【作者】 刘卉琳；

【导师】 谢达平；

【作者基本信息】 湖南农业大学 ， 微生物学， 2012， 博士

【摘要】 对从福建黄酒酒糟中分离出来的产色素菌株进行了鉴定、诱变、培养基和发酵条件的优化研究,并对该菌株所产类胡萝卜素进行了分离纯化与体外转化为维生素A的研究。主要研究结果如下：(1)于酒糟中分离得到一株产色素菌株。对该菌株所产色素进行定性分析,结果表明该色素为类胡萝卜素；对菌株进行常规形态和生理生化特性分析,结果表明该菌株为单细胞,呈椭圆形,芽殖；无子囊孢子；无假菌丝形成。在固体培养基上,菌落呈粉红色,菌落表面湿润、粘稠,边缘整齐,易被挑起；在液体培养基中,产生沉淀。葡萄糖发酵试验为阴性,硝酸钾试验为阳性,耐50%葡萄糖高渗试验为阴性,产类淀粉化合物试验为阴性,在37℃下可正常生长。利用26SrDNA D1/D2区域序列分析法对该菌株进行序列比对鉴定,结果表明,该酵母菌的序列与粘性红圆酵母(Rhodotorula mucilaginosa)模式菌株的序列同源性为100%。因此,结合该菌株形态特征和生理生化特性,确定该菌株为粘性红圆酵母(Rhodotorula mucilaginosa),命名为RM-1。(2)为获得类胡萝卜素高产菌株,利用N+离子注入法对粘性红圆酵母RM-1进行诱变处理,结果表明,当注入能量为10keV,注入剂量为2.0×1014ion/cm2时,经二苯胺的初筛和复筛所得突变株RM-127类胡萝卜素产量最高,与出发菌株RM-1相比,增加了66.79%。再经紫外诱变,得突变株RM-213,发酵试验结果表明,该菌株的类胡萝卜素产量达到10.59mg/L,较出发菌株RM-1提高了89.67%,传代试验表明,突变株RM-213遗传性状稳定,可作为工业生产菌种。(3)以农业废弃物和副产物为发酵原料进行粘性红圆酵母RM-213发酵产类胡萝卜素的研究,经单因素试验、均匀设计试验和正交试验,获得了最佳的发酵工艺与参数。由单因素试验,得到最佳碳源为玉米秸秆水解液(50g/L),最佳氮源为玉米浆(40g/L)。通过均匀设计试验,得到影响发酵液中类胡萝卜素产量的几种添加物的回归方程为：Y=3.94+29.6X1+20.9X2-11.1X3-1.99X6,据此优化得到添加物及其最佳用量为：MgSO4·7H2O0.7g/L,K2HPO40.2g/L,乙醇0.7%,VB21.0g/L。通过正交试验,得出变温培养的最佳条件为0-40h培养温度30℃,40-72h培养温度24℃。通过正交试验对装液量、接种量、初始pH、摇床转速四个因素进行优化,优化结果为：装液量50mL/250mL、初始pH5.5、接种量10.0%、摇床转速170r/min。经过优化后的生物量(17.56g/L)、类胡萝卜素含量(1378.70gg/g)及产量(24.21mg/L)较未优化时(11.85g/L、891.34μg/g、10.56mg/L)分别提高48.19%、54.68%和129.26%。(4)利用大孔树脂对粘性红圆酵母RM-213所产类胡萝卜素进行了分离纯化,得到了最佳的吸附和解吸条件。静态吸附试验结果表明,最佳吸附树脂为X-5树脂,当温度为25℃,吸附时间为1h时该树脂对类胡萝卜素吸附率最大,可达71.01%；最佳洗脱剂为乙醚,当温度为30℃解吸1h时,解吸率高达95.32%。动态吸附试验结果表明粘性红圆酵母RM-213产类胡萝卜素的最佳分离纯化工艺参数为：上样质量浓度以β-胡萝卜素计为111.82μg/mL,以1mL/min的流速通过径高比为1：9的层析柱,再用乙醚以0.5mL/min的流速洗脱。经纯化,类胡萝卜素纯度达到33.29%。(5)对粘性红圆酵母液态发酵提取液成分β-胡萝卜素进行体外酶法转化试验表明该β-胡萝卜素经专一的β-胡萝卜素-15,15’-单加氧酶可转化为维生素A。通过对转化条件的优化研究,得出最佳转化体系为123mg/Lβ-胡萝卜素,3.5mmol/L脱氧胆酸钠,0.25%Tween40(w/v),pH8.0和0.5mmol/L d-a-生育酚。在此条件下,粘性红圆酵母液态发酵所产β-胡萝卜素在酶活力为2.69nmol mg-1h-1的β-胡萝卜素-15,15’-单加氧酶作用下于37℃水浴振荡反应7h,可再经硼氢化钠还原,转化成40.1mg/L维生素A,转化率达到61.11%(mol/mol)。更多还原

【Abstract】 A strain which produces pigment was isolated from yellow wine lees in Fujian,then was identified. To obtain a high-yield strain of carotenoid, the mutation breeding and optimization of media ingredients and culture conditions on the strain were studied, and then the isolation and purification of carotenoid and the cleavage from carotenoid into retinol with β-carotene15,15’-monooxygenase were investigated. The main results were as follows:(1) A strain which produces pigment was isolated from yellow wine lees. The pigment was identified as carotenoid by qualitative analysis. The morphology, physiological and biochemical characteristics of the strain were analyzed, the results showed that the cell of the strain was single, oval and budding, and the strain had no ascospore and pseudohypha. Its colony was of regular edge, humid and sticky pink surface in solid medium and deposition in liquid medium. The strain showed negative response to glucose fermentation and positive response to potassium nitrate. It could neither endure hypertonic solution nor produce amyloid, it could grow at37℃. All of these results showed that the strain was a kind of Rhodotorula. The homology of the26S rDNA D1/D2domain sequence from the Rhodotorula showed that the genetic relationship between the strain and Rhodotorula mucilaginosa was closest and they were100%similarity at nucleotide acid sequence. According to its morphological, physiological and biochemical characteristics and molecular identification, the strain was identified as Rhodotorula mucilaginosa, and named RM-1after the strain.(2) In order to obtain a high-yield strain of carotenoid, R.mucilaginosa RM-1was mutated by10keV N+implantation with dose of2.0×1014ion/cm2implantation and then screened by diphenylamine resistance. Mutant RM-127was obtained, its production of carotenoid reached9.31mg/L, increased by66.79%over the5.58mg/L from the original strain. And then mutant RM-213was obtained by UV mutagenesis of RM-127and produced a large quantity of carotenoids (10.59mg/L), which was higher (with89.67%) than in the original strain RM-1. Its hereditary property was stable after ten times of subculture, which suggested a bright prospect of application.(3) Optimization of carotenoid production by RM-213using agricultural waste and by-product as raw matearial were studied. The single-factor method was employed for the optimization of growth and carotenoid production using corn straw hydrolysate (50g/L) as carbon source and corn steep liquor (40g/L) as nitrogen source. The experimental parameters of seven kinds of additives were added by uniform design experiment, the results showed that the optimum additives and quantity were as follows:MgSO4·7H2O0.7g/L, K2HPO40.2g/L, ethanol0.7%, VB21.0g/L, respectively. The corresponding regression equation was:Y=3.94+29.6X1+20.9X2-11.1X3-1.99X6. The optimum culture conditions were obtained as follows by orthogonal test:250mL Erlenmeyer flasks containing50mL fermentation liquid, initial pH5.5,10%inoculum size with agitation rate of170r/min. The temperature was varied during incubating, the first40h incubated at30℃, then kept32h at24℃. Under these conditions, the cell biomass, carotenoid content and carotenoid yield were respectively. Increased by48.19%,54.68%and129.26%over that before optimization.,(4) The carotenoid which were produced by RM-213was purified by macroporous adsorption resins and the optimum conditions of adsorption and desorption were investigated.. The static experiment results showed that X-5resin was the best adsorbent, the adsorption rate was up to71.01%when absorbed1h at25℃, and aether was the best eluent, the desorption rate was up to95.32%when desorbed1h at30℃. The dynamic experiment results showed that the optimum dynamic adsorption and desorption conditions were as follows:the concentration of sample111.82u.g/mL, ratio of column diameter to length1:9, rate of adsorption flow1mL/min, and rate of desorption flow0.5mL/min. Under optimum conditions, the purity of carotenoid was up to33.29%.(5) The conversion of β-carotene produced by R.mucilaginosa in vitro was conducted, the results showed that β-carotene15,15’-monooxygenase from chicken intestinal mucosa can cleaves β-carotene into retinal. The optimum reaction conditions for conversion were123mg/L β-carotene,3.5mmol/L sodium deoxycholate,0.25%(w/v) Tween40, pH8.0and0.5mmol/L d-a-tocopherol, respectively. The enzyme reaction was conducted at37℃for7h under optimum conditions with2.69nmol mg-1h-1enzyme activity,40.1mg/L retinol was obtained by reducing retinal with the NaBH4procedure, the conversion efficiency come up to61.11％(mol/mol).更多还原