高温湿热酸法破壁提取法夫酵母胞内虾青素

法夫酵母是一种能积累虾青素的红酵母, 对其进行破壁是当前虾青素工业化提取生产的瓶颈工艺。研究在高温湿热条件下,低浓度盐酸对法夫酵母破壁而提取其胞内虾青素的工艺。探讨了不同破壁温度、盐酸浓度、液料比与破壁处理时间等因素对法夫酵母破壁后虾青素及类胡萝卜素提取率的影响, 确定了高温湿热酸法破壁提取虾青素的最佳条件为: 饱和蒸汽压力 0.1 MPa (121°C), 盐酸浓度0.5 mol/L, 液料比 (V/W)30 mL/g, 破壁时间2 min。在最佳条件下进行中试放大实验, 可得到虾青素与类胡萝卜素的提取率分别为: (84.8±3.2)%, (93.3±2)%。经优化后的新破壁工艺安全高效, 不会有毒性残留, 具有良好的工业应用前景。     

胶红酵母虾青素纯品提取及分离纯化方法优化

以胶红酵母ZTHY2作为提取虾青素的出发菌株,通过对其破壁方法、浸提溶剂、皂化方法及提取条件的优化,筛选出胶红酵母虾青素纯品的提取方法。用酸热法、冻融超声波裂解法与二甲基亚砜无水乙醇法等法处理干菌体细胞,用KOH结合甲醇A、甲醇B与乙醇法分别对虾青素粗提液进行皂化处理,分别使用六种展开剂,通过薄层层析鉴定游离的虾青素,用200~300目硅胶对游离的虾青素进行柱层析,获得其纯品,最后用高效液相色谱仪测定虾青素纯品的含量。实验结果表明:胶红酵母ZTHY2干菌体细胞的最佳破壁方法是酸热法,浸提溶剂为乙酸乙酯+乙醇(2∶1)溶液,皂化液为20 g/L KOH的乙醇溶液,皂化条件是40℃、30 min;最佳展开剂和淋洗剂是正己烷+丙酮(5∶2)溶液;高效液相色谱仪测定的游离虾青素比率为35%,提取率为96. 8%。     

超临界CO2萃取雨生红球藻中虾青素工艺的研究

本工艺以雨生红球藻粉为原料,采用超临界CO2萃取技术,萃取雨生红球藻浸膏,可有效地将雨生红球藻颗粒中的虾青素萃取出来,使萃余物(残渣)中总虾青素含量的平均值为0.224%;提取物得率(以油浸膏的总量计)可达28.5%;虾青素的提取率可达66.69%;雨生红球藻油浸膏中虾青素含量为5.710%.     

非水介质大孔树脂分离纯化虾壳中虾青素

通过7种大孔树脂对虾青素的静态吸附容量和解析率等指标的考察,筛选出AB-8大孔吸附树脂,用于分离虾壳中虾青素,同时利用高效液相色谱（HPLC）法测量虾青素的含量。结果表明,AB-8树脂对虾青素的吸附量为（24．17±0．5）mg／g,解吸率为95．2％,最大上样量（每g干树脂）以虾青素计为（23．07±0．2）mg,并确定用8倍量柱床体积的乙酸乙酯为洗脱剂,纯化所得虾青素的纯度为14．73％。     

温度对雨生红球藻(Haematococcus pluvialis)生物量和虾青素产量的影响

在用环形培养池模拟系统培养雨生红球藻的过程中,研究了温度对雨生红球藻生物量及虾青素产量的影响。结果表明,在15～25℃的范围内,不同温度下雨生红球藻生物量和虾青素含量及产量都经历了一个上升—最高—下降的过程。25℃与22℃时红球藻的虾青素产量、虾青素含量(干重)均显著高于其他温度的(P<0.01),但两者间差异不显著(P>0.05)。15℃时,红球藻生物量、虾青素含量和虾青素产量均最低,分别为1.4g、0.54%和2.49mg/L;25℃时,红球藻生物量和虾青素产量最高,分别为2.68g和13.53mg/L;22℃时,虾青素含量最高,为1.52%。     

虾青素对超低温冷冻大黄鱼精子具有细胞膜保护作用

为研究虾青素对冷冻损伤的大黄鱼精子细胞的保护作用,探讨其内在机制,我们将大黄鱼精子稀释于含10%二甲基亚砜和各浓度虾青素的冷冻稀释液中,冷冻并解冻后,显微镜观察发现,1.43×10-4mol/L的虾青素使大黄鱼精子寿命、运动时间和激活率分别增加13.8%、36.0%和5.5%,接近于鲜精;单细胞凝胶电泳显示,核DNA受损程度明显减少,彗星拖尾变短;流式细胞术结果显示,质膜和线粒体明显受到保护,线粒体质量提高了19.27%(P<0.05)。通过双层类脂膜法测定不同浓度虾青素对H+的传递能力,发现2%的虾青素能较高效率地传递H+。结果表明适当浓度的虾青素能通过传递H+维持线粒体功能,从而保护冷冻损伤的大黄鱼精细胞。     

佐夫色绿藻高产虾青素突变体的筛选及虾青素合成机理研究

为了提高佐夫色绿藻(Chromochloris zofingiensis)细胞内虾青素含量,研究通过甲基磺酸乙酯诱变构建了含有20000个单克隆的突变体库,并筛选出一株高产虾青素的突变体12C10。在异养正常培养条件下,当葡萄糖耗完时, 12C10虾青素含量比野生型提高74%;缺氮诱导第4天时,虾青素含量比野生型高25%。利用广泛靶向代谢组学分析在正常培养条件下12C10与野生型在代谢物水平上的差异。与野生型相比, 12C10中除谷氨酸外的氨基酸及脂肪酸的合成普遍下降,但是谷氨酸的水平显著提高。氨基酸和脂肪酸合成减少为虾青素合成提供更多的碳骨架、NADPH和ATP。谷氨酸的积累可能一方面刺激了磷酸戊糖途径中6-磷酸葡萄糖脱氢酶的活性促进NADPH的产生,另一方面导致氧自由基产生促进虾青素合成。代谢物组分析结果还表明12C10中虾青素合成的增强可能与乙烯合成的增强有关。研究为进一步通过代谢调控提高C. zofingiensis虾青素含量奠定了基础,对指导C. zofingiensis虾青素积累新工艺的开发具有重要意义。     

法夫酵母生产虾青素发酵条件的研究

方法：分别进行了接种时间、摇床转速、接种量和装液量对法夫酵母细胞生产虾青素摇瓶发酵过程影响的实验,比较了DMSO法、酸热法、碱法和自溶法等破壁方法和提取溶剂之间的差别,测定了法夫酵母生长过程中的生物量、类胡萝卜素产量和培养基中的残糖。结果：确定了最佳的摇瓶发酵条件为：种瓶至发酵摇瓶的接种时间为40h,摇床转速为160r/min,接种量为10%,装液量为50mL;DMSO法和丙酮分别为合适的破壁方法和提取溶剂。结论：初步确定发酵的基本条件,为进行法夫酵母高产虾青素菌种的筛选以及发酵培养基的优化奠定了基础。     

大鲵油的水酶法提取及精制过程中脂肪酸组成的变化

为研究大鲵油脂肪酸的营养价值,通过水酶法提取大鲵油,采用气相色谱-质谱法(GC-MS)分析粗大鲵油及其精制(脱胶、脱酸、脱臭)过程中脂肪酸组成的变化以及精制草鱼油中脂肪酸组成的差异。研究结果表明:水酶法提取大鲵油的最佳条件为酶解温度60℃,pH6.5,酶添加量0.75%,酶解时间90 min。所得粗大鲵油达到SC/T 3502-2016粗鱼油二级标准。研究发现,在粗大鲵油中共检出脂肪酸20种,精制过程中脂肪酸组成基本不变,精制后大鲵油中不饱和脂肪酸和必需脂肪酸的相对含量分别达74.76%和25.17%,均高于精制草鱼油中不饱和脂肪酸(61.08%)与必需脂肪酸(18.90%)的相对含量,由此可知,当前方法对大鲵油的提取较为有效,且大鲵油营养价值较草鱼油高。     

缺氮胁迫下雨生红球藻虾青素积累过程中的基因组MSAP分析

虾青素具有多种生物学活性,雨生红球藻为天然虾青素的最佳来源,缺氮胁迫会导致雨生红球藻积累虾青素。为了解缺氮条件下雨生红球藻虾青素积累的分子机制,该研究通过对雨生红球藻进行缺氮胁迫,结合MSAP法,研究了雨生红球藻在缺氮胁迫下虾青素积累过程中基因组甲基化水平的变化,结果表明:缺氮胁迫0~72 h期间,雨生红球藻生长速度减慢,而虾青素积累主要发生在缺氮处理12~24 h期间,随后积累速度减慢。同时,对缺氮胁迫0、24、72 h的雨生红球藻基因组DNA进行甲基化敏感扩增多态性分析,共得到了291个甲基化多态性位点,其中发生甲基化变化的位点在0~24 h和24~72 h分别占总位点的29.90%和53.95%。在缺氮胁迫24 h处DNA半甲基化率最大(为12.71%),全甲基化率最低(为26.80%);缺氮胁迫72 h处DNA全甲基化率最高(为28.52%),半甲基化率最低(为1.72%)。这表明DNA甲基化调节方式的改变是虾青素积累过程中的一种重要调控模式。     

Effects of different fungal elicitors on growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous

Six fungal elicitors prepared from Rhodotorula rubra, Rhodotorula glutinis, Panus conchatus, Coriolus versicolor, Mucor mucedo, Mortieralla alpina M-23 were examined to determine their effects on the growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous. The results showed that different fungal elicitor could cause diversely stimulating effects. Among the fungal elicitors tested, the M. mucedo elicitor concentration of 30 mg l(-1) promoted the biomass and total carotenoids yield most remarkably, resulting in 69.81+/-6.00% and 78.87+/-4.15% higher than the control, respectively. At the concentration of 30 mg l(-1), R. glutinis elicitor stimulated the highest astaxanthin yield with a 90.60+/-5.98% increase compared to the control. The R. rubra elicitor concentration of 30 mg l(-1) resulted in the optimal total carotenoids and astaxanthin content to be 42.24+/-0.49% and 69.02+/-0.72% higher than the control, respectively. At the concentration of 30 mg l(-1), R. rubra elicitor gave the highest increase in the ratio of astaxanthin in total carotenoids by 18.85+/-0.11% of the control.     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素*

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

基于动力学模型的法夫酵母发酵生产虾青素的补料策略优化

对法夫酵母的不同补料发酵方式进行了研究.基于底物抑制模型,提出了一种优化的两阶段补料策略,用于法夫酵母产虾青素的高密度发酵.在发酵的延迟期和对数生长期早期,糖浓度控制在25 g/L左右,在此条件下,生物量可以达到最大,且时间缩短.在对数生长期后期及稳定期,糖浓度控制在5 g/L,虾青素的合成时间可以有效延长.与传统的补料方式相比,采用此补料策略取得了较好的发酵效果.发酵终点细胞干重达到23.8g/L,虾青素产量达到29.05 mg/L,分别比分批发酵提高了52.8%和109%.     

Cadmium-resistance in growing Rhodotorula sp. Y11

The uptake of cadmium and responses to this metal were studied in growing Rhodotorula sp. Y11. In presence of cadmium, Y11 showed a similar lag phase with decreased mu(max) in comparison to the control cultures in the absence of cadmium. Different changes in contents of cell elemental composition (carotenoids, total protein, total soluble sugar, and phosphate content) were observed under cadmium pressure. Growing Rhodotorula sp. Y11 took up cadmium in a biphasic mode, involving an initial energy-independent biosorption to the cell surface, followed by a slower energy-dependent intracellular accumulation. In the presence of metabolic inhibitors, intracellular cadmium uptake of growing Y11 was significantly influenced.     

Selective extraction of free astaxanthin from Haematococcus culture using a tandem organic solvent system

A novel tandem solvent process of dodecane and methanol was developed for the selective extraction of free astaxanthin from red encysted Haematococcus culture. The process consists of dodecane extraction for astaxanthin mixture from the culture (stage 1) and methanol extraction for free astaxanthin from the dodecane extract (stage 2). In the first stage, astaxanthin mixture was directly extracted to dodecane from the culture broth without cell harvest process, followed by a rapid separation of the dodecane extract and the culture medium containing cell debris by simple settling. In the second stage, free astaxanthin was selectively collected to methanol from the dodecane extract, accompanied with saponification of astaxanthin-esters by the addition of NaOH to methanol. During saponification, use of the optimum NaOH concentration (0.02 M) and low temperature (4 degrees C) reaction minimized the degradation of free astaxanthin, resulting in a total recovery yield of free astaxanthin of over 85%. The free-astaxanthin-containing methanol extract was also simply separated from dodecane by gravity settling, after which the astaxanthin-free dodecane was effectively recycled to the first stage, yielding a stable extractability of astaxanthin mixture during repeated extraction. Our results indicate the potential of the proposed tandem solvent process as an alternative extraction technology for the high-value antioxidant Haematococcus astaxanthin.     

An improved process for cell disruption and astaxanthin extraction from Phaffia rhodozyma

Phaffia rhodozyma is one of the most important natural sources of the carotenoid astaxanthin, and the key process for extracting intracellular astaxanthin is disrption of the thick cell wall. In this work, an improved process for cell disruption and astaxanthin extraction from Phaffica rhodozyma was studied using an autoclave at low acid concentration. Under the optimum conditions (HCl 0.5 M and autoclave pressure 0.1 Mpa, 15 min), the relative residual astaxanthin and astaxanthin extractability reached 90.4 ± 3.5% and 84.8 ± 3.2%, respectively. The scanning electron microscopy pictures showed that all yeast cells shattered into fragments after autoclave treatment at low acid concentration condition, whereas cells were intact or partly broken after treatment by some other physical and chemical processes. This new method left no residual toxin and gavehigher extraction recovery, with good prospects for industrial use.     

Engineering a beta-carotene ketolase for astaxanthin production

A new beta-carotene ketolase gene (crtW) was cloned from an environmental isolate Sphingomonas sp. DC18. A robust and reliable color screen was developed for protein engineering to improve its activity on hydroxylated carotenoids for astaxanthin production. Localized random mutagenesis was performed on the crtW gene including the upstream ribosomal binding site (RBS). Six mutations (H96L, R203W, A205V, A208V, F213L and A215T) in the crtW gene were isolated multiple times that showed improved astaxanthin production. These mutations were localized near the conserved histidine motifs, which were proposed for binding iron required for enzymatic activity. Combination of two of the mutations (R203W/F213L) further improved astaxanthin production. One mutation at the RBS (a438t) was shown to have additional effect on improving astaxanthin production. Most of the mutants still retained high activity on beta-carotene, however, the F213L single mutant and the R203W/F213L double mutant that yielded the highest improvement for astaxanthin production showed decreased activity for canthaxanthin production.     

海洋红酵母产虾青素培养基优化的初步研究

为了提高海洋红酵母发酵虾青素的产量水平,对海洋红酵母的培养基成分进行了初步研究。试验结果表明,海洋红酵母能利用葡萄糖、淀粉水解糖、糖蜜等多种碳源,用淀粉水解糖为碳源培养海洋红酵母所获得的虾青素体积产率最大;用牛肉膏为氮源有利于提高海洋红酵母的生物量,以(NH4)2SO4、NH4Cl和蛋白胨为氮源有利于提高海洋红酵母的虾青素体积产率,用KNO3、草酸铵、蛋白胨、尿素有利于提高海洋红酵母的虾青素细胞产率;在海洋红酵母的培养基中添加Mn2 、Cd2 、Zn2 、Fe2 能增加生物量,添加Zn2 、Fe3 、Mn2 能增加海洋红酵母的虾青素体积产率,添加Fe3 能提高海洋红酵母的虾青素细胞产率。