红酵母产类胡萝卜素提取工艺的优化研究

目的:从提取溶剂及提取条件等方面对红酵母产类胡萝卜素提取工艺进行了优化研究。方法:用单因子实验对提取溶剂进行了筛选;用析因试验对提取条件进行了分析研究;通过最陡爬坡实验和中心组合设计实验,优化得到了最佳的类胡萝卜素提取条件。结果:结果表明丙酮和二甲亚砜组成的复合体系是理想的提取溶剂。溶剂添加量、提取温度、提取时间、丙酮与二甲亚砜组分比例都对红酵母产类胡萝卜素提取产生一定影响,其中溶剂添加量和丙酮与二甲亚砜组成比例的影响最为显著(P<0.001)。最佳优化提取条件为:溶剂添加量为42.66%(V/V),丙酮与二甲亚砜组成比例为62.47%(V/V),温度为20℃,提取时间为90min,在此条件下红酵母产类胡萝卜素的最大提取量为5.51μg/ml。结论:实验得到了红酵母发酵产类胡萝卜素溶剂提取条件的一个非常合适的模型。     

粘红酵母发酵生产类胡萝卜素培养条件的优化

目的是通过测定不同条件下类胡萝卜素的产量找出粘红酵母发酵生产类胡萝卜素的最优条件。探讨了不同碳源、氮源对粘红酵母菌体生长和色素形成的影响,并通过正交实验确定了最佳条件组合。实验结果表明,最适发酵培养条件为：蔗糖40g/L、酵母粉20g/L、转速150r/min、装液量30mL/500mL、发酵时间84h。在此条件下,粘红酵母摇瓶发酵的生物量、类胡萝卜素含量及产量分别达15.17g/L、718.6μg/g、10.9mg/L,依次比初始发酵提高了1倍、7.4倍和15.8倍。发酵过程动态分析表明,84h色素产量达最高峰。     

油菜种子类胡萝卜素总量测定方法的研究

类胡萝卜素是植物中最重要的一类色素,具有抗氧化、预防心血管疾病和癌症、延缓衰老等生物学功能,与人类健康关系密切.培育高类胡萝卜素油菜品种能有效地提升菜籽油的营养保健价值和稳定性,是今后油菜品质育种的一个新方向.研究建立快速、准确和简便的油菜种子类胡萝卜素总量测定方法,是发掘高类胡萝卜素油菜种质资源和开展高类胡萝卜素油菜育种的前提条件.本实验研究了不同提取溶剂、时间、料液比等因素对油菜种子中类胡萝卜素的提取效果和测定结果的影响,确定了最佳的提取和测定条件.建立了快速、准确和简便的油菜种子类胡萝卜素总量测定方法,并测定了30份不同类型油菜种质资源种子中类胡萝卜素总量,为进一步发掘高类胡萝卜素油菜种质资源和开展高类胡萝卜素油菜育种创造了条件.     

氮源对蛹虫草生长及类胡萝卜素产生的影响

【背景】蛹虫草是一种珍稀食药用菌,类胡萝卜素不仅为其重要活性成分,而且影响子实体的外观品相,但是类胡萝卜素产生的影响因素不明。【目的】揭示氮源对蛹虫草生长和类胡萝卜素产生的影响。【方法】测定不同氮源培养基中菌株生长速度、分生孢子产生及类胡萝卜含量,筛选菌株生长的最适氮源,进一步研究不同浓度的氮源对蛹虫草生长及类胡萝卜素产生的影响,并测定不同光照条件下氮源浓度对蛹虫草子实体类胡萝卜素产生的影响。【结果】蛹虫草在不同的氮源培养基中菌落形态和类胡萝卜素产生存在明显差异。麦麸和黄豆粉培养基中菌株生长速度最快,但是菌落稀疏,正面分别呈现荧光黄色和极微弱红色;蛋白胨和酵母提取物培养基中菌落致密,产孢量极显著高于其它氮源培养基(P0.01),菌落正面为橙黄色;甘氨酸和柠檬酸为氮源时完全没有色素产生,其它无机和氨基酸氮源培养基平板背面有微量色素产生。固体和液体静置培养条件下均发现蛋白胨浓度在0-3%的范围内,随着浓度的增加,类胡萝卜素含量增加;子实体栽培中不同的氮源浓度均表现为蓝光光照条件下类胡萝卜素含量显著高于白光;而在白光和蓝光光照条件下均表现为蛋白胨浓度为1%时类胡萝卜素含量最高,分别为2 809.38±386.24μg/g和4 093.75±518.37μg/g。【结论】氮源种类和浓度显著影响蛹虫草类胡萝卜素的产生,蓝光光照和1%蛋白胨浓度为子实体类胡萝卜素产生的最佳条件,这为栽培富含类胡萝卜素的蛹虫草子实体提供了试验依据。     

沼泽红假单胞菌中类胡萝卜素的提取与分析

目的:研究沼泽红假单胞菌中类胡萝卜素的提取工艺条件。方法:对细胞收集、前处理、色素初浸、二次萃取,皂化和溶剂回收等方面进行了系统的研究,并通过紫外分光光度法和高效液相色谱法对所提取的类胡萝卜素进行了分析。结果:实验结果表明,类胡萝卜素的最佳提取工艺条件为:碱性氯化钙絮凝法收集细胞,乙醇浸泡法进行前处理,49℃下丙酮初浸提浸提4h(搅拌并加抗氧化剂),丙酮用量为5mL/g菌泥,回收丙酮,乙醚二次萃取,30℃下皂化1.5h,洗涤,浓缩并回收乙醚,在此提取工艺条件下不仅能够有效地提取红螺菌中的类胡萝卜素,而且还能分离细菌叶绿素。结论在本实验条件下,根据标准曲线计算出类胡萝卜素的含量高达6.148mg/L菌泥。通过HPLC分析表明:类胡萝卜素提取液中主要成分至少有10种。     

逆境对红酵母发酵生产类胡萝卜素的促进作用

类胡萝卜素广泛存在于细菌、藻类、真菌和植物中,是一类具有重要的生理保健功能的呈黄色、橙红色或红色的多烯类化合物。考察了不同逆境条件,包括低温、低温处理时间、酸处理及高浓度盐处理对促进一株红酵母(Rhodotorulasp.)发酵生产类胡萝卜素的影响,并利用响应面分析方法研究了其交互作用。结果表明,低温,酸处理、高浓度盐的影响以及低温和酸处理、低温和高浓度盐以及低温处理时间和酸处理的交互作用对类胡萝卜素产量的影响均显著,在温度为15℃处理48 h,pH3.5,NaCl浓度为2 mol/L的条件下,类胡萝卜素最高产量达到31.04 mg/L,说明逆境对红酵母发酵生产类胡萝卜素具有促进作用。     

香蕉皮类胡萝卜素提取工艺条件的研究

为探究香蕉皮类胡萝卜素提取及综合利用的可能性,采用单因素试验与正交试验相结合的方法优化香蕉皮类胡萝卜素的提取工艺条件。结果表明：其最佳提取工艺条件为：以丙酮为提取溶剂、料液比1∶25、提取温度45℃、提取时间75min,在此工艺条件下可获得最佳提取效果,类胡萝卜素提取量平均值为12.541 mg/g。另用超声波辅助提取法,得到香蕉皮中类胡萝卜素提取量平均值为13.740mg/g,在提取时间仅为20min的情况下,比普通浸提法的提取量增加了9.56%,提取效率大大提高。     

产类胡萝卜素酵母菌原生质体的制备、再生与诱变

确定了1株产类胡萝卜素红酵母Y-35的原生质体最佳制备条件和再生条件,以及在此基础上的诱变育种,通过实验,初步确定Y-35菌株原生质体形成和再生的适宜条件为;菌龄16h,蜗牛酶浓度1%,30℃处理60min。红酵母Y-35原生原体经紫外线诱变后得到18株诱变菌株,分别测定其生物量,类胡萝卜素含量及产量,获得2株类胡萝卜素产量明显提高的变异菌株RY-10和RY-19。其产量分别比出发菌株提高49%和54%。     

诺卡氏菌属5205菌株产生类胡萝卜素研究

对诺卡氏菌属5205菌株形成类胡萝卜素的培养基和培养条件进行了初步研究。结果表明,该菌在培养基,蛋白胨10g,牛肉膏3g,葡萄糖20g,NaCl 5g,水1000mL,pH 7.5,500mL三角瓶装量100mL培养基于旋转式摇床上30℃,170r/min振荡培养72h,菌体生物量和类胡萝卜素含量分别为7．26mg/mL和471．45ug/干菌体,添加核黄素明显促进该菌的生长和类胡萝卜素的形成。     

苹果类胡萝卜素研究进展

类胡萝卜素是苹果果实色泽形成的一个重要影响因子,其种类和含量决定果实是否具有良好的外观和丰富的营养。本文综述了近年来有关苹果果实类胡萝卜素方面的研究进展,并对苹果类胡萝卜素的种类和含量,苹果发育和贮藏过程中类胡萝卜素含量的变化规律,生物合成途径中相关基因的表达,以及环境因子对类胡萝卜素积累的影响等方面进行了阐述。     

Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants

As a proof of concept, the qualitative and quantitative engineering of carotenoid formation has been achieved in crop plants. Successful reports in tomato, potato, rice, and canola all describe the enhancement of carotenoid with nutritional value, while in model systems such as tobacco and Arabidopsis the engineering of carotenoid to confer abiotic stress has been described. For all the successful applications there have been many examples of unintended/unpredicted phenotypes and results. Typically this has resided from our lack of understanding of carotenoid formation and its regulation. In the present article, we will review advances in carotenoid formation and its regulation to illustrate how metabolic engineering experiments have shed light on regulatory mechanisms.     

Engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

The basidiomycetous yeast, Xanthophyllomyces dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 μg/g dry wt within 4 days of growth.     

Carotenoid responses to environmental stimuli: integrating redox and carbon controls into a fruit model

Carotenoids play an important role in plant adaptation to fluctuating environments as well as in the human diet by contributing to the prevention of chronic diseases. Insights have been gained recently into the way individual factors, genetic, environmental or developmental, control the carotenoid biosynthetic pathway at the molecular level. The identification of the rate‐limiting steps of carotenogenesis has paved the way for programmes of breeding, and metabolic engineering, aimed at increasing the concentration of carotenoids in different crop species. However, the complexity that arises from the interactions between the different factors as well as from the coordination between organs remains poorly understood. This review focuses on recent advances in carotenoid responses to environmental stimuli and discusses how the interactions between the modulation factors and between organs affect carotenoid build‐up. We develop the idea that reactive oxygen species/redox status and sugars/carbon status can be considered as integrated factors that account for most effects of the major environmental factors influencing carotenoid biosynthesis. The discussion highlights the concept of carotenoids or carotenoid‐derivatives as stress signals that may be involved in feedback controls. We propose a conceptual model of the effects of environmental and developmental factors on carotenoid build‐up in fruits.     

The triplet state of reaction center bacteriochlorophyll: Determination of a relative quantum yield

The low temperature EPR signal of the excited triplet state of bacteriochlorophyll has been quantitatively studied in reaction centers from Rhodopseudomonas spheroides (carotenoid free R 26 mutant). Using laser flash excitation the light saturation curve of the triplet signal has been compared with that of the free-radical formation due to photooxidation of P870 under identical optical conditions. This comparison shows that the quantum yield of triplet formation is nearly the same as that of the photochemical bleaching of bacteriochlorophyll.     

Movement of Ions and Change in the Electrical Potential Profile across the Membrane of the Rhodopseudomonas sphaeroides Chromatophore Studied by the Absorbance Change of Carotenoid

The relationship between the light-induced movements of electronsand ions and the change in the electrical potential profileacross the chromatophore membranes of a green mutant of Rhodopseudomonassphaeroides was studied by measuring the carotenoid absorbancechange. The light-induced absorbance change of the carotenoid, i.e.,the change of electrical field within the membrane, was shownto be affected by the following factors: (1) the formation ofa membrane potential by the electrogenic electron transfer;(2) the decay of the membrane potential due to the electrophoreticmovements of ions; (3) the development of a diffusion potentialinduced by the ion movements; and (4) the change of surfacepotential of the inner surface of the chromatophore membranedue to the proton uptake. The kinetics of the absorbance change of carotenoid during illuminationwere semiquantitatively explained by considering these factors,and the light-induced change in the electrical potential profileacross the membrane was estimated under various conditions. ¹ Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received October 21, 1981; Accepted March 18, 1982)     

法夫酵母高产虾青素的选育及发酵条件的优化

对法夫酵母原始菌株进行了紫外线－氯化锂复合诱变,获得了一株遗传性状稳定、在25℃条件下虾青素高产的菌株UL-40。其类胡萝卜素产量为7.10mg/L,经HPLC测定,其虾青素产量为643.97μg/g,比出发菌株提高了249.87%。利用SAS软件中的Plackett-Burman设计对发酵温度、初始pH值以及发酵培养基的八种组分进行优化组合,从中选出发酵温度、初始pH值和Corn steep liquor浓度为重要因素,通过响应面分析法建立了模型并从该模型中计算出在发酵温度为16.78℃、初始pH为4.73和CSL浓度为7.06mg/L时预测的最大响应值为3.9407mg/L,经实验证实此点的实测值为3.9261mg/L,证明模型是有效的并且存在极大值点。采用优化后的发酵条件及发酵培养基,法夫酵母生产虾青素的产量较原始发酵培养基提高了20.4%。     

Chromoplast biogenesis and carotenoid accumulation

Chromoplasts are special organelles that possess superior ability to synthesize and store massive amounts of carotenoids. They are responsible for the distinctive colors found in fruits, flowers, and roots. Chromoplasts exhibit various morphologies and are derived from either pre-existing chloroplasts or other non-photosynthetic plastids such as proplastids, leucoplasts or amyloplasts. While little is known about the molecular mechanisms underlying chromoplast biogenesis, research progress along with proteomics study of chromoplast proteomes signifies various processes and factors important for chromoplast differentiation and development. Chromoplasts act as a metabolic sink that enables great biosynthesis and high storage capacity of carotenoids. The formation of chromoplasts enhances carotenoid metabolic sink strength and controls carotenoid accumulation in plants. The objective of this review is to provide an integrated view on our understanding of chromoplast biogenesis and carotenoid accumulation in plants.     

Can charge recombination as caused by pH-dependent donor-side limitation in PS 2 account for high-energy-state quenching?

Schreiber and Neubauer (Photosynthesis Research 25: 279–293, 1990) have proposed a model which explains energy quenching by enhanced triplet formation as caused by charge recombination due to pH-dependent donor-side limitation. Quenching under these conditions is assumed to result from two mechanisms. Firstly, there is the withdrawal of excited states by charge recombination and formation of triplet states. Secondly, these triplet states can result in carotenoid triplets in the antenna which are supposed to quench excitons. Here, it is shown that quenching caused by both mechanisms can account for only about 25% of the experimentally observed energy quenching even under extremely favorable conditions. More likely, this number is less than 15%, as the contribution of the second step in the proposed triplet cycle is expected to be low as the life times of the carotenoid triplets are not long enough to cause the assumed quenching of excitons in the antenna.     

Comparison of carotenoid content,gene expression and enzyme levels in tomato (Lycopersicon esculentum) leaves

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.     

The effects of dietary astaxanthin on the carotenoid pattern of the prawn Penaeus japonicus during postlarval development

Penaeus japonicus postlarvae, reared under laboratory conditions, were fed an astaxanthin enriched diet to the investigate carotenoid metabolic capabilities during the postlarval development. Animals fed astaxanthin were found to absorb this carotenoid. The decrease of pigment concentration in the carotenoid starved group is related to the duration of the experimental feeding conditions; the carotenoid depletion depends upon the postlarval status at the starting point. As has been shown for adult prawns, the carotenoid pattern of postlarval stages, regardless of the diet, consists mainly of free and esterified astaxanthin; the relative amounts of these fractions undergo slight variations depending on the diet.