锁掷酵母破壁技术的研究

分析比较了酸热法、二甲基亚砜法、细胞自溶法、高压均质法及酶促法对锁掷酵母的破壁效果,及对类胡萝卜素提取率的影响。结果表明,与其他4种破壁方法相比,高压均质法具有破壁效果好（破壁率达72.4%）、类胡萝卜素提取率高（67.3%）、破壁时间短、工艺简单、安全无污染等优点。通过均质压力、均质次数和菌液浓度这3个因素对高压均质法进行工艺优化,发现均质压力80 MPa、均质3次、菌液浓度为8%时,细胞破壁及类胡萝卜素提取效果最佳,分别是78.3%和82.5%。该研究结果为锁掷酵母的开发利用及类胡萝卜素的提取提供了理论依据及技术支持。     

掷孢酵母类胡萝卜素的提取与鉴定

将一株从草莓果实上分离得到的能够积累类胡萝卜素的掷孢酵母利用二甲基亚砜法进行色素提取,经过薄层层析法（thin layer chromatography,TLC）、分光光度计法及高效液相色谱法（high performance liquidchromatography,HPLC）检测,与粘红酵母标准菌株R.glutinis NBRC 1125T、掷孢酵母标准菌株S. pararoseus CBS0376T及β-胡萝卜素标准品进行对比,结果表明：掷孢酵母野生型NGR主要积累3 种色素,分别为红酵母红素、圆酵母素和β-胡萝卜素,其中圆酵母素和β-胡萝卜素及其异构体（推测为13-顺式-β-胡萝卜素）含量最多（分别约为653.22 μg/g和635.10 μg/g）,红酵母红素积累量较少,约为150.37 μg/g。野生型NGR的紫外诱变突变株Y9为高产β-胡萝卜素突变株,β-胡萝卜素积累量可占总类胡萝卜素比例的54%,W1为不产β-胡萝卜素,红酵母红素和圆酵母素积累量减少的粉色突变株,W4为不产色素的白化突变株。在常规酵母培养基培养下,野生型NGR积累总色素含量可达1 438.69 μg/g。     

Assessment of Cell Disruption and Carotenoids Extraction from Sporidiobolus salmonicolor (CBS 2636)

The increasing demand for carotenoids by industries has drawn attention to their bio-production. Since pigments are intracellular, extraction steps are then needed after cell cultivation. In this work, different strategies for extraction of carotenoid pigments from Sporidiobolus salmonicolor (CBS 2636) were studied. Different solvents (dimethyl sulfoxide, petroleum ether, hexane, ethyl acetate, chloroform, and acetone), liquid N₂, and diatomaceous earth were used to disrupt the cell and thus release the intracellular carotenoids. The results of this study showed that when multiple solvents were used, a synergistic effect on the extent of carotenoids recovery was obtained. Maximum concentration of total carotenoids (913 μg/L) was obtained in the treatment using liquid N₂ and dimethyl sulfoxide to disrupt the cell, followed by the extraction with a solution of acetone/methanol (7:3, v/v).     

拟粉红锁掷孢酵母降解展青霉素的机制

展青霉素(PAT)是一类主要由曲霉属和青霉属等真菌产生的有毒次级代谢产物,主要污染水果及其制品,严重威胁着人类的健康。为了寻找一种安全高效的PAT脱毒方法,近年来采用生物法清除PAT已成为主要的研究方向。作者研究了拟粉红锁掷孢酵母(Sporidiobolus pararoseus)对PAT降解的影响及其降解机制。结果显示,S.pararoseus可以显著降低苹果伤口处PAT的积累量,体外实验发现S.pararoseus与PAT(5μg/mL)共同培养18 h,PAT可以被S.pararoseus完全降解。S.pararoseus对PAT的降解作用既不是酵母细胞壁的吸附作用,也不是细胞的吸收作用,而是细胞正常代谢下产生的胞内酶对PAT具有降解作用。本研究有助于了解S.pararoseus降解PAT的作用机制,为真菌毒素的生物降解提供了新的理论依据。     

Study of the bio‐production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) using pre‐treated agro‐industrial substrates

BACKGROUND: The increasing industrial demand for carotenoids has aroused interest in their bio‐production, and the need to reduce production costs has encouraged the use of low cost industrial substrates, such as agro‐industrial residues. Thus the objective of this research was the bio‐production of carotenoids by Sporidiobolus salmonicolor using agro‐industrial substrates (corn steep liquor and sugarcane molasses), pre‐treated with acids (sulphuric and phosphoric). RESULTS: Bio‐production was carried out in an orbital shaker using a 10% (v/v) inoculum, incubation at 25 °C, and agitation at 180 rpm for 120 h in a non‐illuminated environment. The carotenoids were recovered using liquid N₂ combined with dimethylsulphoxide for cell rupture, and an acetone/methanol mixture (7:3 v/v) for extraction. CONCLUSION: The complete second‐order design allowed for optimisation of the carotenoid concentration obtained from industrial substrates pre‐treated with acids (sulphuric and phosphoric), obtaining a total carotenoid content of 541.5 µg L^?1 using 10 g L^?1 sugarcane molasses, 5 g L^?1 corn steep liquor and 5 g L^?1 yeast hydrolysate at 25 °C, with agitation at 180 rpm and an initial pH of 4.0. Copyright © 2008 Society of Chemical Industry     

Evaluation of the conditions of carotenoids production in a synthetic medium by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor

This work studied the cultivation conditions for the production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor. A Plackett–Burman design was used for the screening of the most important factors, followed by a complete second order design, to maximise the concentration of total carotenoids. The maximum concentration of 3425.9 μg L^?1 of total carotenoids was obtained in a medium containing 80 g L^?1 glucose, 15 g L^?1 peptone and 5 g L^?1 malt extract, with an aeration rate 1.5 vvm, 180 r.p.m., 25 °C and an initial pH of 4.0. Fermentation kinetics showed that the maximum concentration of total carotenoids was reached after 90 h of fermentation. Carotenoid bio‐production was partially associated with cell growth. The specific carotenoid production (Y_P/X) was 238 μg carotenoids/g cells, whereas Y_P/S (substrate to product yield) was 41.3 μg g^?1. The specific growth rate (μ_x) was 0.045 h^?1. The highest cell and total carotenoid productivity were 0.19 g L^?1 h^?1 and 56.9 μg L^?1 h^?1, respectively.     

Sporidiobolus pararoseus WZ012产类胡萝卜素发酵条件的优化

以响应面方法对Sporidiobolus pararoseus WZ012产类胡萝卜素的摇瓶发酵条件进行了优化。在Plackett-Burman实验的基础上,利用中心组合设计研究了4个主要因素(葡萄糖、酵母膏、KH2PO4、接种量)对产类胡萝卜素的影响。结果表明,较高浓度的酵母膏和较大的接种量有助于类胡萝卜素的生产,而较高浓度的葡萄糖和KH2PO4有助于生物量的提高,生物量与类胡萝卜素产量之间没有必然的联系。优化得到的发酵培养基组成(g/L)为:葡萄糖60.23、酵母膏12.21、KH2PO41.68、接种量为8.38%。在此条件下红酵母产类胡萝卜素的最大产量为29.3mg/L,较优化前提高了54.1%。类胡萝卜素成分分析表明主要含有β-胡萝卜素、圆酵母素和红酵母红素这3种类胡萝卜素物质,其中β-胡萝卜素占62.5%。     

产胞外多糖酵母菌的分离鉴定及其发酵条件优化

为筛选新型高产胞外多糖(exopolysaccharides,EPS)的酵母菌株,满足工业化生产的要求,该研究利用WL选择培养基和产糖基础培养基筛选出高产EPS的酵母菌,通过形态特征观察、生理生化试验和18S rDNA技术对其进行鉴定,并利用响应面方法对菌株产胞外多糖条件进行优化。结果表明,从土壤中筛选得到一株高产EPS的酵母菌,经鉴定,该酵母菌为近粉红锁掷孢酵母(Sporidiobolus pararoseus),命名为S.pararoseus PFY-Z1,当葡萄糖17.34 g/L、硫酸镁1.98 g/L、初始pH值5.3时,EPS的含量为(6.74±0.13)g/L,是优化前的1.63倍。不仅为EPS的生产提供菌种来源,也为今后利用S.pararoseus大规模生产EPS提供理论基础。     

A novel β-glucosidase from Sporidiobolus pararoseus: characterization and application in winemaking

For the first time, the production of an extracellular β-glucosidase (Sp-β-gl) by a Sporidiobolus pararoseus yeast strain is reported. The Sp-β-gl activity was quantified, characterized, and assessed for its efficiency in releasing aroma-enhancing compounds in wines. The maximum enzymatic synthesis was after 72 h of growth in a complex media with 20 g/L of cellobiose. The optimal pH and temperature were 5.5 and at 50 °C, respectively. It showed a wide range of pH stability and exhibited quite high thermostability at low temperatures. In addition, this β-glucosidase revealed tolerance to wine-associated inhibitory compounds (sugars and ethanol), showing suitable characteristics for all the stages of alcoholic fermentation. The hydrolysis of the glycosidic terpenes by Sp-β-gl was studied by gas chromatography, and its ability to efficiently release free terpenols has been demonstrated. The concentrations of geraniol, linalool, α-terpineol, and nerol were significantly increased in treated wines. These results suggest the potential application of this new yeast β-glucosidase as an aroma-enhancing enzyme in winemaking. PRACTICAL APPLICATION: The search for new β-glucosidase from yeast sources is important to improve the quality of wines. In this work, an S. pararoseus yeast strain has shown to be capable to produce a β-glucosidase with suitable combination of properties for functionality in wines and with potential to increase the concentration of free aroma compounds, showing good prospects for an industrial application.     

锁掷酵母产胞外多糖的研究

研究了氮源的种类和添加量对锁掷酵母产胞外多糖的影响,发现(NH4)2SO4有利于胞外多糖的积累,并且随着(NH4)2SO4加入量的增多胞外多糖的分子量趋于降低,在(NH4)2SO4添加量为1.5 g/L时多糖积累量最高为6.89 g/L。采用补料分批发酵时,控制发酵的pH和碳源,72 h多糖积累接近最大值为16.2 g/L。锁掷酵母胞外多糖主要由半乳糖和葡萄糖组成,其组成比例约为1∶2,细胞壁组成主要以葡萄糖、半乳糖和甘露糖为主,其比例接近于2∶1∶1。