【作者】 蒋运钢；

【导师】 桂仲争；

【作者基本信息】 江苏科技大学 ， 特种经济动物饲养， 2011， 硕士

【摘要】 1-脱氧野尻霉素（1-deoxynojirimycin,DNJ）是一种多羟基哌啶生物碱,因其结构类似单糖也称为亚胺基糖。DNJ具有多种生物活性,如降血糖、抗病毒、抗肿瘤等,在食品、医药等领域具有重要的应用价值而倍受关注。桑树中含有丰富的DNJ,其DNJ的含量已经作为判断蚕桑生物质降血糖等效果的重要指标之一。本研究以高效提取蚕桑生物质DNJ和通过发酵法提高其含量为出发点,探讨响应面法优化蚕粉DNJ稀酸浸提工艺、筛选出能大幅度提高蚕桑生物质DNJ的发酵菌种并对发酵条件进行优化、研究优势微生物菌种发酵蚕桑生物质DNJ产量的应用前景,以期为蚕桑资源的精深加工和DNJ产品的开发利用提供理论和技术指导。主要取得如下研究结果。采用响应面法优化出稀酸浸提家蚕DNJ的数学模型为:Y= 0.47+ 0.054X₁ - 0.03X₂- 0.00925X₃+ 0.03X₁X₂- 0.07X₁X₃- 0.059X₂X₃- 0.013X₁² - 0.027X₂² + 0.024X₃² ,该模型显著,能够解释各因素对家蚕DNJ提取效果的影响。通过响应面法优化出稀酸浸提家蚕DNJ的最佳工艺参数为:提取时间3.3h,浸提温度72.9℃,料液比分数为1:282。在此模型影响的稀酸浸提DNJ的理论提取率为0.493%,实际提取率为0.487%,理论值和实际值基本一致。模型的响应面和等高线分析发现,浸提时间、浸提温度和料液比分数之间存在明显的交互作用。本文通过比较8种起始发酵菌株,筛选出灵芝菌种为发酵桑叶DNJ产量的最合适菌种。经PB设计,在比较8个不同因素对灵芝菌种发酵桑叶产DNJ的实际影响分析得出,微环境pH值,无机氮源KNO3含量和菌体接种量作用明显。进一步的单因素分析,并结合采用响应面法优化出的灵芝菌种发酵条件数学模型为:Y=0.054-9.37×10-4X1+1.25×10-5X2+5×10-5X3-9.75×10-4X1X2+1.5×10-4X1X3+6.5×10-4X2X3-7.32×10-3X 12 -9.27×10-3X 22 -7.25×10-3X 32 ,该模型显著,能够解释各因素对桑叶DNJ提取效果的影响。通过响应面法优化出灵芝菌种发酵桑叶DNJ的最佳发酵为:pH值6.97,KNO3含量0.81%,菌体添加量2.00 mL。在此模型影响的灵芝菌发酵液DNJ提取率为0.548%,与发酵前比较,经灵芝菌种发酵后DNJ含量大幅度提高。模型的响应面和等高线分析显示,pH值、KNO3含量和菌体添加量之间交互作用明显,进一步验证模型可靠。在比较桑树不同部位DNJ含量发现:桑叶DNJ的分布规律为:上位叶>侧根>中位叶≥桑枝皮>下位叶,由此可见桑嫩叶可能是DNJ的主要来源生产部位;比较蚕粉不同干燥工艺得出:超低温冷冻干燥>喷雾干燥>热风干燥,显示出超低温冷冻干燥有利于保持生物活性物质DNJ分子结构特性,而喷雾干燥过程中有瞬时加热的过程,通风干燥则加热时间较长,从而影响其检测量。经灵芝菌发酵后,桑叶（上位叶）DNJ含量提高20.6%;冻干燥蚕粉DNJ含量为51.2%,提高了6.9%;调查三龄第1天至五龄第7天每天蚕粪发酵前后DNJ含量的变化趋势,结果显示,发酵前后变化趋势一致;三龄第1天蚕粪DNJ含量最高,然后逐渐下降,从五龄第2天至五龄第7天趋于平缓,其中五龄第4天含量最低。发酵后蚕粪DNJ含量平均提高20%,最高为五龄第4天,提高40.8%。关于家蚕对DNJ的吸收和排泄机制有待于进一步深入研究。更多还原

【Abstract】 1-Deoxynorijimycin （DNJ） as a kind of polyhydroxy piperidines was also called iminosugar for its configuration similar with sugar. DNJ appeared to be useful in food and medicine for its potential biological activities, such as inhibition of abnormal elevation of plasma glucose, anti-virus and anti-tumor. Therefore, DNJ had long been of interest for its suppressing the plasma glucose content with general consents. Mulberry resource enriched in DNJ, which can prohibit the bioactivities of intestinal alpha-glucosidase in vivo, and whose content in sericulture biomass was considered as one of significant indexes for effect on inhibition of postprandial glycemia. From the perspectives of extraction of DNJ from the silkworm with proficiency and elevation of DNJ content in sericulture resource by fermentation, this study focused on diluted acid extraction of DNJ from silkworm with surface response methodology （RSM）, investigated a advantageous strain with fermentation conditions optimized by PB design and RSM, which could enhanced the DNJ content to a large extent from mulberry leaves, and groped for a prospect for production of DNJ from other sericulture resource fermented by the specific microorganism. Based on the results of this study, it is foreseen that production of DNJ and sericulture development of deep level would have the performance improved. Main results are as the following:The formula of diluted acid extraction of DNJ from silkworm with RSM could expressed as Y= 0.47+ 0.054X₁ - 0.03X₂- 0.00925X₃+ 0.03X₁X₂- 0.07X₁X₃- 0.059X₂X₃- 0.013X₁² - 0.027X₂² + 0.024X₃² , Which was significant and could elucidate impact of extraction factors on DNJ content from silkworm. The optimal extraction conditions were defined as: 72.9℃, 3 hours for 18 minutes and 1:282 of solid to liquid. The DNJ extracted after optimization was 0.493%, while predicted value was 0.487%, reflecting the both was consent with each other. There were obvious mutual interactions among the extraction time, temperature and the ratio of material to liquid throughout Contour line plot and response surface graph of DNJ yield.In compare with eight strains as starter cultures, Ganoderma lucidum was defined as the candidate for the fermentation by conversion of mulberry leaves into DNJ. And then analyzing eight factors effects on yield of DNJ fermented by Ganoderma lucidum by Plackett-Burman design, the range of pH, content of potassium nitrate and inoculum size was utilized for the following study, that is to say, for the single factor assay. The regression model for the optimizing the fermentation conditions was expressed as Y=0.054-9.37×10-4X1+1.25×10-5X2+5×10-5X3- 9.75×10-4X1X2+ 1.5×10-4X1X3+6.5×10-4X2X3-7.32×10-3X 12 -9.27×10-3X 22 -7.25×10-3 X 32 , Which was significantly appropriate and could elucidate impact of fermentation factors on DNJ content from mulberry. The highest amount of DNJ yield （0.548%） from the incubation broth by Ganoderma lucidum, in which fermentation broth showed better performance than origin before fermentation, would be calculated by first order local deviation with equivalence to zero, when X1, X2 and X3 received 6.97, 0.81% and 2.00 ml, respectively. There were obvious mutual interactions among the ph range, content of potassium nitrate and the inoculum size throughout Contour line plot and response surface graph of DNJ yield, suggesting the model was reliable and feasible.The distribution of DNJ in mulberry was shown that most important independent parts were upper leaves, roots, middle leaves （equaled to cortex）, bottom leaves in order by content, suggesting DNJ mostly was produced by the young leaves. In compare with three methods of drying silkworm powders, indicating that lyophilization was the best approach to deal with it, suggesting its structure would be greatly maintained by lyophilization and DNJ molecule would be degraded or denatured under hot-air involved circumstance the elevation of DNJ content fermented by microorganism from mulberry was more than from silkworm. In compare with the unfermented, the DNJ contents increased by 20.8% in mulberry and 6.9% in silkworm powder, respectively. The content of DNJ in feces before or after fermentation decreased swiftly during the 6 days, followed mildly for the rest of days till mounting, appearing to be lowest in feces on 4th day of fifth-instar larvae. And the DNJ yield in feces fermented by given microorganism was higher than one in feces primitive, 20 percent promotion in average. However, DNJ content in feces on 4th day of fifth-instar larvae advanced most, up to 40.8%. The circulation and excretion mechanism of DNJ accumulated by the larvae of Bombyx mori will be investigated for the further study.更多还原