. to isolate SW from fermentation broth,use the thin layer chromatography to detect timely,use the thin-layer chromatography and gas chromatograph to identify and analyse the isolate product.
2.Use the Bauer test and tube dilution method,choose common representative bacteria of GN and GP to test the bacteriostasis of fermentation broth extract from different polar solvent in vitro qualitatively,then on the basis of the results of qualitative bacteriostatic experiment to do the quantitative bacteriostatic experiment,test the MIC of each abstract.
3.Study the effect to chicken's immunologic function of Rhizoctonia leguminicola,inject the Rhizoctonia leguminicola fermentation broth (the content of SW were 1、2、4 mg·kg~(-1)) before and after the chikens was vaccinated Newcastle disease vaccine three days ,through test the chikens' antibodytiter、Immune organs index、Peripheral leukocyte count and Spleen lymphocyte proliferation to confirm the using dosage and time.
Result:1.The growth state of each Rhizoctonia leguminicola that was preserved regularly and grafted was good;isolated the white powder from the 3.2871A Rhizoctonia leguminicola fermentation broth, and identified that there was the SW in it through the thin-layer chromatography and gas chromatograph.
2.Four part extraction from fermentation broth have the different bacteriostatic efficacy.the bacteriostatic efficacy of the extraction from the n-butanol parts was obvious,Reinecke's salt extraction also had the good bacteriostatic efficacy,because of the low concentration of the SW Standard liquids the bacteriostatic efficacy didn't display obviously.Through the further tests we found that the MIC of the extraction from the n-butanol parts to escherichia coli was 50mg/mL,to staphylococcus aureus、Listeria monocytogenes were all 25mg/mL,to the rest two kinds of bacteria was 12.5mg/mL;the MIC of Reinecke's salt extraction to Streptococcus agalactiae、Bacillus cereus were all 50mg/mL,to the rest three kinds of bacteria was 25mg/mL.
3.Inject the the Rhizoctonia leguminicola fermentation broth(the content of SW was 2 mg·kg~(-1)) after the chickens was vaccinated Newcastle disease vaccine three days,use it as a kind of adjuvant,could improve the chickens' specific serum antibody titer obviously,the number of peripheral blood was increase,has the definite facilitation effect to the upgrowth of chickens' immune organs;when the Rhizoctonia leguminicola secondary metabolite(the content of SW was 2 mg·kg~(-1))stimulate the lymphocyte independently or synergeticly, lymphocyte has the obvious proliferation.
Conclusion:Improved the method of isolate SW from 3.2871A Rhizoctonia leguminicola fermentation broth,confirmed that there was SW through the thin-layer chromatography and gas chromatograph,but didn't obtain SW puregrad,the experiment need the further study and improvment;four part extraction from fermentation broth have the different bacteriostatic efficacy,the bacteriostatic efficacy of the extraction from the n-butanol parts was most obvious,illustrated that SW had the definite bacteriostatic efficacy;Use the regular dose of Rhizoctonia leguminicola fermentation broth can improve the chickens' humoral immunity and cellular immunity function.
方法:1.豆类丝核菌保藏与移接,用马铃薯葡萄糖琼脂(PDA)培养基,发酵培养用改良的查氏(Czapek’s)培养基,温度25℃,温箱培养14d,收集发酵液并对其进行预处理。采用减压浓缩、分段萃取、D101大孔树脂吸附、硅胶柱层析、冷冻干燥、减压升华等方法从发酵液中提取分离SW,薄层色谱适时监测,薄层色谱、气相色谱对分离产物进行鉴定分析。
2.采用纸片扩散法与试管稀释法,选用金黄色葡萄球菌(Staphyloccocus aureus)、大肠埃希菌(Escherichia coli)、无乳链球菌(S.agalactiae)、李斯特菌(L.monocytogenes)、蜡状芽孢杆菌(Bacillus cereus)对发酵液各段萃取物进行定性体外抑菌试验,然后根据定性体外抑菌试验结果再进行定量体外抑菌试验,测定各段萃取物的最低抑菌浓度(MIC)。
3.研究豆类丝核菌发酵液对雏鸡免疫功能的影响时,在雏鸡免疫新城疫疫苗前、后3天对其注射SW含量为(1、2、4 mg·kg-1)3种剂量的豆类丝核菌发酵液,通过测定雏鸡的抗体效价、免疫器官指数、外周血白细胞数及脾脏淋巴细胞增殖情况来分析免疫效果,确定其使用剂量和使用时间。
结果:1.定期保藏与移接的各豆类丝核菌菌株生长状态良好;从3.2871A豆类丝核菌菌株发酵液中提取分离到白色粉末,经薄层色谱、气相色谱鉴定分析确定其中含有苦马豆素。
2.发酵液4个部分提取物均呈现不同程度的抑菌效果。豆类丝核菌发酵液正丁醇萃取部分抑菌作用明显,雷氏盐提取物也具有较好的抑菌效果,由于SW标准液浓度太小没能表现出明显的抑菌效果。经进一步试验发现正丁醇萃取部分对大肠埃希菌MIC为50mg/mL,对金黄色葡萄球菌、李斯特杆菌MIC均25mg/mL,对其他2种菌MIC均为12.5mg/mL;雷氏盐提取物对无乳链球菌、蜡状芽孢杆菌MIC均为50mg/mL,对其他3种菌MIC均为25mg/mL。
3.雏鸡免疫新城疫疫苗前后3天,注射中剂量豆类丝核菌发酵液,作佐剂,可使雏鸡血清特异性抗体效价极显著增高(P<0.01),外周血白细胞数增加(P<0.01),雏鸡免疫器官指数差异极显著(P<0.01);中剂量豆类丝核菌发酵液单独或协同刺激淋巴细胞时,淋巴细胞增殖极显著(P<0.01)。
结论:改进从3.2871A豆类丝核菌菌株发酵液中提取分离苦马豆素的方法,经薄层色谱、气相色谱鉴定分析确定其产物中含有苦马豆素,但未得到纯品SW,试验方法仍需进一步的研究与改进;发酵液4个部分提取物均呈现不同程度的抑菌效果,豆类丝核菌发酵液正丁醇萃取部分抑菌作用最明显,说明SW具有一定的抑菌作用;一定剂量的豆类丝核菌发酵液能增强雏鸡的免疫功能。
Objective: Improve the method of isolate SW from Rhizoctonia leguminicola fermentation broth;Study the bacteriostasis in vitro of fermentation broth extract from different polar solvent, widen the use scope of Rhizoctonia leguminicola fermentation broth,improve its utilization value;Study the effect to chicken's immunologic function of 3.2871A Rhizoctonia leguminicola fermentation broth。
Method:1.Use the potato dextrose agar(PDA) to preserve regularly and graft the Rhizoctonia leguminicola ,fermented culture use the improved Czapek's medium,temperature 25℃,Thermostate culture 14d,collect the fermentation broth and pretreat.Use the method of Reduced pressure concentration、stage extraction、D101macroporous resin adsorption、Silica gel column chromatography、freeze drying、Decompression sublimation etc. to isolate SW from fermentation broth,use the thin layer chromatography to detect timely,use the thin-layer chromatography and gas chromatograph to identify and analyse the isolate product.
2.Use the Bauer test and tube dilution method,choose common representative bacteria of GN and GP to test the bacteriostasis of fermentation broth extract from different polar solvent in vitro qualitatively,then on the basis of the results of qualitative bacteriostatic experiment to do the quantitative bacteriostatic experiment,test the MIC of each abstract.
3.Study the effect to chicken's immunologic function of Rhizoctonia leguminicola,inject the Rhizoctonia leguminicola fermentation broth (the content of SW were 1、2、4 mg·kg~(-1)) before and after the chikens was vaccinated Newcastle disease vaccine three days ,through test the chikens' antibodytiter、Immune organs index、Peripheral leukocyte count and Spleen lymphocyte proliferation to confirm the using dosage and time.
Result:1.The growth state of each Rhizoctonia leguminicola that was preserved regularly and grafted was good;isolated the white powder from the 3.2871A Rhizoctonia leguminicola fermentation broth, and identified that there was the SW in it through the thin-layer chromatography and gas chromatograph.
2.Four part extraction from fermentation broth have the different bacteriostatic efficacy.the bacteriostatic efficacy of the extraction from the n-butanol parts was obvious,Reinecke's salt extraction also had the good bacteriostatic efficacy,because of the low concentration of the SW Standard liquids the bacteriostatic efficacy didn't display obviously.Through the further tests we found that the MIC of the extraction from the n-butanol parts to escherichia coli was 50mg/mL,to staphylococcus aureus、Listeria monocytogenes were all 25mg/mL,to the rest two kinds of bacteria was 12.5mg/mL;the MIC of Reinecke's salt extraction to Streptococcus agalactiae、Bacillus cereus were all 50mg/mL,to the rest three kinds of bacteria was 25mg/mL.
3.Inject the the Rhizoctonia leguminicola fermentation broth(the content of SW was 2 mg·kg~(-1)) after the chickens was vaccinated Newcastle disease vaccine three days,use it as a kind of adjuvant,could improve the chickens' specific serum antibody titer obviously,the number of peripheral blood was increase,has the definite facilitation effect to the upgrowth of chickens' immune organs;when the Rhizoctonia leguminicola secondary metabolite(the content of SW was 2 mg·kg~(-1))stimulate the lymphocyte independently or synergeticly, lymphocyte has the obvious proliferation.
Conclusion:Improved the method of isolate SW from 3.2871A Rhizoctonia leguminicola fermentation broth,confirmed that there was SW through the thin-layer chromatography and gas chromatograph,but didn't obtain SW puregrad,the experiment need the further study and improvment;four part extraction from fermentation broth have the different bacteriostatic efficacy,the bacteriostatic efficacy of the extraction from the n-butanol parts was most obvious,illustrated that SW had the definite bacteriostatic efficacy;Use the regular dose of Rhizoctonia leguminicola fermentation broth can improve the chickens' humoral immunity and cellular immunity function.
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