高密度固态培养产朊假丝酵母菌剂的工艺优化

利用粮油副产物豆粕、稻壳和秸秆混合固态发酵制备饲料级酵母益生菌剂,采用正交试验确定培养基最佳配比,中心组合试验及响应面分析法建立最优发酵条件试验模型。结果表明:豆粕∶秸秆∶稻壳=5∶2∶2,40g原料添加葡萄糖4g、硫酸铵0.2g和1倍微量元素组合,接种量3.5%,原料厚度1.3cm,水分添加量∶原料=1∶1(m∶m),温度30.6℃,发酵时间28h,酵母菌活菌数可达(2.92±0.05)×109CFU/g。     

基于两阶段氨基酸添加的谷胱甘肽发酵高产方法

为提高产朊假丝酵母(Candida utilis SZU 07-01)发酵生产谷胱甘肽(glutathione,GSH)的产量,分别考察L-蛋氨酸和L-半胱氨酸在GSH分批发酵和流加发酵中的作用,结果发现L-蛋氨酸提升了酵母细胞在生长期的GSH合成能力,而L-半胱氨酸显著提高了细胞生长结束后的GSH产量。在此基础上,提出了两阶段氨基酸添加策略,即在分批发酵初始时(0 h)添加60 mmol/L L-蛋氨酸,在流加发酵过程中细胞干质量达到最大值时(第27小时)一次性添加30 mmol/L L-半胱氨酸。最终,GSH产量和胞内GSH含量进一步提高,分别达到1 247.1 mg/L和24.1 mg/g。该两阶段氨基酸添加策略在GSH发酵高产中的成功应用,对其他类似化合物的高效生产具有一定的借鉴意义。     

高生物量富硒产朊假丝酵母菌株的选育

从5株优良的发酵酵母菌中筛选出耐硒和富硒能力较好的产朊假丝酵母,采用耐酸驯化和梯度浓度的耐硒驯化增加菌株的抗性。研究表明,产朊假丝酵母Ⅰ的富硒能力更好。在此基础上,经复合诱变、亚硒酸钠抗性平板初筛和摇瓶复筛,筛选出生物量和含硒量都较高的菌株D-5,再将突变株D-5进行紫外诱变,筛选出1株高生物量和高含硒量的菌株U-16,其菌株生物量为5.86 g/L,总硒量为1 575.40 μg/g,有机硒量为1 500.90 μg/g,其有机硒占胞内总硒比重的95.26%。与出发菌株相比,筛选菌株的生物量提高了67.90%,有机硒量增加了95.95%。     

产朊假丝酵母利用有机酸的研究

在有机酸为唯一碳源的培养中培养产朊假丝酵母(Candida utilis)时,以L-苹果酸、乳酸、琥珀酸或柠檬酸为碳源的培养基经过48h后,有机酸浓度均由初始浓度5.0g/L下降到0.0g/L。以乙酸、草酸和富马酸为碳源的培养基有机酸浓度始终没有明显变化,说明产朊假丝酵母能够利用细胞外的L-苹果酸、乳酸、琥珀酸和柠檬酸,不能利用乙酸、草酸和富马酸。当葡萄糖和L-苹果酸、乳酸、琥珀酸和柠檬酸中的某种有机酸共同做碳源时,葡萄糖浓度均可以在32h内从20.0g/L降到0.0g/L,而各有机酸在0~24h内浓度变化不大,24~48h浓度均有不同程度的下降,说明当培养基中有葡萄糖时,有机酸的利用受到抑制。当浓度均为2g/L的L-苹果酸、乳酸、琥珀酸和柠檬酸同时做碳源培养产朊假丝酵母时,乳酸大约经过40h浓度首先降到0.0g/L,L-苹果酸、柠檬酸、琥珀酸浓度在0~16h过程中没有明显变化,16~48h下降趋势明显,最后也都被菌体完全利用,说明乳酸比较容易被菌体利用,而L-苹果酸、琥珀酸和柠檬酸在被菌体利用先后顺序上没有明显区别。     

提高产朊假丝酵母富硒能力的工艺条件研究

在5L发酵罐水平上研究不同培养条件对富硒产朊假丝酵母细胞生长、谷胱甘肽合成和富硒能力的影响,通过分批发酵试验确定较优的培养方案:在发酵培养的第18小时加入20mg/L亚硒酸钠,发酵罐转速450r/min,L-蛋氨酸的添加浓度10mmol/L.该条件下,酵母细胞干重为11.32g/L,胞内谷胱甘肽和有机硒含量分别达到11.5mg/g和1352μg/g,该方案为高性能(高胞内谷胱甘肽含量、高有机硒含量)富硒产朊假丝酵母的高效制备奠定了基础.     

Yeast derived from lignocellulosic biomass as a sustainable feed resource for use in aquaculture

The global expansion in aquaculture production implies an emerging need of suitable and sustainable protein sources. Currently, the fish feed industry is dependent on high‐quality protein sources of marine and plant origin. Yeast derived from processing of low‐value and non‐food lignocellulosic biomass is a potential sustainable source of protein in fish diets. Following enzymatic hydrolysis, the hexose and pentose sugars of lignocellulosic substrates and supplementary nutrients can be converted into protein‐rich yeast biomass by fermentation. Studies have shown that yeasts such as Saccharomyces cerevisiae, Candida utilis and Kluyveromyces marxianus have favourable amino acid composition and excellent properties as protein sources in diets for fish, including carnivorous species such as Atlantic salmon and rainbow trout. Suitable downstream processing of the biomass to disrupt cell walls is required to secure high nutrient digestibility. A number of studies have shown various immunological and health benefits from feeding fish low levels of yeast and yeast‐derived cell wall fractions. This review summarises current literature on the potential of yeast from lignocellulosic biomass as an alternative protein source for the aquaculture industry. It is concluded that further research and development within yeast production can be important to secure the future sustainability and economic viability of intensive aquaculture. © 2016 Society of Chemical Industry     

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。     

高生物量富硒产朊假丝酵母培养基优化

对富硒产朊假丝酵母发酵培养基进行优化,获得高生物量富硒酵母。从6种发酵培养基中确定G改良培养基为最佳培养基。利用Plackett-Burman设计法从影响富硒产朊假丝酵母生长的12个因子中筛选出葡萄糖、蛋白胨、KH2PO4、CuSO4添加量这4个关键因子。再利用Box-Behnken试验设计及响应面分析法确定关键因子的最佳水平及交互作用。试验得出各关键因子的最优组合为葡萄糖30 g/L、蛋白胨17 g/L、KH2PO4 6.5 g/L、CuSO4 0.01 g/L。结果表明,培养基优化后酵母生物量为12.01 g/L,有机硒含量为1 337.46 μg/g,谷胱甘肽为134.27 mg/L。将培养基中亚硒酸钠添加量由25 μg/mL提高至30 μg/mL,酵母生物量为11.21 g/L,有机硒为1 673.32 μg/g,谷胱甘肽为126.80 mg/L。     

产朊假丝酵母富硒及产谷胱甘肽的培养工艺

为生产高生物活性（高谷胱甘肽、高有机硒含量）的富硒产朊假丝酵母,在100 L发酵罐规模水平研究了菌体培养、无机硒的添加工艺,结果表明指数连续流加是富硒酵母最佳的培养方法,在补料的同时添加125 mg/L的无机硒（Na₂SeO₃）和3 mmol/L的半胱氨酸,菌体浓度、胞内谷胱甘肽浓度及无机硒转化率分别达到72 g/L、1080 mg/L和86%,胞内有机硒含量达到1493 μg/g,研究结果对富硒酵母大规模工业化生产具有一定的实践指导意义。      

大曲丢糟生产假丝酵母饲料蛋白的研究

白酒酿造产生大量的酒糟废弃物,其可转化为营养丰富、易被动物吸收的高效价生物活性蛋白饲料。以大曲丢糟为主要原料,假丝酵母为菌种进行饲料蛋白的发酵研究,通过正交试验得到最佳的饲料生产工艺条件为丢糟:麸皮=4:1,30℃培养3d,发酵终产物的粗蛋白和还原糖的含量分别达到了17.5%和1.13%。     

高性能富硒产朊假丝酵母的制备

在5 L发酵罐上,分别考察了亚硒酸钠添加时间和L-蛋氨酸对富硒产朊假丝酵母制备过程中的细胞量、谷胱甘肽合成、胞内谷胱甘肽含量以及硒有机转化率的影响。以高性能(高有机硒含量、高GSH含量)富硒酵母的制备为目标,得到一种比较有效的发酵培养策略,即发酵初期(0 h)添加L-蛋氨酸以及葡萄糖耗尽时(15h)添加亚硒酸钠,最终硒有机转化率达到87.5%。     

Isolation of the YAP1 homologue of Candida utilis and its use as an efficient selection marker

The industrially important yeast Candida utilis is widely used in the production of food and medical materials, but its practical host-vector system has not been well developed. In order to construct a food-grade host-vector system, we isolated the YAP1 homologue, CuYAP1, of C. utilis IAM4264 and evaluated its use as a selection marker in transformation. A DNA probe was obtained by PCR using degenerate primers and the CuYAP1-encoding 438 amino acid protein was isolated by hybridization. Although the amino acid identity of Yap1 and CuYap1 was 28.7% as a whole, the characteristic bZip region and two cysteine-rich domains (CRDs) showed a higher homology. CuYAP1 was inserted in a CuGAP1 expression cassette of the C. utilis ARS vector pRI177, and C. utilis AHU3053 was transformed with this plasmid. A number of transformant colonies grew in the presence of cycloheximide, which indicated that CuGAP1-CuYAP1 is an effective selection marker. The transformant also showed higher resistance to other agents, including cadmium and fluconazole. The overexpression of CuYAP1 in S. cerevisiae also resulted in increased resistance to various types of drugs.     

Reconstitution of lactate proton symport activity in plasma membrane vesicles from the yeast Candida utilis

Lactic acid transport was studied in plasma membrane vesicles from the yeast Candida utilis IGC 3092 which were fused with liposomes containing cytochrome c oxidase. After the addition of an electron donor system, these hybrid membrane vesicles were able to generate a proton-motive force of about −150mV, inside alkaline and negative. In vesicles prepared from lactic acid-grown cells, the uptake of labelled lactic acid, at pH 6·2, under energized conditions, was expressed by a kinetics consistent with the involvement of a mediated transport system. This carrier exhibited a substrate specificity pattern identical to the one found for the lactate-proton symport in intact cells. The transport of labelled lactic acid was accumulative and strongly sensitive to the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, consistent with the involvement of the proton-motive force in acid uptake, hence with the presence of a proton symport for lactate. Dissipation of the transmembrane electric potential by valinomycin did not have a significant effect on lactate accumulation, whereas abolishing the transmembrane pH gradient (ΔpH) by nigericin prevented the accumulation and led to a rapid efflux of the accumulated acid. The data support that the ΔpH is the main component of the proton-motive force involved in the transport of the acid and its accumulation. The lactate-proton symport stoichiometry was 1:1, being independent of the pH. Vesicles prepared from glucose-grown cells did not display the capacity to transport and accumulate lactate. However, activity for the carrier was also reconstituted in vesicles obtained from glucose-grown cells after incubation in buffer containing lactic acid. These results were consistent with those obtained in intact cells, which demonstrated that the lactate-proton symport of the yeast C. utilis is inducible.     

Isolation and structural analysis of efficient autonomously replicating sequences (ARSs) of the yeast Candida utilis

The industrially important yeast Candida utilis is widely used in production of food and medical materials, but its host-vector system has not been well developed. We screened for compact and efficient ARSs to construct practically useful vectors. The C. utilis strain AHU3053 was found to be efficiently transformed by the conventional lithium acetate method and was used as the host. The C. utilis IAM4264 genomic library was constructed by inserting the partial Sau3AI digests in pRI51, which has a kanMX gene expressible in C. utilis. By examining 98 C. utilis G418-resistant transformants, five plasmids had the highest ARS activity. By trimming of the inserts, the 1490 and 552 bp fragments with transformation activity of over 10(3)/microg DNA were obtained from ARS3 and ARS4, respectively. Although several sequences identical to S. cerevisiae ARS consensus sequences (ACSs) were found in ARS3 and ARS4, our deletion analysis indicated that these were not essential for the activity. Because the minimal functional ARS fragment was also several-fold larger than that of S. cerevisiae, the C. utilis ARSs have some unique characteristics resembling the Sz. pombe ARSs. These ARSs were functional in other C. utilis strains tested and useful for constructing practical vectors.     

两阶段pH控制方式提高富硒产朊假丝酵母的性能

为了提高富硒产朊假丝酵母的性能,本文在摇瓶培养和分批培养的基础上,考察了不同pH控制方式对流加培养条件下富硒产朊假丝酵母细胞生长、谷胱甘肽（GSH）合成和有机硒转化的影响,结果发现：在12 h将pH从3.5切换至5.5（pH 3.5→5.5）的两阶段pH控制方式最有利于富硒产朊假丝酵母胞内GSH和有机硒含量的提高。在pH 3.5→5.5条件下,富硒产朊假丝酵母胞内GSH含量、有机硒含量和有机硒转化率分别达到最大值13.09 mg/g、1.88 mg/g和94.69%。通过对酵母胞内GSH合成关键酶活性、氧化还原酶活性和能量代谢物质ATP水平进行测定,发现pH 3.5→5.5两阶段pH控制方式提高了富硒酵母胞内过氧化氢酶活性,降低了丙二醛含量,为酵母进行有机硒富集与转化以及GSH合成与积累提供了合适的氧化还原环境,并最终提高了富硒酵母的性能。     

弱酸胁迫提升富硒产朊假丝酵母性能及其作用机理

以一株耐亚硒酸钠的产朊假丝酵母为研究对象,考察了该菌株在不同p H环境下的富硒和谷胱甘肽（GSH）合成能力及抗氧化性能。结果表明,在弱酸胁迫（p H3.5）条件下,富硒产朊假丝酵母胞内有机硒和GSH含量均达到最高水平,分别为1.08 mg/g和18.48 mg/g。通过对酵母胞内γ-谷氨酰半胱氨酸合成酶、过氧化氢酶、超氧化物歧化酶的活性以及丙二醛含量进行测定,发现弱酸胁迫不仅有利于增强GSH的合成能力,也有助于提高酵母细胞的抗氧化能力。该研究结果为发酵法制备高性能富硒产朊假丝酵母提供了可行的技术参考。      

Isolation and sequence of the MIG1 homologue from the yeast Candida utilis

The Mig1p repressor from the food yeast Candida utilis has been isolated using a homologous PCR hybridization probe. This probe was amplified with two sets of degenerate primers designed on the basis of highly conserved motifs in the DNA-binding region (zinc-finger domain) from yeast Mig1p and fungi CreA repressors. The cloned gene was sequenced and found to encode a polypeptide of 345 amino acids which shows significant identity with other yeast and fungus repressors in the DNA-binding domain and also with the yeast Mig1 proteins in the C-terminal region (effector domain). The MIG1 repressor gene from C. utilis was able to complement functionally the mig1 mutation of S. cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession No. AJ277830.     

Transient responses of Candida utilis to oxygen limitation: Regulation of the Kluyver effect for maltose

The facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar-limited growth to oxygen-limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose-grown cultures to oxygen limitation was very similar to that of glucose-grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D-lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose- and glucose-grown cultures diverged: ethanol disappeared from the maltose-grown cultures, whereas fermentation continued in steady-state, oxygen-limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen-limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.     

A comparative study on the transport of L(-)malic acid and other short-chain carboxylic acids in the yeast Candida utilis: Evidence for a general organic acid permease

Cells of the yeast Candida utilis grown in medium with short-chain mono-, di- or tricarboxylic acids transported L(-)malic acid by two transport systems at pH 3·0. Results indicate that probably a proton symport for the ionized form of the acid and a facilitated diffusion for the undissociated form were present. Dicarboxylic acids such as succinic, fumaric, oxaloacetic and α-ketoglutaric acids were competitive inhibitors of the malic acid for the high-affinity system, suggesting that these acids used the same transport system. In turn, competitive inhibition uptake studies of labelled carboxylic acid in the low-affinity range indicated that this system was non-specific and able to accept not only carboxylic (mono-, di- or tri-) acids but also some amino acids. Additionally, under the same growth conditions, C. utilis produced two mediated transport systems for lactic acid: a proton symport for the anionic form which appeared to be a common monocarboxylate carrier and a facilitated diffusion system for the undissociated acid displaying a substrate specificity similar to that observed for the low-affinity dicarboxylic acid transport. The mediated carboxylic acid transport systems were inducible and subjected to repression by glucose. In glucose-grown cells the undissociated dicarboxylic acids entered the cells slowly by simple diffusion. Repressed glucose-grown cells were only able to produce both transport systems if an inducer, at low concentration (0·5%, w/v), was present during starvation in buffer. This process was inhibited by the presence of cycloheximide indicating that induction requires de novo protein synthesis. If a higher acid concentration was used, only the low-affinity transport system was detectable, showing that the high-affinity system was also repressed by high concentrations of the inducer.