桑树中产1-脱氧野尻霉素微生物的研究
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摘要
1-脱氧野尻霉素(DNJ)是一种α-葡萄糖苷酶抑制剂,能有效抑制糖吸收,降低血糖值。桑作为具有良好降血糖效果的中药,其降血糖的主要有效成分DNJ的含量已经被测定。前人已经对不同桑品种、不同叶位、不同蚕品种以及不同发育阶段的蚕体、蚕沙、蛹等的DNJ含量做了大量的研究。目前许多蚕桑原料已被开发为降血糖产品用于临床。
目前已从多种微生物和植物中检测到DNJ及其类似物,植物中以桑树中的DNJ含量最高,家蚕体内的DNJ来源于桑叶。但关于桑树中DNJ的确切来源还没有定论,一般认为桑树中的DNJ是由桑树自身合成的,但研究表明植物体内的很多次生代谢产物都是由体内的共生微生物产生的,DNJ也是在链霉菌中首次被发现的。为探讨桑树中的DNJ来源,本文通过对桑树内生菌(细菌、真菌、放线菌)的分离,利用RP-HPLC-UV法对分离株能否产生DNJ进行检测,发现桑树体内的确存在能够产生DNJ的内生菌。进一步对分离获得的产DNJ性质稳定的菌株进行了鉴定、对其中一菌株产DNJ的发酵条件和发酵培养基进行了优化,对发酵产物进行了初步的分离鉴定,最后通过动物试验,对产DNJ内生菌发酵液的安全性及其降血糖效果进行检测,主要取得以下结果。
1分离桑树、组培苗及桑籽中的内生菌。利用RP-HPLC-UV法检测分离株中DNJ的产生情况,共分离得到10株产DNJ的内生细菌分离株,经Rep-PCR鉴定,归类为6株能够产生DNJ的内生细菌,其中菌株Y1、Y2产DNJ的性质稳定。
2对菌株Y1、Y2进行了系统的分类学鉴定。主要包括:菌株形态观察、生理生化指标测定、自动细菌鉴定仪鉴定、脂肪酸分析及16S rDNA序列分析。结果表明, Y1菌株属于嗜麦芽寡养假单孢杆菌(Stenotrophomonas maltophilia),菌株的16S rDNA序列已在Genbank中注册,登录号为GQ268318;Y2菌株属于藤黄微球菌(Micrococcus luteus),菌株的16S rDNA序列已在Genbank中注册,登录号为GU591156。
3通过摇瓶培养方法,探讨了发酵条件和发酵培养基对Y1菌株产DNJ的影响。
利用单次单因子试验确定了摇瓶培养的最佳发酵条件为:接种量2%(v/v)、摇瓶装液量50mL/250mL、发酵温度30℃、初始pH6.0。在此基础上通过单次单因子试验确定了发酵培养基的最佳碳源、氮源、碳氮比、无机盐。运用PB试验分析了培养基组分:麦芽糖、蛋白胨、尿素、硫酸镁、氯化钠对Y1菌株产DNJ的影响。结果表明,蛋白胨和硫酸镁对Y1菌株产DNJ的影响达到极显著水平;尿素对Y1菌株产DNJ的影响达到显著水平;而麦芽糖和氯化钠对Y1菌株产DNJ的影响不显著。通过最陡爬坡路径逼近最大响应区域,最后用中心组合设计和响应面分析,确定了发酵培养基中主要影响因子的最佳浓度。
最优发酵培养基配方为:麦芽糖40.00g、蛋白胨14.29g、尿素7.39g、MgSO4 0.77g、NaCl 1.00g、蒸馏水1000mL。
运用最佳发酵条件和最佳发酵培养基进行摇瓶培养,利用RP-HPLC-UV法测得衍生化后DNJ的峰面积达到了102.9mAU*min,较基础发酵培养基提高了227.21%。
4对Y1菌株产生的DNJ进行了初步分离鉴定。获得了DNJ粗提物,并采用RP-HPLC-UV以及MS对所得DNJ粗提物进行了检测。RP-HPLC-UV结果显示,发酵液中的DNJ得到初步纯化;MS分析结果表明,Y1菌株发酵产生的DNJ与桑叶中DNJ性质相同(离子峰的出峰位置一致)。
5通过动物试验对Y1菌株发酵液的安全性及降血糖效果进行了研究。试验结果表明,Y1菌株的发酵液对小鼠安全,并且具有明显的降血糖作用,能够有效缓解糖尿病小鼠“三多一少”的症状。
1-deoxynojirimycin is an alpha-glycosidase inhibitor, which can inhibit the absorption of sugar, and then lower the blood glucose effectively. Mulberry has an excellent hypoglycemin effect as a traditional Chinese medicine, and its active composition has been identified as DNJ. Former studies has determined the DNJ content from these materials as follows: different mulberry varieties,different leaf positions, different silkworm species as well as silkworm body, silkworm excrement and pupa in different developing stages. At present, the silkworm and mulberry resources have been developed into hypoglycemic products in clinical practice.
Now DNJ and its analogs have been detected from many microorganisms and plants. Mulberry has the highest DNJ content among the plants and the DNJ of the silkworm is obtained from mulberry. Although there is no conclu- sion of the exact origin of DNJ in mulberry, people generally consider that DNJ is synthesized by the mulberry. Meanwhile some studies show that many secondary metabolism products are generated by the associated microorganis- ms in the plants and DNJ is firstly discovered in Streptomyces. In order to explore the DNJ origin, we have isolated the endophytes in mulberry, and then determined the DNJ content of the endophytes by RP-HPLC-UV. We have discovered the endophytes that could produce DNJ. Further researches were as follows: the identification of the endophytes producing DNJ stably, the optimization of fermenting conditions, the initial identification and character- ization of DNJ produced by the endophytes. At last we studied the security and the hypoglycemic effect of the fermentation broth.
1 The endophytes from mulberry, tissue cultured seedlings and mulberry seeds were isolated and determined the DNJ content with the method of RP-HPLC-UV. Nine endophytes producing DNJ were obtained, clustered into seven endophytes by the analysis of rep-PCR. Among of the seven endophytes, strain Y1 and Y2 showed stable quality in the DNJ production.
2 Taxonomic determinations of bacteria strain Y1 and Y2 were carried out, including morphological observation, physiochemical analyses, test of biolog- ical antomated bacterial identification system, analysis of the fatty acids and 16S rDNA sequence analyses. The results showed strain Y1 was fell to Stenotrophomonas maltophilia and its 16S rDNA sequence was registered in GenBank (GenBank accession NO.GQ268318); Strain Y2 fell to Micrococcus luteus and its 16S rDNA sequence was registered in GenBank (GenBank accession NO.GU591156).
3 Through the cultivation in shake-flask fermentation, the effects of environmental conditions and nutrition of strain Y1 producing DNJ were studied.
The optimal shaking flask fermentation conditions were confirmed by one-factor-at-a-time experiment. The optimized fermentation conditions for DNJ production were: inoculum volume 2%(v/v), solution amount 50mL/250mL, fermentation temperature 30℃, initial pH6.0.
Based on the results above, the effects of nutrition on production of DNJ were studied involving the optimal carbon, the optimal nitrogen, C/N, inorga- nic salt by one-factor-at-a-time experiment. In the optimization of medium, the influences of corn meal, maltose, peptone, urea, MgSO4, (NH4)2SO4 and NaCl on DNJ production were first evaluated using Plackett Burman design. Among of the test components, the effect of peptone and MgSO4 both reach extremely significant level and the urea shows significant effect on the DNJ production. However, maltose and NaCl have no significant effects on DNJ production. The path of steepest ascent was used to approach the optimal region of the medium composition. In the last step, the optimal concentrations of test components were determined by central composite design and response surface analysis.
The optimum fermentation medium for production of DNJ were: maltose 40.0g/L, peptone 14.285 g/L, urea 7.391 g/L, MgSO4 0.7706 g/L, NaCl1g/L,corn meal 20.0g/L, maltose 20.0g/L, soybean meal 29.5g/L, (NH4)2SO4 1.0g/L, CaCO3 3.8g/L.
Based on the optimal fermentation condition and medium, the spectrum peak area of DNJ after derivatization reached 102.9mAU*min, 227.21% higher than that of basal medium.
4 The initial extraction, purification of DNJ produced by strain Y1 was studied by Rep-HPLC-UV and MS analysis. The results showed that the DNJ produced by strain Y1 was initial purified. The analysis of MS showed that the DNJ produced by Y1 had similar properties to the DNJ from Mulberry leaves. However, there were differences between the DNJ extraction and DNJ standard sample. It was concluded that the DNJ from strain Y1 and Mulberry leaves was analogue of DNJ sample.
5 The security and hypoglycemic effect of fermention broth were studied by animal experiments. The results showed that the fermention broth was safty for test mice and had excellent hypoglycemin effect, so it could relieve diabetes symptoms effectively.
目前已从多种微生物和植物中检测到DNJ及其类似物,植物中以桑树中的DNJ含量最高,家蚕体内的DNJ来源于桑叶。但关于桑树中DNJ的确切来源还没有定论,一般认为桑树中的DNJ是由桑树自身合成的,但研究表明植物体内的很多次生代谢产物都是由体内的共生微生物产生的,DNJ也是在链霉菌中首次被发现的。为探讨桑树中的DNJ来源,本文通过对桑树内生菌(细菌、真菌、放线菌)的分离,利用RP-HPLC-UV法对分离株能否产生DNJ进行检测,发现桑树体内的确存在能够产生DNJ的内生菌。进一步对分离获得的产DNJ性质稳定的菌株进行了鉴定、对其中一菌株产DNJ的发酵条件和发酵培养基进行了优化,对发酵产物进行了初步的分离鉴定,最后通过动物试验,对产DNJ内生菌发酵液的安全性及其降血糖效果进行检测,主要取得以下结果。
1分离桑树、组培苗及桑籽中的内生菌。利用RP-HPLC-UV法检测分离株中DNJ的产生情况,共分离得到10株产DNJ的内生细菌分离株,经Rep-PCR鉴定,归类为6株能够产生DNJ的内生细菌,其中菌株Y1、Y2产DNJ的性质稳定。
2对菌株Y1、Y2进行了系统的分类学鉴定。主要包括:菌株形态观察、生理生化指标测定、自动细菌鉴定仪鉴定、脂肪酸分析及16S rDNA序列分析。结果表明, Y1菌株属于嗜麦芽寡养假单孢杆菌(Stenotrophomonas maltophilia),菌株的16S rDNA序列已在Genbank中注册,登录号为GQ268318;Y2菌株属于藤黄微球菌(Micrococcus luteus),菌株的16S rDNA序列已在Genbank中注册,登录号为GU591156。
3通过摇瓶培养方法,探讨了发酵条件和发酵培养基对Y1菌株产DNJ的影响。
利用单次单因子试验确定了摇瓶培养的最佳发酵条件为:接种量2%(v/v)、摇瓶装液量50mL/250mL、发酵温度30℃、初始pH6.0。在此基础上通过单次单因子试验确定了发酵培养基的最佳碳源、氮源、碳氮比、无机盐。运用PB试验分析了培养基组分:麦芽糖、蛋白胨、尿素、硫酸镁、氯化钠对Y1菌株产DNJ的影响。结果表明,蛋白胨和硫酸镁对Y1菌株产DNJ的影响达到极显著水平;尿素对Y1菌株产DNJ的影响达到显著水平;而麦芽糖和氯化钠对Y1菌株产DNJ的影响不显著。通过最陡爬坡路径逼近最大响应区域,最后用中心组合设计和响应面分析,确定了发酵培养基中主要影响因子的最佳浓度。
最优发酵培养基配方为:麦芽糖40.00g、蛋白胨14.29g、尿素7.39g、MgSO4 0.77g、NaCl 1.00g、蒸馏水1000mL。
运用最佳发酵条件和最佳发酵培养基进行摇瓶培养,利用RP-HPLC-UV法测得衍生化后DNJ的峰面积达到了102.9mAU*min,较基础发酵培养基提高了227.21%。
4对Y1菌株产生的DNJ进行了初步分离鉴定。获得了DNJ粗提物,并采用RP-HPLC-UV以及MS对所得DNJ粗提物进行了检测。RP-HPLC-UV结果显示,发酵液中的DNJ得到初步纯化;MS分析结果表明,Y1菌株发酵产生的DNJ与桑叶中DNJ性质相同(离子峰的出峰位置一致)。
5通过动物试验对Y1菌株发酵液的安全性及降血糖效果进行了研究。试验结果表明,Y1菌株的发酵液对小鼠安全,并且具有明显的降血糖作用,能够有效缓解糖尿病小鼠“三多一少”的症状。
1-deoxynojirimycin is an alpha-glycosidase inhibitor, which can inhibit the absorption of sugar, and then lower the blood glucose effectively. Mulberry has an excellent hypoglycemin effect as a traditional Chinese medicine, and its active composition has been identified as DNJ. Former studies has determined the DNJ content from these materials as follows: different mulberry varieties,different leaf positions, different silkworm species as well as silkworm body, silkworm excrement and pupa in different developing stages. At present, the silkworm and mulberry resources have been developed into hypoglycemic products in clinical practice.
Now DNJ and its analogs have been detected from many microorganisms and plants. Mulberry has the highest DNJ content among the plants and the DNJ of the silkworm is obtained from mulberry. Although there is no conclu- sion of the exact origin of DNJ in mulberry, people generally consider that DNJ is synthesized by the mulberry. Meanwhile some studies show that many secondary metabolism products are generated by the associated microorganis- ms in the plants and DNJ is firstly discovered in Streptomyces. In order to explore the DNJ origin, we have isolated the endophytes in mulberry, and then determined the DNJ content of the endophytes by RP-HPLC-UV. We have discovered the endophytes that could produce DNJ. Further researches were as follows: the identification of the endophytes producing DNJ stably, the optimization of fermenting conditions, the initial identification and character- ization of DNJ produced by the endophytes. At last we studied the security and the hypoglycemic effect of the fermentation broth.
1 The endophytes from mulberry, tissue cultured seedlings and mulberry seeds were isolated and determined the DNJ content with the method of RP-HPLC-UV. Nine endophytes producing DNJ were obtained, clustered into seven endophytes by the analysis of rep-PCR. Among of the seven endophytes, strain Y1 and Y2 showed stable quality in the DNJ production.
2 Taxonomic determinations of bacteria strain Y1 and Y2 were carried out, including morphological observation, physiochemical analyses, test of biolog- ical antomated bacterial identification system, analysis of the fatty acids and 16S rDNA sequence analyses. The results showed strain Y1 was fell to Stenotrophomonas maltophilia and its 16S rDNA sequence was registered in GenBank (GenBank accession NO.GQ268318); Strain Y2 fell to Micrococcus luteus and its 16S rDNA sequence was registered in GenBank (GenBank accession NO.GU591156).
3 Through the cultivation in shake-flask fermentation, the effects of environmental conditions and nutrition of strain Y1 producing DNJ were studied.
The optimal shaking flask fermentation conditions were confirmed by one-factor-at-a-time experiment. The optimized fermentation conditions for DNJ production were: inoculum volume 2%(v/v), solution amount 50mL/250mL, fermentation temperature 30℃, initial pH6.0.
Based on the results above, the effects of nutrition on production of DNJ were studied involving the optimal carbon, the optimal nitrogen, C/N, inorga- nic salt by one-factor-at-a-time experiment. In the optimization of medium, the influences of corn meal, maltose, peptone, urea, MgSO4, (NH4)2SO4 and NaCl on DNJ production were first evaluated using Plackett Burman design. Among of the test components, the effect of peptone and MgSO4 both reach extremely significant level and the urea shows significant effect on the DNJ production. However, maltose and NaCl have no significant effects on DNJ production. The path of steepest ascent was used to approach the optimal region of the medium composition. In the last step, the optimal concentrations of test components were determined by central composite design and response surface analysis.
The optimum fermentation medium for production of DNJ were: maltose 40.0g/L, peptone 14.285 g/L, urea 7.391 g/L, MgSO4 0.7706 g/L, NaCl1g/L,corn meal 20.0g/L, maltose 20.0g/L, soybean meal 29.5g/L, (NH4)2SO4 1.0g/L, CaCO3 3.8g/L.
Based on the optimal fermentation condition and medium, the spectrum peak area of DNJ after derivatization reached 102.9mAU*min, 227.21% higher than that of basal medium.
4 The initial extraction, purification of DNJ produced by strain Y1 was studied by Rep-HPLC-UV and MS analysis. The results showed that the DNJ produced by strain Y1 was initial purified. The analysis of MS showed that the DNJ produced by Y1 had similar properties to the DNJ from Mulberry leaves. However, there were differences between the DNJ extraction and DNJ standard sample. It was concluded that the DNJ from strain Y1 and Mulberry leaves was analogue of DNJ sample.
5 The security and hypoglycemic effect of fermention broth were studied by animal experiments. The results showed that the fermention broth was safty for test mice and had excellent hypoglycemin effect, so it could relieve diabetes symptoms effectively.
引文
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