【作者】 汪文俊；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2006， 博士

【摘要】 虾青素（3,3’-二羟基-β,β’-胡萝卜素-4,4’-二酮）是一种重要的类胡萝卜素,具有优良的色素沉积作用并能促进动物发育,更具有超强的抗氧化活性及较强抗肿瘤活性。由于化学合成的虾青素在应用和安全性上的局限,人们对天然来源虾青素需求量越来越大,而利用微生物（如红法夫酵母）发酵生产虾青素是当前研究的热点。红法夫酵母是唯一能利用多种碳氮源并积累虾青素为主要类胡萝卜素的酵母,本文首先采用各种物化因子对红法夫酵母原始菌株进行诱变,获得了虾青素高产突变株,研究了该突变株生产虾青素的发酵条件,建立了虾青素检测的快速方法和类胡萝卜素大规模提取方法,并进行了不同发酵规模培养条件的研究,进行了基于红法夫酵母虾青素生物合成途径的代谢调控研究。本文采用各种物化因子如紫外线（UV）、甲基磺酸乙酯（EMS）和60Coγ射线多次诱变红法夫酵母原始菌株,获得了一株稳定高产虾青素的突变株,随后在摇瓶条件下对此菌株的发酵条件进行了研究。种子、通气量、温度和pH值影响红法夫酵母生长、类胡萝卜素和虾青素的合成,种龄为48 h的种子在接种量为9%、摇床转速为250 r min–1、250 ml三角瓶装液量为30 ml、培养温度为16¹8℃、培养基初始pH值为5.0时有利于发酵。研究了培养基成分如碳源、氮源和无机盐对生长、类胡萝卜素和虾青素合成的影响,并对培养基成分进行了优化,最佳培养基为（g l-1）:葡萄糖30、蔗糖30、酵母浸粉6、（NH₄）₂SO₄ 3、KH₂PO₄2、Na₂HPO₄ 1、MgSO₄ 5。研究了虾青素快速和大规模提取条件,采用二甲基亚砜对湿菌体直接提取类胡萝卜素后492nm处比色测定是一种快速的虾青素近似定量方法。在优化破壁条件即50℃用4M的盐酸和6 ml的溶菌酶处理8 h,然后中和到pH值为3.0,再经喷雾干燥,用丙酮提取类胡萝卜素,提取率最高达到99. 23%。随后对红法夫酵母生物反应器间歇发酵培养工艺进行了研究,进行了5 L、100 L、2000 L、15000 L等不同规模发酵放大实验,发酵工艺研究主要在5 L全自动罐中进行。结果表明搅拌转速、通气量对溶氧、生长和产物合成有着显著影响,当搅拌速度为300 r min-1、通气量为1.5vvm时发酵结果最佳。研究了补水工艺和氧载体对发酵和溶解氧水平的影响,结果表明在大量少次补水、二甲基硅油为氧载体添加量为2%时最佳。进行的红法夫酵母15000 L规模发酵生产尚未见报道。结果表明发酵罐规模越大,发酵周期越短,推断氧的供给是红法夫酵母发酵产虾青素的一个重要影响因子。基于红法夫酵母虾青素生物合成途径下,研究了不同代谢中间产物如番茄红素、胡萝卜素、柠檬酸钠、谷氨酸钠、乙醇等物质对于红法夫酵母生长、类胡萝卜素和虾青素合成的影响,它们在一定浓度下对红法夫酵母的生长、类胡萝卜素和虾青素合成都有促进作用。研究了真菌、化学诱导子对红法夫酵母生长、类胡萝卜素和虾青素合成的影响,将由Rhodotorula rubra、Rhodotorula glutinis、Panus conchatus、Coriolus versicolor、Mucor mucedo和Mortieralla alpina制备的真菌诱导子和用氯化镉、二氧化钛制备的化学诱导子以不同的浓度添加到发酵培养基中,结果表明一定浓度下真菌诱导子对红法夫酵母的生长、类胡萝卜素和虾青素合成有促进作用,在较高添加浓度下生物量、类胡萝卜素产量和虾青素产量比对照高,而在较低浓度下类胡萝卜素和虾青素含量比对照高。化学诱导子对生长有一定的抑制作用,而类胡萝卜素和虾青素产量相对对照有较大提高。在添加P. conchatus、C. Versicolor、氯化镉和二氧化钛诱导子后,诱导产生的红法夫酵母胞内活性氧含量随时间而升高,在诱导后24⁴0 h时胞内活性氧含量最高,如二氧化钛和氯化镉在诱导后第40 h胞内活性氧均达到最高值,分别为299和286 nmol g-1 FCW（fresh cell weight,湿菌体重量）,证明胞内活性氧对于虾青素合成的确有促进作用,推断活性氧激活了虾青素生物合成途径中相关酶或基因表达。更多还原

【Abstract】 Astaxanthin（3,3’-dihydroxy-β,β’-carotene-4,4’-dione） is an interesting carotenoid which is the main carotenoid pigment found in aquatic animals such as lobsters, crab, shrimp, trout and salmon and plays a role in delaying or preventing degenerative diseases. Moreover, due to its special structure, astaxanthin is a more powerful scavenger of singlet oxygen （1O2） and peroxyl radicals （H2O2） thanβ-carotene, cantaxanthin, and zeaxanthin （3,3’-dihydroxyl-β-carotene）. Its antioxidant activity is much stronger than all other carotenoids. Furthermore, astaxanthin may exert antitumor activities through the enhancement of immune responses. However, consumer and governmental concerns regarding chemical additives in foods have stimulated research in biological systems to produce astaxanthin by biotechnology. These systems are mainly focused on algae, bacteria or yeast such as Xanthophyllomyces dendrorhous. Using different physical and chemical methods, a mutant was isolated and its biological characteristics and production processes of carotenoids were studied. Fast method to determine the astaxanthin and scale-up extract of carotenoids were established, then the parameters of different scale-up fermentation were studied. Based on the astaxanthin biosynthesis pathway by X. dendrorhous, effects of different chemicals and fungal elicitors added into the broth on the growth, total carotenoids and astaxanthin were investigated.To isolate the astaxanthin-hyperproducing yeast, the methods such as different low-dosages of 60Co gamma irradiation, UV and Ethyl Methanesulfonate（EMS） were used to treat X. dendrorhous during exponential phase. Effects of inoculum age, inoculum size, agitation speeds, temperature and initial pH value on the growth, total carotenoids and astaxanthin by X. dendrorhous with optimal conditions of 48 h, 9%, 250 r min–1, 16¹8℃and pH 5.0, respectively. After main nutrition compositions including carbon source, nitrogen source and minerals on carotenoids by X. dendrorhous were determined and optimized, the optimal concentrations of media ingredients such as gluose, sucrose, yeast extract, （NH₄）₂SO₄、KH₂PO₄, Na₂HPO₄ and MgSO₄ were 30,30,6,3,2,1 and 5 g l-1, respectively. Using DMSO as direct extract agent of carotenoids and determination on a spectrophotometer at 492 nm resulted in a fast and approximate method to measure astaxanthin yields by X. dendrorhous. Under the optimal conditions of cell wall disruption which the yeast was treated with 50℃,2M,9ml, 4h and neutralized to pH 3.0, the maximal carotenoid extractability （99. 23%） was obtained.The parameters of different scale-up fermentations were studied in 5, 100, 2000 and 5000 L fermentors, respectively. Influences of operation parameters including agitation speed and aeration were mainly studied in a 5 L fermentor. The results showed that the agitation speed and aeration could significantly affect the growth, total carotenoids, astaxanthin formation and dissolved oxygen by X. dendrorhous. The optimal results were obtained when agitation speed and aeration were 300 r min-1 and 1.5 vvm, respectively. When adding mass water by less times and using dimethyl silicone oil as oxygen vector at concentration of 2% （v/v）, the optimal results were obtained by enhancing growth, total carotenoids, astaxanthin formation and dissolved oxygen by X. dendrorhous. 15000 L scale-up experiments which hadn’t been reported by any other researcher in this filed was performed in a pharmacy plant. The results of scale-up experiments showed the bigger the fermentor was the shorter the culvitation time was and the dissolved oxygen was a key factor to astaxanthin biosynthesis by X. dendrorhous.Based on astaxanthin biosynthesis by X. dendrorhous, some intermediates such as tomato juice, carrot juice, tri-sodium citrate, ethanol and glutamine added into the culture media could enhance the growth, total carotenoids and astaxanthin formation at different concentrations. Six fungal elicitors prepared from Rhodotorula rubra, Rhodotorula glutinis, Panus conchatus, Coriolus versicolor, Mucor mucedo, Mortieralla alpina stimulate the growth, total carotenoids and astaxanthin formation by X. dendrorhous. Chemical elicitors prepared from TiO₂ and CdCl2 were advantage for total carotenoids and astaxanthin formation but slightly restrained the growth by X. dendrorhous. After being treated with P. conchatus, C. versicolor, TiO₂ and CdCl₂, each elicitor showed a clear positive influence on the reactive oxygen species （ROSs） in the yeast, and there was a steady increase of H2O2 accumulation along with the concentrations, reaching the maximum at certain time at 24 to 40 h with highest H₂O₂ concentration of 299 and 286 nmol g-1 FCW （fresh cell weight） concerning TiO₂ and CdCl₂ elicitor, respectively. The maximal astaxanthin yield was obtained at 96 h after treated by elicitors, which indicated ROSs were due to the sitimulating effects on astaxanthin formation by activating some enzymes and genes involved in astaxanthin biosynthesis pathway by X. dendrorhous.更多还原