废糖蜜培养高核酸酵母提取RNA的研究

利用糖蜜做碳源连续发酵培养高核酸酵母来提取RNA,以期综合利用制糖业的废物糖蜜。结果表明:利用糖蜜和葡萄糖母液做碳源培养酵母,前者酵母对糖的转化率达43.8%,后者转化率最高只有29.7%,且前者的菌体量和RNA量均高于后者;安装空气分布器的发酵罐酵母对糖的转化率为47.5%,没有空气分布器的转化率只有20.2%在稀释率不断提高的条件下,发酵液中RNA的含量不断提高,稀释率为0.75时,发酵液中RNA含量最高达13.8%,干菌浓度达19g/L;利用浓盐法提取酵母中的RNA得率达80.4%,纯度达92.3%。     

风味酵母A10-2的高密度培养及其生长动力学

为实现一株自分离风味酵母的高密度培养及生长动力学预测模型构建,本文对从酱油发酵醪中筛选的产4-乙基愈创木酚的风味酵母A10-2进行了高密度培养工艺的研究,对培养基种类（氮、碳源）、浓度进行了研究及优化,并对酵母的生长、基质消耗过程进行了数学模型的构建和验证。结果表明:磷酸二氢铵作为无机氮源,浓度0.2 g/100 mL为最适氮源流加补料方案;使用糖蜜作为唯一碳源时,培养液中持续流加保持总糖浓度为0.4~0.6 g/100 mL可获得最佳菌体生长速度;A10-2酵母的生长符合Logistic方程S形曲线,基质（总糖）消耗符合Leudeking-Priet方程,比生长速率最大值μ_m为0.4764 h?1,最大菌体生长得率系数Y_G值为0.5879 g/g,维持系数m_s=0.0127 g·L?1·h?1。所建立模型能够较好的描述酵母在高密度培养过程中的生长和耗糖情况,具有预测指导意义。     

利用废糖蜜生产SCP菌种性能的测定

分别以白地霉、热带假丝酵母、皮状丝孢酵母和产朊假丝酵母为研究对象,以糖蜜废液为培养基,通过恒温培养,测定活细菌数、细胞干重和菌体蛋白质含量等指标比较,从而选育生长快、蛋白质含量高的菌种生产SCP。结果表明,热带假丝酵母生长较快、粗蛋白含量较高,为生产SCP的最佳菌种。     

耐高糖面包酵母发酵工艺优化

为了优化耐高糖酵母发酵工艺,基于培养温度、摇床转速、装液量、初始p H、菌体接种量五个因素的单因素实验的结果,之后对装液量、初始p H、菌体接种量进行响应面优化实验。结果表明,最佳工艺条件为:培养温度30℃,摇床转速200 r/min,装液量8.4%,p H4.7,接种量7.4%,在此条件下,酵母细胞干重提高了55.72%。同时,利用5.0 L机械搅拌罐以溶氧反馈流加、恒速流加、间歇流加三种糖蜜补充方式进行酵母细胞培养,绘制生长曲线,发现以溶氧反馈流加方式补充糖蜜更有利于酵母繁殖,最大酵母湿重达到143.95 g/L,达到了耐高糖面包酵母高密度发酵的实验水平,确定了酵母最佳的流加补料方式。     

拟指数—DO-stat两阶段补料策略在糖蜜酵母高密度培养中的应用

高密度培养(high cell density cultivation,HCDC)是酵母生产实现高效高产的途径。文中以菌体量、菌体得率和生产强度为评价指标,探讨了拟指数和DO-stat两种补料策略的优缺点,提出了1种两阶段补料策略:在21 h的分批培养结束后,首先采用比生长速率为0.03 h~(-1)的拟指数补料,然后在溶氧跌到20%以下后将补料方式切换为DO 40%的DO-stat补料。结果显示,两阶段补料策略达到了菌体量123.57 g/L、总菌体得率0.51 g/g、生产强度1.15 g/(L·h)。与拟指数补料策略相比,菌体量提高了23.8%、菌体得率提高6.25%;与DO-Stat补料策略相比,培养周期缩短20.4%、生产强度提高了14.5%。研究结果表明,两阶段补料策略可以有效地提高酵母生长能力。     

活性干酵母扩大培养的研究

酵母的生长繁殖与其培养的温度和通风量有着密切的关系，以活性干酵母为出发菌体进行了以下试验。1、活性干酵母的活化；2、培养温度对酵母生长繁殖的影响；3、通气量对酵母细胞生长的影响。     

三七渣发酵生产蛋白饲料的菌体生长动力学

对黑曲霉(Aspergillus niger sp.)/产朊假丝酵母(Candida utilis 2.281)固态发酵三七渣生产蛋白饲料的过程进行了研究,考察了发酵培养物真蛋白含量、酶活、干重、核酸提取液OD260等参数的动态变化趋势,并建立了菌体生长动力学模型。研究结果表明,发酵培养物真蛋白含量、酶活、核酸提取液OD260变化趋势基本一致:它们先随发酵时间的延长而逐渐增大,然后又逐渐降低并趋于稳定;发酵培养物水含量变化较大,而水活度变化较小。菌体生长动力学适合采用对数模型(R2=0.992 9)进行描述,其中,比生长速率常数μ=0.041 31 h-1,最大干物质减重率Xm=52.22%,菌体生长速率在发酵66 h后达到最大值。该模型可以用来预测固态发酵过程中菌体的生长过程。     

产朊假丝酵母核酸高产株的选育及其发酵性能研究

为了获得一株发酵性能优良的高核酸酵母,以产朊假丝酵母(Candida utilis)CL1501为出发菌株,采用紫外-亚硝基胍复合诱变,筛选获得一株高核酸含量的突变株CL15013,经测定其核酸含量占菌体干质量的16.8%,高于出发菌47.8%。对比研究其流加及分批培养工艺,流加培养细胞收获量为16.9 g/L,比分批培养提高94.3%;正交试验设计优化流加培养条件后,CL15013的收获量达21.3 g/L,比分批培养提高144.8%,具有良好的生产应用潜力。     

高浓度培养YE乳液的研究

在酵母抽提物乳液的培养中,通过在甘蔗糖蜜流加糖液中添加一定比例的果葡萄糖浆来降低发酵液的渗透压,减轻高渗透压对酵母细胞生长的抑制作用,以达到高浓度培养酵母细胞的目的。通过正交试验优化发酵工艺条件,在试验中取得了酵母培养浓度高达320g/L的效果。     

利用废糖蜜发酵生产单细胞蛋白的动力学研究

为探明以废糖蜜为原料发酵生产单细胞蛋白的过程,在4L 自动生物发酵罐中对白地霉、热带假丝酵母和产朊假丝酵母组成的混合菌种进行了扩大培养,采用 Monod 模型对混合菌的发酵动力学进行了研究。通过建立出混合菌生长、底物(糖蜜)消耗相关动力学模型参数,阐述了混合菌发酵动力学特征。结果表明,混合菌发酵前期生长速度缓慢,20h 后进入对数生长期,60h 后生长趋于稳定,到60h 时生物量达到最高,为23g/L。基于 Monod 方程,得到了描述发酵过程的动力学模型和模型参数。该模型反应了菌体生长和底物消耗之间的关系,模型的拟合结果与试验值吻合较好,误差小于10%。     

紫外照射培养对酵母细胞壁葡聚糖的影响

在研究紫外伤害培养对酵母菌生理性能影响的基础上,利用紫外伤害培养后的酵母细胞提取葡聚糖。比较紫外伤害培养和未经紫外伤害培养的酵母细胞的胞壁葡聚糖得率,分别为22.60％和18.85％,相对纯度分别为81.67％和75.76％;葡聚糖得率比未经紫外伤害培养的酵母细胞提高19.90％,葡聚糖含量提高29.25％。经红外光谱分析多糖为葡聚糖。     

High-level production of prourokinase-annexin V chimeras in the methylotrophic yeast Pichia pastoris

Prourokinase (proUK)-annexin V chimeras expressed by the methylotrophic yeast Pichia pastoris in a synthetic medium as part of a system designed to yield a novel thrombolytic agent are degraded, as it is thought, by various yeast proteases present in the culture supernatant. Minimization of proteolysis was therefore investigated to increase the yield of intact proUK-annexin V. Protease inhibitor screening study indicated several proteases including at least serine protease like chymotrypsin were involved in the proteolysis. Addition of more than 10% of peptone or more than 0.2 mol l(-1) of arginine to the medium was effective in minimizing proteolysis in shake-flask culture. Culture condition of higher pH was also effective, however, induced a cell death. Cell improvement by increasing the methanol utilization ability yielded greater tolerance to high pH. As a result, the culture condition with highly concentrated peptone solution fed under controlled conditions of pH 8.0 was established, which greatly reduced proteolytic degradation in fed-batch fermentation. These optimal conditions, which enabled fibrinolytic activity to reach 7800 IU ml(-1), could easily be applied in industrial scale production.     

热带假丝酵母发酵生产木糖醇的研究

对热带假丝酵母 (C tropicalis )As2 1 776发酵木糖醇的营养条件进行了初步研究。初始木糖浓度在 80g/L附近时木糖醇转化率较高 ,限制性供氧条件下有利于木糖醇积累。酵母膏和蛋白胨是比较适合产木糖醇的有机氮源 ,而酵母膏更利于酵母细胞生长。培养基中添加 2 g/L的(NH4 ) 2 HPO4 、2～ 6g/L的NaCl、1～ 3g/L的KH2 PO4 、0 1～ 0 3 g/L的MgSO4 ·7H2 O能提高木糖醇的转化率     

酸马奶中乳酸菌与酵母菌的共生发酵特性

对内蒙古锡盟地区酸马奶中分离出的1株乳酸菌和1株酵母菌进行混合培养,初步确定双菌混合发酵的最佳培养条件：双菌发酵计数乳酸菌活菌数的最佳发酵温度为30℃摇床培养12h再转到37℃静置培养,最佳发酵时间为20h,脱脂乳中添加的营养成分最优配方为蛋白胨1g/100mL、蔗糖0.5g/100mL、酵母浸粉0.5g/100mL;双菌发酵计数酵母菌活菌数的最佳发酵温度为37℃静置培养8h再转到30℃摇床培养,最佳发酵时间为32h,脱脂乳中添加的营养成分最优配方为蛋白胨0.5g/100mL、蔗糖0.5g/100mL、酵母浸粉0.5g/100mL;选用乳酸菌与酵母菌质量比1:1作为菌种配比。 同时在最佳生长条件下探讨乳酸菌与酵母菌的相互作用关系以及后发酵对二者共生作用的影响,结果表明,促进乳酸菌生长的活性物质生成的时间为12h以前(即将酵母菌在5号配方中30℃摇床培养),促进酵母菌生长的活性物质生成的时间应为16h以前(即将乳酸菌在1号配方中37℃静置培养),在后发酵过程中,乳酸菌与酵母菌双菌培养的活菌数都极显著高于单菌培养(P＜0.01)。     

黄酒淋饭酒母中一株酵母菌发酵特性的研究

从传统黄酒生产的淋饭酒母中分离出酵母,并对纯化的酵母菌株的培养条件进行了研究。试验结果表明：适宜培养温度是28～30℃,适宜发酵pH为4.5～6.0。     

酵母对无机锌的富集及其影响因素研究

在培养基中添加无机锌，利用生物转化技术，通过酵母的生命活动，将无机锌有效地富集到细胞内，研究锌离子浓度对酵母生长繁殖、细胞含锌量、锌富集率及酵母发酵力的影响，并通过控制锌离子浓度，使酵母细胞既能富集锌，又对其发酵力不产生显著的影响，以便将仍具有发酵活力的富锌酵母用于食品加工之中。研究结果表明，锌离子浓度对酵母的生长繁殖和富集能力影响较大，浓度越高，酵母细胞含锌量越大。但无机锌对酵母细胞有显著的损伤，且这种损伤是非遗传性的，当无机锌去掉后，酵母细胞形态又恢复正常。     

十种添加物对红曲菌monacolin K产量的促进效果分析

为了提高红曲菌液态发酵产monacolin K的能力,本文对紫色红曲菌(Monascus Purpureus)M1进行培养基成分优化从而提高monacolin K产量。在实验室原培养基成分中添加山药粉、桔皮粉、酵母菌液、酵母上清液、酵母破壁液、破壁后的酵母菌液、亮氨酸、谷氨酸、乙醇等物质,对不同培养基中红曲菌的次级代谢产物产量进行测定。结果表明:添加谷氨酸可以极显著(p<0.01)提高monacolin K产量,提高了5.60倍;添加山药粉、酵母菌液、酵母上清液、酵母破壁液、破壁后的酵母菌液能提高红曲红和红曲黄色素的色价,其中添加酵母菌液对红曲红色素提高效果极显著(p<0.01),提高51.4%;通过扫描电镜检测谷氨酸培养基和普通培养基中红曲菌菌丝体的差异发现添加了谷氨酸的菌丝体更膨大,其细胞壁与原生质体之间缝隙增大,推测其细胞膜的通透性增加,进而导致细胞内积累的monacolin K能转移到胞外,使发酵液中monacolin K产量提高。     

Inhibitory effect of mineral ion accumulation on high density growth of the hyperthermophilic archaeon Sulfolobus solfataricus

A fed-batch operation for high density cultivation of Sulfolobus solfataricus (DSM 1617) in a bench-top fermentor using a feed medium composed of glucose and yeast extract was investigated. The highest maximal cell density obtained in controlled fed-batch cultures was 21.7 g/l. Although higher yeast extract concentrations in the medium favored greater cell biomass yield, cell growth ceased with low cell densities. It was observed that large amounts of inorganic ions, such as sulfate, ammonium, potassium and phosphate ions, were accumulated in the culture broth at higher yeast extract concentrations. This was due to either the addition of the titrant or feeding of yeast extract during cultivation. Fed-batch cultures with additional mineral salts in the feed medium showed much lower cell biomass, indicating that accumulation of inorganic ions has a significant inhibitory effect on the growth of S. solfataricus. Inhibition of cell growth by the presence of mineral ions was further confirmed by the batch culture experiments. Some plausible mechanisms which can account for the growth inhibition at higher mineral ion concentrations have been suggested.     

A simple colony‐formation assay in liquid medium,termed ‘tadpoling’, provides a sensitive measure of Saccharomyces cerevisiae culture viability

Here we describe the first high‐throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high‐throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10⁸ dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high‐throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space‐saving method, amenable to high‐throughput screens, for accurately measuring yeast cell viability. Copyright © 2013 John Wiley & Sons, Ltd.     

用于微生物培养基有机氮源的废酵母自溶液研究

为进一步降低聚 β 羟基丁酸酯 (PHB)的生产成本 ,考察并优化了酵母自溶的影响因素与条件 ,探索采用廉价的废酵母自溶液作为有机氮源替代酵母浸粉的可行性。结果表明 ,自溶反应的最佳初始 pH为 7 0 ;加水比不仅影响酵母细胞的自溶速度和自溶效率 ,而且影响自溶产品的氨基氮终浓度 ,因此最佳加水比的确定取决于最终的经济核算。乙酸乙酯可以成功替代甲苯作为自溶促进剂 ,其优化用量为 3 %～ 4%。进一步以自制的酵母自溶液对重组大肠杆菌VG1( pTU 14 )进行摇瓶培养 ,结果表明 ,当以酵母自溶液为有机氮源替代酵母浸粉时 ,PHB浓度可以提高 2 2 7%以上。因此 ,废啤酒酵母自溶液在PHB的大规模生产中具有广泛的应用前景。