Molecular Cloning and Characterization of Fruit Softening Related Gene β-Mannanase from Banana Fruit

A 1 250 bp cDNA fragment encoding β-mannanase, named MaMAN, was cloned from banana （Musa spp cv. Baxi） fruit using degenerate primers designed with reference to the conserved nucleic acid sequences of known β-mannanase genes by RT-PCR. Sequence analysis showed that MaMAN cDNA encompassed a 1 085 bp open-reading frame （ORF）, encoding a predicted polypeptide of 395 amino acids. Alignment of the deduced amino acid sequence of MaMAN and other putative β-mannanases showed that MaMAN has an identity of 86, 70, 69, 54, and 57%, respectively, to β-mannanases from tomato, lettuce, arabidopsis, carrot and oryza sativa. The catalytic residues： Asn203, Glu204, Glu318 and the active site residues： Arg86, His277, Tyr279, and Trp360, which were strictly conserved in the glycoside hydrolase family 5 to which all β-mannanases belonged, were found in MaMAN. Semi-quantitative RT-PCR revealed that the level of MaMAN transcript in the pulp increased during banana fruit ripening, suggesting that MaMAN was likely to be involved highly in banana fruit softening.

Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.

Development of two methods for rapid screening of recombinant Pichia pastoris strains with high-level expression of β-mannanase

The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.

Improving Semi-Dried Brown Rice Noodle Quality via Mixed Fermentation of Lactobacillus and Yeast

To address the coarse texture and poor cooking quality of brown rice flour,we employed fermentation using lactobacillus and yeast in varying proportions.The fermented flour from early indica rice Pear 13 was then processed into semi-dried brown rice noodles.

Effect of β-Glucan (Angel Yeast) Compared to a Placebo on Cold and Flu Incidence and Symptoms in an Adult Population—A Double Blind, Randomised Controlled Trial

Background: 1-3, 1-6 β-glucan derived from Baker’s yeast (Saccharomyces cerevisiae) has been widely studied for its immune stimulatory capabilities and safety. Previous studies found β-glucan to have efficacy at reducing incidence of URTIs as well as being a low risk for negative side effects. The current study aimed to examine the effects of yeast β-glucan (Angel Yeast) on cold and flu incidences and symptoms in healthy adults. Methods: Two hundred and thirty-one males and females aged 18 to 65 years old supplemented with either β-glucan or a placebo for 3-months. Participants completed a general health questionnaire every 4 weeks and in addition, if participants experienced any cold or flu symptoms, these were recorded daily (along with severity) until resolved or up to 2 weeks. Results: Supplementation with β-glucan reduced the self-reported severity of sore throats and improved sleep quality compared to the placebo group. Conclusions: Yeast β-glucan supplementation appears to be able to help reduce certain symptoms experienced during a cold or flu episode and is safe and well tolerated.

Mathematical Modeling of Cell Polarity Establishment of Budding Yeast

The budding yeast Saccharomyces cerevisiae is a powerful model system for studying the cell polarity establishment.The cell polarization process is regulated by signaling molecules,which are initially distributed in the cytoplasm and then recruited to a proper location on the cell membrane in response to spatial cues or spontaneously.Polarization of these signaling molecules involves complex regulation,so the mathematical models become a useful tool to investigate the mechanism behind the process.In this review,we discuss how mathematical modeling has shed light on different regulations in the cell polarization.We also propose future applications for the mathematical modeling of cell polarization and morphogenesis.

Influence of nitrogen status on fermentation performances of non-Saccharomyces yeasts:a review

Nitrogen,one of the most crucial nutrients present in grapes and musts,plays a key role in yeast activities during alcoholic fermentation.Such influences are imposed on yeast growth and fermentation performances including the formation of secondary metabolites.Saccharomyces cerevisiae,the main yeast responsible for fermentation,has been studied extensively regarding nitrogen impacts.On the other hand,a similar study for non-Saccharomyces yeasts,whose contributions to winemaking have gradually been acknowledged,remains to be fully explored,with a few studies being reported.This review starts by discussing nitrogen impacts on non-Saccharomyces yeast growth and fermentation kinetics in different case scenarios,then proceeds to summarize the nitrogen preferences of individual yeast strains with regulation mechanisms elucidated by recent studies.Detailed discussions on the influences on the production of volatile compounds and proposed pathways therein are made,followed by future work suggested as the final section.In summarizing the nitrogen impacts on non-Saccharomyces yeasts throughout alcoholic fermentation,this review will be helpful in obtaining a more comprehensive view on these non-conventional wine yeasts in terms of nutrient requirements and corresponding volatile production.Research gaps will therefore be elucidated for future research.

Impact of Storage Temperature on Microbial Diversity and Probiotic Effect of Liquid Brewers’ Yeast

Using probiotics as animal feed additives instead of antibiotics is gaining momentum to avert adverse negative effects on human health. Liquid brewers yeast (LBY) is an industrial by-product containing probiotic microorganisms and is also used as a protein supplement for dairy animals. Nevertheless, value chain actors lack of appropriate handling practices compromises the by-products quality and safety. This study aimed to determine the effect of variation in temperature on microbial diversity and probiotic effects during the storage time of LBY sampled from distributors and farmers from Githunguri sub-county of Kenya. The samples were stored at 20C, 25C and 30C, then tested on 0, 5, 10, 15 and 20 days. The studys parameters involved determining the pH levels, lactic acid bacteria (LAB), total coliform count (TCC), mould, and yeast in LBY. The rate (k) of the reaction kinetics model was used to extrapolate the expected probiotic shelf life. The LAB and yeast populations were reduced in a first-order reaction at all storage temperatures. The rate of reduction in the numbers of LAB reduced with an increase in temperature (k = 0.019 and 0.023) at 20C and 30C, respectively. Yeasts highest rate of growth reduction was 25C (k = 0.009) and least at 30C (k = 0.043). The minimum effective concentration for probiotics of 106 CFU/mL needed to observe the beneficial physiological impact on farm animals was achieved between 34.9 and 35.5 days at the tested storage temperatures. The study provides insight into the unexploited low-cost probiotic potential of LBY in dairy production. Conversely, handling practices and environmental microbial contamination along the value chain can compromise product quality and safety. There is a need to advocate its use in dairy for improved productivity and sensitize farmers to appropriate hygienic measures along the LBY value chain.

Microbiological Quality of “Rabilé”, a Yeast Used for Fermentation of Dolo, a Local Beer in Dédougou, Burkina Faso

Sorghum beer or dolo is part of the eating habits of part of the population of Dédougou because of its low price compared with industrial beers. Its production is an ancestral tradition that uses traditional equipment and gives dolo organoleptic properties that are not found in industrial beers. The production process involves several stages, including fermentation, which itself comprises natural lactic fermentation followed by alcoholic fermentation using traditional yeasts, which are not controlled in any way. The general aim of this study is to assess the microbiological quality of these fermentative yeasts in the town of Dédougou, in order to contribute to the health safety of the population and the promotion of these local beers. Twenty samples of fermenting yeast were analyzed according to ISO standards, to isolate enterobacteria, total and faecal coliforms according to standard procedures for isolating these micro-organisms. The isolated strains were identified using the API20E gallery. Microbiological analyses revealed the presence of 51.17% enterobacteria, 45.38% total coliforms and 3.45% thermotolerant coliforms. We counted 40% Escherichia coli, 20% Enterobacter cloacae, 20% Klebsiella pneumoniae and 20% Klebsiella spp. All the strains detected are capable of surviving in hostile conditions and could harm the quality of the dolo, consumer health and cause real collective food poisoning in the town of Dédougou. This enabled us to assess the microbial quality of these yeasts and to propose more suitable measures for producing and preserving dolo under hygienic conditions to protect consumer health.

Immobilization techniques for beverage production using yeast cell systems: challenges, types, and future perspectives - a mini review

Yeast immobilization is a process of physical entrapment of yeast cells using different techniques while maintaining their biological activity.Continuous fermentation systems have significant advantages over conventional methods.Research highlights that immobilized yeast cell systems have several benefits as compared to free yeast cells.The immobilized yeast cell systems improve fermentation rates,especially when paired with continuous fermentation and appropriate immobilization techniques.Understanding various immobilization techniques,continuous fermentation processes,yeast metabolic activity related to beverage flavor production,and bioreactor designs is vital for optimizing the use of immobilized yeast cells systems on industrial scale.This review provides an overview of recent basic research on immobilized yeast cell systems,with a focus on continuous beverage fermentation.In this study,different reactor configurations and immobilization techniques are explored.The study focus on the impacts of immobilization on the yeast cells,and discuss the recent advancements in these techniques.The review concludes with a discussion on the practical applications of immobilized yeast cells and continuous fermentation in beverage production.

以分子生物学鉴定结果为“金标准”评估Rapid ID Yeast Plus及API20C AUX对酵母样真菌的鉴定效能

目的以分子生物学方法为"金标准"对两种商品化酵母样真菌鉴定产品Rapid ID Yeast Plus(简称RapID YST)及API20C AUX(简称API20C)的鉴定效能进行评估。方法从2010年中国医院侵袭性真菌感染监测网菌株库中筛选酵母样真菌25种,共计194株。其中,包含临床最常见的5种酵母样真菌(白念珠菌、热带念珠菌、光滑念珠菌、近平滑念珠菌、新生隐球菌)共130株,占研究总菌株数的67.0%。所有菌株已经过分子生物学方法准确鉴定至种水平。菌株复苏分纯后,严格按照产品操作指南,平行进行RapID YST和API20C鉴定。结果所研究菌株中,有181株(18种)在RapID YST鉴定菌种数据库中,所有在库菌株种及复合体鉴定正确率为87.8%(159/181)。相比,API鉴定菌种库包含菌株174株(18种),在库菌株种及复合体鉴定正确率为92.0%(160/174)。RapID YST与API20C对于5种临床常见的酵母样真菌的种鉴定正确率分别为93.1%和97.1%。对于库外菌株,RapID YST的鉴定错误率分别为23.1%(3/13),相比API20C的鉴定错误率为60.0%(12/20)。综合此次评估结果,二者对酵母样真菌的鉴定效能无显著差异(McNemar检验,P>0.05)。结论两种商品化产品对酵母样真菌的鉴定效能基本一致;相较而言,RapID YST在操作便捷性、检测时间方面具有较大优势。

Yeast基因下游二级结构与多聚腺苷作用信号

形成真核生物mRNA 3′末端的多聚腺苷 (poly (A) )作用涉及前体mRNA下游的三个元件 :效率元件(EE)、定位元件 (PE)以及实际的剪切和 poly (A)作用位点 ,实验研究提出了一些EE和PE的碱基序列组成 .对 180个Yeast基因下游 (终止密码子后 2 0 0个碱基 )二级结构进行的详细分析显示 ,约 86 %的EE、 89%的PE与二级结构中碱基非配对的环 (发夹环、膨胀环、内环或多分支环 )区或连接单链区有关 .这个结果提示 ,反式因子对EE和PE的识别和作用在一定程度上有赖于EE和PE的二级结构特征 .

Yeast基因组编码区特征参数的研究

以碱基成分偏移量D值[1]为基本参数定义参数d ,以d为Yeast编码区的特征参数 ,对Yeast的第1、2、3类ORF(openreadingframe)进行了统计 ,得到d的特征参数区间。并且 ,以此区间为标准对Yeast的6类ORF ,以及5′帽、3′尾、内含子、组分随机序列等非编码序列进行了检验。结果表明 ,用d作编码区的特征参数是可行的 ,它可以很好地区分编码序列和非编码序列。另外 ,又讨论了参数d与基因表达水平 (用CAI值来衡量 )的关系。发现 ,参数d与基因表达水平成很好的正相关关系 ;发现密码子的第1位点和第2位点的某些碱基分布与基因表达水平有关。

Detection for Transcriptional Activity of Alternaria Tenuissim Protein Elicitor in Yeast Two-hybrid System

The peaT1 gene fragment was amplified from pGEM-6p-l-peaT1 by PCR, and recovered target gene was cloned into pLexA vector. After digestion and sequencing, the bait vector pLexA-peaT1 was transformed into yeast strain EGY48 [p8op-lacZ] by PEG/LiAC, and the transcriptional activity of bait vector was detected. The results showed that recombinant bait plasmid pLexA-PEMG1 was constructed, for the two bands of recombinant bait plasmid in agarose gel eleetrophoresis were expected after digesting by restriction endonuclease EcoR I and Xho I. Therefore, the recombinant bait plasmid could be used in yeast two-hybrid system to screen a cDNA library.

The Self-activated Experimental of T_(1083) Substitution Mutation Vector pGBKT7-TS in Yeast

[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.

Screening of Extracellular Binding Proteins of Rice Receptor-like Kinase CR4 by the Yeast Two-hybrid

[Objective] The research aimed to find the extracellular binding proteins of CR4.[Method] The extracellular domain of OsCR4 was as the bait protein,and the yeast two-hybrid was used to screen cDNA library of seedling which was cultivated 14 d.[Result] A lot of proteins which included a peroxide B（D26484）,a methionine thioredoxin reductase（ABF96078）and an unknown function protein were gained.[Conclusion] It provided the theory basis for studying the signal transduction mechanism of CR4.

RapIDYeast Plus系统在鉴定常见酵母中的临床应用

目的：探讨RapIDYeast Plus（RYP）系统在鉴定临床常见酵母中的作用。方法：将所试菌株在沙氏培养基上传代2次，然后30℃孵育24-48h后，用RYP系统进行试验。结果：150株临床分离株中有139株被正确鉴定到种的水平；有8株得到了具有2种或2种以上可能性的鉴定结果，而在使用了附加试验后都得出了正确的鉴定结果；有3株鉴定错误。RYP系统在不用附加试验的情况下鉴定的准确率达到92％，采用附加试验后鉴定的准确率达到98％。结论：RYP系统适用于临床微生物实验室的常规鉴定，在不需要特殊仪器的条件下，就能准确地鉴定临床上40多种酵母菌和酵母样菌。

黄萎病菌诱导下陆地棉酵母双杂交文库的构建及GhMAPKKK2互作蛋白的筛选

为了探究棉花响应黄萎病菌侵染的分子机制并筛选GhMAPKKK2的互作蛋白,以陆地棉XLZ35为材料,基于Gateway克隆技术构建了棉花黄萎病菌诱导的陆地棉酵母双杂交文库,其中初级文库和次级文库的库容分别为1.2×10^(7)和2.0×10^(7)CFU,插入片段重组率均为100%且平均长度大于1000 bp。次级文库质粒浓度为953 ng·μL^(-1),符合构建酵母文库的标准,可用于酵母双杂交筛选互作蛋白。构建了pGBKT7-GhMAPKKK2表达载体,并验证了该载体对酵母细胞无毒性且不存在自激活活性。以GhMAPKKK2为诱饵蛋白,利用酵母文库初步筛选,获得81个阳性互作蛋白,经测序对比选取6个候选蛋白,通过酵母双杂交及双分子荧光互补试验(BiFC)验证,明确了候选蛋白GhFLA与GhMAPKKK2存在互作关系。本研究结果为解析GhMAPKKK2调控棉花抗黄萎病反应分子机制及调控网络提供了重要参考。

生香酵母Yeast-1产香物质的GC-MS分析

从洋河大曲中成功筛选到一株生香酵母 Yeast-1。采用 GC-MS 联用技术，对该株酵母葡萄糖发酵产生的香气成分进行定性分析。结果表明：发酵产物中的风味组分多达 27 种，包含醇类（4 种）、酮类（2 种）、醛类（2 种）、有机酸类（4 种）、酯类（4 种）等典型的香味成分；此外还有一些特有的生理活性组分，如 3 种吡咯并吡嗪酮类、4 种哌嗪二酮类，这些发酵代谢产物也具有特殊的香气。所有风味组分独特优雅的香气将会赋予白酒独特的风味香型。

Physiologicalandbiochemicalcharacteristicsofactivedryyeast

建立一种酿酒活性干酵母生理活性的简易评价方法，比较并筛选一株适合以木薯粉水解液发酵发酵生产燃料酒精的活性干酵母。优化2株酿酒活性干酵母的活化条件、生长温度和生长pH值，以木薯粉水解液为发酵液，在最适生长条件下比较两株酵母的生长曲线、发酵强度、耐糖能力、耐温能力和耐酒精能力。选择酿酒活性干酵母Ⅰ作为木薯粉水解液发酵酵母，该酵母在前36h发酵强度高于1g／（L．h），36h）后发酵强度迅速下降；耐糖能力为20％，耐受温度是55℃以及耐酒精浓度是7％。