如何控制啤酒酵母质量的稳定

啤酒跟其他产品一样,一旦被广大消费者认可、推崇,就被确认为品牌啤酒。每个品牌啤酒都有其独特的风味,如何保证其良好的啤酒风味稳定性及一致性是应该关注的问题。而良好的风味来自优良酵母的发酵,只有保证酵母质量的稳定才能保证啤酒风味的稳定性和一致性,我们认为应从以下几方面来控制酵母质量的稳定。

黏质红酵母的鉴定及链格孢菌抑菌机制的研究

从柿子椒中分离得到一株具有抑菌效果的革兰氏阳性红酵母属菌株,经16SrDNA序列分析,该菌株鉴定为黏质红酵母。对该菌株的菌液乙酸乙酯提取物和75%乙醇沉淀物的抑菌机制进行研究,结果表明:其75%乙醇沉淀物和乙酸乙酯提取物对茄科链格孢菌产孢、萌发和菌丝的生长等方面都具有较好的抑菌效果,将其应用于食品(辣椒、番茄)防腐上也有一定的效果,乙酸乙酯提取液浓度为100 mg/mL时,辣椒腐烂率下降了51.879%,番茄腐烂率下降了58.47%,75%乙醇沉淀提取液浓度为100 mg/mL时,辣椒腐烂率下降了47.5%,番茄腐烂率下降了50.47%。strain-39菌液75%乙醇沉淀物和乙酸乙酯提取物经过不同酸碱和温度处理后,抑菌效果存在差异,紫外诱变处理后对链格孢菌抑菌效果基本没有影响。

酵母泥的检测和回收

1.前言:酵母的培养、发酵和回收利用是啤酒生产重要的一环,它对啤酒的风味稳定性和非生物稳定性都有着非常重要的作用。2.酵母泥的外观2.1 观察酵母泥是否洁白,若颜色发暗,说明麦汁过滤质量差或冷、热凝固物的排放不彻底。2.2 观察酵母泥的稀稠,一般情况下,排出的中间酵母应较稠,若较稀,可能污染杂菌。

Physiological functions of Atg6/Beclin 1： a unique autophagy-related protein

The most striking morphological feature of eukaryotic cells is the presence of various membrane-enclosed compartments. These compartments, including organelles and transient transport intermediates, are not static. Rather, dynamic exchange of proteins and membrane is needed to maintain cellular homeostasis. One of the most dramatic examples of membrane mobilization is seen during the process ofmacroautophagy. Macroautophagy is the primary cellular pathway for degradation of long-lived proteins and organelles. In response to environmental cues, such as starvation or other types of stress, the cell produces a unique membrane structure, the phagophore. The phagophore sequesters cytoplasm as it forms a double-membrane cytosolic vesicle, an autophagosome. Upon completion, the autophagosome fuses with a lysosome or a vacuole in yeast, which delivers hydrolases that break down the inner autophagosome membrane along with its cargo, and the resulting macromolecules are released back into the cytosol for reuse. Autophagy is therefore a recycling process, allowing cells to survive periods of nutrient limitation; however, it has a wider physiological role, participating in development and aging, and also in protection against pathogen invasion, cancer and certain neurodegenerative diseases. In many cases, the role ofautophagy is identified through studies of an autophagy-related protein, Atg6/Beclin 1. This protein is part of a lipid kinase complex, and recent studies suggest that it plays a central role in coordinating the cytoprotective function ofautophagy and in opposing the cellular death process of apoptosis. Here, we summarize our current knowledge ofAtg6/Beclin 1 in different model organisms and its unique function in the cell.

Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid

AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.

Marine Yeasts and Their Applications in Mariculture

The terrestrial yeasts have been receiving great attention in science and industry for over one hundred years because they can produce many kinds of bioactive substances. However, little is known about the bioactive substances of marine yeasts. In recent years, it has been found that marine yeasts have wide applications in mariculture and other fields. Therefore,marine yeasts, the bioactive substances from them and the applications of marine yeasts themselves and the bioactive substances they produced are reviewed in this paper.

Improvement of chromium biosorption through protoplast electrofusion between Candida tropicalis and Candida lipolytica

Protoplasts from Candida tropicalis and Candida lipolytica were fused under an optimized electrofusion （electrical pulse strength 6 kV/cm, pulse duration time 40μs and pulse times 5） and then regenerated on YEPD media for achieving new genotypes with higher chromium loading capacity. A target fusant RHJ-004 was screened out by its chromium resistance and chromium-sorbing capacity tests for further research. The comparative study of applicability shows that the fusant has better performance than its parent strains in respect of solution pH, biomass concentration and chromium loading capacity. Especially for treating low concentration Cr（VI） （〈20 mg/L）, above 80% chromium is sequestered from the aqueous phase at pH 1-9. Atomic force microscopy （AFM） visualizes the distribution of chromium on the binding sites of the cells, suggesting that the altered surface structure and intracellular constitutes of the fusant associate with its increased biosorption capacity. The rapid biosorption processes of chromium foUow the Langmuir model well.

Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1

AIM： To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells （PBMCs）.METHODS： HBcAg region was amplified by polymerase chain reaction （PCR） and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH109 yeast strains （Western blot analysis）, yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His- Ade） （QDO） and synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade） （TDO）. The second screening was performed with the LacZ report gene （ yeast cells were grown in QDO medium containing X-a-gal）. The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods.RESULTS： Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 （3 colones）, eukaryotic translation elongation factor 2 （2 colones）, acetyl-coenzyme A synthetase 3 （1 colone）, DNA polymerase gamma （1 colone）, putative translation initiation factor （1 colone）, chemokine （C-C motif） receptor 5 （1 colone）, mitochondrial ribosomal protein L41 （1 colone）, kyot binding protein genes （1 colone）, RanBPM （1 colone）, HBeAg-binding protein 3 （1 colone）, programmed cell death 2 （1 colone）. Four new genes with unknown function were identified.CONCLUSION： Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.

Mapping the human protein interactome

Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.

Effect of Low-pressure Carbonation on the Heat Inactivation and Cytoplasmic Acidification of Saccharomyces cerevisiae

Effect of low-pressure carbonation （LPC） on heat inactivation of Saccharomyces cerevisiae was investigated. The cell suspension was carbonated at 1 MPa and 4℃ for 15 min and subsequently heated from 51 to 61 ℃ and 5 s to 5 min （heating with LPC）. As a control experiment, cell suspension was heat-treated under atmospheric pressure without LPC （heating）. The inactivation ratio of heating at 53℃ and 55℃ for l rain with LPC was approximately 1 log order higher than heating alone. Extending heating time to 5 min did not widen the difference in the inactivation ratio between heating with LPC and heating alone at both heating temperatures. At 57℃, the difference in inactivation ratio increased from 1 to 2.5 log order with extending treatment time from 5 to 15 s. The results suggested that the enhanced inactivation effect by LPC was obtained at the higher temperature with short time treatment than the lower temperature with longer time treatment. Under fluorescence microscope observation of LPC-treated cell stained with LysoSensor probe, it seemed that LPC was hardly able to acidify the cytoplasm ofS. cerevisiae. It is considered that the ability orS. cerevisiae ceils to keep their cytoplasmic pH during LPC resulted in the inferior increase in heat inactivation ratio by LPC as compared with bacteria in the previous studies.

Influences of different substrates on simulated lignite biogas production

Using lignite samples, selected from Zhaotong basin, Yunnan province, China, as the parent source, simulating experiments of lignite biogas were conducted with 0.1% methanol, 5 mg/L yeast extract and 0.2 mol/L sodium acetate solutions as the exogenous substance respectively. Variation characteristics of gas production, gas composition, VFA content and activity of coenzyme 1：42o in the simulated process were analyzed to discuss the influence of different substrates on lignite biogas generation. The results show that 0.1% methanol and 5 mg/L yeast extract solutions increase VFA contents in the biogas generation system （p 〈 0.05） and inhibit coenzyme F420 and methanogen activities significantly, so they decrease both gas amounts （p 〈 0.05） and CH4 contents （p 〈 0.05）. 0.2 mol/L sodium acetate solution activates coenzyme F42o and methanogen activities and improves the efficiency of enzymatic reaction, so the gas quantity （p 〈 0.05） and the CH4 content （p 〈 0.01） increase significantly. Therefore, sodium acetate can be one kind of good exogenous substance for the generation of lignite biogenic gas.

Construction and Expression of Serratia Marcescens ECU1010 Lipase （lipA） in Pichia Pastoris

Serratia marcescens ECUI010, as an extracellular lipase and a significant catalyst, which had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications, was previously mostly expressed intracellularly as inclusion bodies in Escherichia coli. Denaturation and renaturation of inclusion bodies had a significant influence on the lipase activity. Thereupon, our present work described the secretion expression of gene encoding of this lipase in Pichia pastoris GS 115 and characterization of the recombinant enzyme. Firstly, the obtained lipA gene fragment was introduced into P. pastoris expression vector pPIC9K, the lipA gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOXI promoter, and the recombinant plasmid pPIC9K-lipA was transformed into P. pastoris strain GS115 by electroporation, and this recombinant P. pastoris were identified by PCR. Then lipase activity was detected on BMMY-tributyrin and olive oil agar plates containing Rhodamine B. Transformants with lipase activity by screening were induced 6 days by methanol, one band of 77 kDa protein could be observed by 10% SDS-PAGE. p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The pH and temperature optimum of lipase were pH 8.5 and 40℃ respectively. The stability and effects of metal ions and other reagents were also determined. However, the recombinant fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 4 U of lipase activity per milliliter of culture media supernatant.

Selective Affinity Separation of Yeast Alcohol Dehydrogenase by Reverse Micelles with Unbound Triazine Dye

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol.L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH＞pI with electrostatic attraction between YADH and CTAB. The recovery of YADH activity and purification factor have been improved with addition of a small amount of affinity CB. The recovery of YADH activity obtained was ～99% and the purification factor was about 4.0-fold after one cycle of full forward and backward extraction. The low ionic strength in the initial aqueous phase might be responsible for the YADH transfer into the reversed micellar phase.

Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

AIM： To investigate the biological function of F protein by yeast two-hybrid system. METHODS： We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 （a type）. The transformed yeast AH109 was mated with yeast Y187 （α type） containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-HisAde） containing X-α-gal for selection and screening. After extracting and sequencing plasmids from positive （blue） colonies, we underwent sequence analysis by bioinformatics. RESULTS： Thirty-six colonies were selected and sequenced. Among them, 11 colonies were zymogen granule protein, 5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor I, 1 colony was vitronectin, and 2 colonies were new genes with unknown function. CONCLUSION： The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins. 2005 The WJG Press and Elsevier Inc. All rights reserved

Cloning and expression in Saccharomyces cerevisiae of chit2 gene from Beauveria bassiana

To study recycled trashes from shrimps and crabs in the sea through chitinase secreted by microorganisms,the chitinase gene chit2 was cloned and sequenced from Beauveria bassiana by the polymerase chain reaction(PCR),and was ligated into the yeast expression vector pYES2.The expression vector plasmid was transformed into Saccharomyces cerevisiae H158.Gene expression took place upon induction with 2% galactose.The measurement of enzyme activity shows that the expression production can be expressed in active forms and secreted to the medium.The enzyme activity approaches the peak of 0.63 U/mL when the culture time is 36 h.

Effect of Feeding Different Levels of Dietary Protein and Addition of Baker＇s Yeast （Saccharomyces cerevisiae） on Productive Parameters of Awassi Lambs

Forty eight individually fed Awassi male lambs were used in factorial experiment to investigate their responses to feeding concentrate diets containing three levels of dietary crude protein （CP）, each was offered without or with baker＇s yeast （SC） at rate of 0.5% （on dry matter （DM） basis）. Concentrates were offered at rate of 3% of live body weight with free choice of barley straw. Results revealed that higher （P 〈 0.05） digestible dry matter （DDM） and digestible organic matter （DOM） intakes were achieved due to feeding medium level of CP and to the addition of SC. Addition of SC improved （P 〈 0.05） gain, lambs fed medium and high levels gained higher （P 〈 0.05） than those fed the low level of dietary CP. Feed conversion ratio （FCR） based on DM and organic matter （OM） intakes was not significantly affected by level of dietary CP or addition of yeast. Even though, less amount of N required per unit of gain was achieved with low and medium as compared to high levels. Higher DM, nitrogen free extract （NFE） and hemicellulose （P 〈 0.05）, OM, CP, crude fiber （CF） and cellulose （P 〈 0.01） digestibilities were achieved in lambs fed the medium level of CP, whereas, no significant effect was observed on ether extract （EE）, neutral detergent fiber （NDF） and acid detergent fiber （ADF） digestibilities. Results also revealed that digestibility of almost all nutrients was improved with different extent due to addition of SC. Effect of interaction between levels of dietary CP and addition of SC referred to the preferability of addition of SC with medium level of dietary protein.

Corncobs as Substrate for Oleaginous Yeast-Pretreatment via Steam Explosion and Hydrolysis

Corn cobs are a promising lignocellulosic substrate for the production of biofuels like bioethanol via conventional yeast or biodiesel via oleaginous yeast. Pretreatment of the substrate is essential for further hydrolysis and fermentation steps. This study focused on the steam explosion method as pretreatment. Therefore, different steam explosion severities were evaluated. The content of glucan, xylan and Klason lignin was examined. Xylan degraded with increasing severity from 412.7 g·kg-1 （untreated） to a minimum of 127.3 g-kg1 dry matter （190 ℃/30 min）. Glucan concentrations increased from 315.1 g·kg1 （untreated） to a maximum of 371.6 g·kg-1 dry matter （200 ℃/20 min）. For soluble lignin, an increase could be observed at rising severity, from 145.3 g·kg-l （untreated） to a maximum of 214.9 g·kg-1 dry matter （190 ℃/30 min）. Furthermore, the mass recovery was calculated. At harsher pretreatment conditions, a significant mass loss was observed, estimated by the ash content in the recovered dry matter. The lowest recovery rate was observed for SF = 4.13 （190 ℃/30 min） with 68.39%. The produced inhibitors were evaluated.

Microbiological Quality of Dates in the North Center Region of Morocco

Date palm （Phoenix dactylifera L.） fruit is an important component in arid and semi-arid diet regions of the world. The present study pointed out microbial investigating of date palm collected from National wholesale market in Fez city in the center north of Morocco. The results reveal that samples dates studied are contaminated by bacteria such as TAMF （total aerobic mesophilic flora）, ASR （anaerobic sulfite reducing）, yeasts and molds. The percentage of yeasts isolated is 50% for Saccharomyces cervisae, 10% for Zygosaccharomyces fermentati, 20% for Hansenula anomala, 10% for Lodderomyces elongisporus and 10% for Kluyveromyces fragilis. The frequency of molds distribution is 51% for Aspergillus niger, 33% for Penicillium notatum and 16% for Rhizopus oryzae. Newman and keuls grouping test shows that fungal germs distribution is equal for yeast and molds in all samples analyzed. In the opposite bacterial grouping test shows greet difference between groups of bacteria isolated from date＇s samples.

Recombinant human heparin-binding neurite-promoting factor expressed with yeast stimulates neurites outgrowth

OBJECTIVES: Heparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro. We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro. METHODS: cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free. His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth. RESULTS: In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells. CONCLUSION: Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.