【作者】 王见冬；

【导师】 袁其朋；

【作者基本信息】 北京化工大学 ， 生物化工， 2004， 硕士

【摘要】 番茄红素是一种重要的类胡萝卜素，其消除单线态氧及清除自由基的能力均优于其它类胡萝卜素，同时还具有诱导细胞间信息传递、降低胆固醇等生理功能，因而番茄红素在防癌、抗癌以及防治动脉硬化等方面具有强大的潜力。基于番茄红素卓越的生理功能，其生产方法显得至为重要。迄今为止，番茄红素的生产方法有三种：化学合成法、有机溶剂提取以及发酵法生产，其中，发酵法因其低廉的成本成为研究的重点。目前，利用β-carotene的高产菌株发酵生产番茄红素是实现其工业化生产最有效的方法，本文主要研究了以下几个方面：新型测定方法的建立、最佳阻断剂的选择、分离纯化工艺的完善以及三孢酸生产工艺的初探。番茄红素标品价格昂贵且稳定性很差，随着时间的推移，其浓度下降，造成测量结果不准确，故建立一条精确、有效的HPLC测定方法极为重要。实验发现：用苏丹Ⅰ代替番茄红素标准品测定是完全可行的，精确度高，误差≤5％，换算关系为Y番茄红素峰面积=1.03Y苏丹Ⅰ峰面积。利用?－胡萝卜素高产菌株三孢布拉氏霉菌（Blakeslea trispora）发酵生产番茄红素过程中，可通过加入阻断剂的方法，将代谢控制在番茄红素水平。实验结果表明：化学合成的阻断剂中，1.0 g/L的2，6-二甲基吡啶阻断效果最好，大量积累番茄红素，几乎无?-胡萝卜素生成；加入富含生物碱alkaloid的烟草废弃物为阻断剂且浓度为15g/L<WP=4>时，总胡萝卜素含量增加，有少量?-胡萝卜素生成，番茄红素含量达1090.03 mg/L，较前者提高58％。随后，本文进行了番茄红素下游纯化工艺的研究，建立了从菌体中经萃取、皂化及结晶后得到产品的工艺，具体结果如下：萃取剂石油醚与干菌体的比例为20：1， 70℃下萃取20min，萃取次数为两次且其中所含抗氧化剂BHT达0.5％时，番茄红素回收率为93.5%；皂化过程中，皂化液与萃取液比例为1：1；60℃下皂化 30min时皂化效果最好，番茄红素约为10％；当温度为1－5℃，结晶时间12h，与无水乙醇比例为1：5时，所得晶体为松散、深红色针状晶体。在以上工艺中，萃取到结晶步骤总损失较大，为48.7%，其中结晶步骤搞骤高达39％，几乎20％的产品残留在母液中，故如何寻找一条更佳的结晶路线仍是以后研究的重点。另外也进行了预处理湿菌体后再提取番茄红素工艺的研究，以除去湿菌体中部分醇溶性的脂肪酸及杂质，省略皂化步骤并得到松散的晶体，预处理优化条件为：无水乙醇与湿菌体比例为85：15，常温下处理30min。与未进行预处理相比，菌体中番茄红素含量提高20％，干燥时间缩短为12h。最后我们对发酵过程中的副产物之一——三孢酸生产工艺进行了初步探讨，建立了一条完整的三孢酸生产工艺，并对其稳定性进行了研究，以期为番茄红素高产提供理论依据。更多还原

【Abstract】 Lycopene is an important carotenoid，and the ability to eliminate singlet oxygen quencher and free radical is much better than other carotenoids ；Because lycopene can induce information transfer of cells and reduce simultaneity cholesterin, so beneficial effect in the treatment and inhibitator of diseases such as cancer and atherosclerosis are behaved well and lycopene has received a great attention in recent years .The preparation of lycopene is very significant in the studies of lycopene. So far, there are 3 kinds methods of getting lycopene which are chemosynthesis ,extratration from plant by organic solvent and fermentation by microorganism. Compared with the other two method,fermentation has been widely studied for its lower cost.Now, Fermentation is one of potential methods for producing lycopene by Blakeslea trispora which is the highest yield in β-carotene industrially. In the paper, a new method in determination ,the choice of blocking agents, a systemic separation and purification and trisporic acid have been studied reasonedly.It is essential to establish an accurate and effective method on determining lycopene and β-carotene by HPLC to reduce the metrical <WP=6>error，because they are very instable when exposed to light and heat and very valuable. As a result，it is feasible to determine lycopene and β-carotene in our sample when sudan reagent is standard substance instead of lycopene andβ-carotene and the metrical error is lower than 5%. When they behave the following formula：Ylycopene peakaera =1.03Ysudan peakaera.In order to get lycopene from the fermentation ofβ-carotene, some bloking agents have been chosen. As a result, it is completely possible to achieve only lycopene without ?－carotene almostly in the presence of 2-amino-6-methylpyridine whose concentration is up to 1.0 g/L. When the concentration of tobacco dust employed in medium reaches 15g/L, The yield of lycopene is 58％ higher than the former ，which climbs to 1090.03mg/L , and there is little ?－carotene obtained simultaneity.In order to achieve an optimized route about separation and purification of lycopene by fermentation, the process about extraction , saponification and crystal have been established subsequently. Firstly，the optimal exatration condition has been rearched：the production reclaim is 93.5% when the proportion of extraction solvent Petroleum Ether and dry thalli is 20：1，the temperature is 70℃, the time is 20 min and extractive time is 3；secondly, we studied the different temperature ,time and the proportion of saponificated solvent and extraction.. It is indicated that we can achieved a best result that the proportion was 1:1, the temperature was 60℃ and the time was 30min. At the step，the percent of production losing <WP=7>is about 10；finally, incompact crystal instead of oiled crystal can be attained when the proportion of ethanol and concentrated saponificated solution is 5:1 under 4 ℃ for 12 hours，but about 39％ production has been lost at the process，so the condition of crystal must be optimized in the latter experiment . In addition to，pretreatment of wet thalli has been studied to remove fatty acid and else impurity in the process of lycopene extraction ,which can leave out saponification and get incompact crystal. the proportion of, New aspect of the method involves interaction at normal temperature，for 30 minutes and in the weight ratio ethanol and wet thalli is 85:15. The content of lycopene after pretreatment is improved 20%, and the dry period is storted to 12h . In the process of production, we found trisporic acid which is one of the outgrowths had played an important role in enhancing the yield of lycopene, so the preparation , extraction, stability trisporic acid have been studied in detail.更多还原