Antihypertensive peptides from skimmed milk hydrolysate digested by cell-free extract of Lactobacillus helveticus JCM1004

In the present experiments, the proteinase, aminopeptidase and x-prolyl-dipeptidyl aminopeptidase activitives were measured at 513 ± 11.9, 376 ± 9.3 and 23.6 ± 1.6 units per gramme of cell-free extract of Lactobacillus helveticus JCM1004. ACE-inhibitory activity of skimmed milk hydrolysate produced by cell-free extract of L. helveticus JCM1004 was determined, and the optimum pH and hydrolysis time for the production of ACE-inhibition substances from skimmed milk were 6.5–7.0 and 6–10 h, respectively. Peptides of Val-Pro-Pro and Ile-Pro-Pro with potent ACE inhibitor and antihypertensive activitives were purified from the hydrolysate by three step-reverse-phase high-performance liquid chromatography. The IC₅₀ values of Val-Pro-Pro and Ile-Pro-Pro were 9.13 ± 0.21 and 5.15 ± 0.17 μM, respectively. Significant decrease (p < 0.01) in SBP of SHR was measured after a single gastric intubation of Val-Pro-Pro or Ile-Pro-Pro at 8 or 4 h, respectively.     

Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae

The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post‐translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone‐specific proteases. Here, we employed various biochemical methods to report histone H3‐specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3‐clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of high pressure on the proteolytic activity and autolysis of yeast Saccharomyces cerevisiae

This work explores the effect of High Pressure (HP) treatment on the proteolytic activity and autolytic capacity of Saccharomyces cerevisiae suspensions, based on yeast extract production. Cellular suspensions were treated at 200–600 MPa for 0–120 min and the activity of vacuolar proteases was measured during autolysis. Moreover, the autolytic capacity of yeast was determined based on the physicochemical characteristics of the produced yeast extract. At pressures of 200 and 400 MPa, proteolytic activity was enhanced up to 160%, after 40 and 10 min of HP treatment, respectively, leading to significantly accelerated autolysis, in combination with cellular permeabilization achieved with HP treatment. However, at 600 MPa, despite the greater cell permeabilization, proteolytic enzymes were gradually inactivated (total inactivation after 15 min of HP treatment), leading to inhibition of autolysis. These results highlight HP as an effective process to enhance endogenous proteolytic activity and thus accelerate yeast extract production.     

Breeding of brewer's yeast by hybridization between a top-fermenting yeast Saccharomyces cerevisiae and a cryophilic yeast Saccharomyces bayanus

To improve the fermentability of a top-fermenting yeast at low-temperature, we performed hybridization trials between four top-fermenting Saccharomyces cerevisiae strains and a cryophilic yeast Saccharomyces bayanus YM84 with good fermentability at low-temperature. The hybrids selected using 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside were checked with pulsed-field gel electrophoresis and their brewing performance at the low-temperature of 10.5 degrees C was observed using small-scale (2 l) fermentation trials.     

Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

Autoselection systems allow the selection of a genetically engineered population independently of the growth medium composition. The structure of a Saccharomyces cerevisiae population transformed with an autoselection plasmid, in which a carbon-source-dependent modulation of the plasmid copy number occurs, was analysed. By means of flow cytometric procedures we tested the cell viability, dynamics of growth and heterologous protein production at single cell level. Such analyses allow the identification and the tracking of a specific cellular sub-population with a higher plasmid copy number which arises after the carbon source shift. The effects of the cellular plasmid distribution on the dynamics of growth are also discussed.     

香蕉酒嗜杀野生酿酒酵母的筛选

该研究采用从香蕉酒醪中初筛获得的嗜杀野生酿酒酵母进行热冲击处理和紫外诱变(灯管功率20W,波长254nm,照射距离为50cm);经2,3,5-氯化三苯基四氮唑(TTC)的显色筛选,获得了5株呈深红色菌落的突变株;通过麦芽汁发酵对比,选定一株优良菌株.该菌株在麦芽汁糖度为10°BX,温度为20℃的条件下发酵7d,发酵液最终的糖度为1.71g/L,最终pH值为3.8,可溶性固形物为2.86％,酒精体积分数为4.2％vol.     

Division of labour in the yeast: Saccharomyces cerevisiae

Division of labour between different specialized cell types is a central part of how we describe complexity in multicellular organisms. However, it is increasingly being recognized that division of labour also plays an important role in the lives of predominantly unicellular organisms. Saccharomyces cerevisiae displays several phenotypes that could be considered a division of labour, including quiescence, apoptosis and biofilm formation, but they have not been explicitly treated as such. We discuss each of these examples, using a definition of division of labour that involves phenotypic variation between cells within a population, cooperation between cells performing different tasks and maximization of the inclusive fitness of all cells involved. We then propose future research directions and possible experimental tests using S. cerevisiae as a model organism for understanding the genetic mechanisms and selective pressures that can lead to the evolution of the very first stages of a division of labour. Copyright © 2017 John Wiley & Sons, Ltd.     

啤酒废酵母双酶法生产酱油的工业化探讨

在实验基础上提出了以啤酒废酵母为主要原料,添加溶菌酶与麦芽根酶进行酵母溶解与RNA的降解等工艺方法生产酱油的途径。在设计基础上叙述了啤酒废酵母在生产酱油中的主要设备和经济分析。     

Peptides from the N-terminal end of bovine lactoferrin induce apoptosis in human leukemic (HL-60) cells

To determine the effects of the multifunctional iron-binding glycoprotein, lactoferrin (LF) and related compounds on the growth of leukemic cells, human myeloid leukemic cells (HL-60) were exposed to bovine lactoferrin (bLF) and proteolytic hydrolysates of bLF. Pepsin hydrolysates of bLF showed a greater growth suppressive effect than tryptic hydrolysates or mature bLF. Four peptides with proliferation inhibition activity were purified from pepsin hydrolysates by ion-exchange chromatography, reverse-phase HPLC, and gel-filtration. All peptides were from the N-terminal end, in a region where lactoferricin B (Lfcin B), an antibacterial peptide, is located. Among the four peptides, peptide 1 (pep1) was found to exhibit highest activity and corresponded to residues 17 to 38 of bLF, with a molecular weight of 2753.88. The IC50 value of this peptide was 6.3 micrograms/ml. Three other peptides were less active and corresponded to sequences 1 to 16 and 45 to 48, linked by disulfide-bridge (pep2, molecular mass of 2430.13), 1 to 15 and 45 to 46 linked by disulfide bridge (pep3, molecular mass of 2017,92) and from residues 1 to 13 (pep4, molecular mass of 1558.73). Cell proliferation inhibition activity of the peptides was thought to be due to induction of apoptosis, which was evaluated by DNA ladder formation, DNA fragmentation, enhanced expression of phosphatidyl serine, and morphological changes. The IC50 values of the three peptides were confirmed using synthetic peptides and were consistent with those of purified peptides.     

Induction of a heat-shock-type response in Saccharomyces cerevisiae following glucose limitation

The protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985). In this study, we used two-dimensional gel electrophoresis to characterize the protein events responsible for these alterations. We found that the induction of heat-shock proteins is one of the major events responsible for these changes. This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose-limited cells. It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells. The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at various stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides. The results showed in particular that the synthesis of most of the heat-shock proteins synthesized in glucose-limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted. Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion. It was thus concluded that the observed induction of heat-shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation.     

Systematic disruption of 456 ORFs in the yeast Saccharomyces cerevisiae

In the framework of the European Network for Functional Analysis (EUROFAN), five packages of 96 ORFs from chromosomes III, IV, VII, XIII, XIV and XV were subjected to systematic deletions in an isogenic derivative of strain S288c. Deletions were constructed in diploid and haploid strains. Two questionable ORFs overlapping with larger ORFs and seven TY ORFs were discarded. A total of 456 heterozygous and 385 homozygous deletant diploids were obtained. Sixty-nine deletions, 25 of which had never been published before, were lethal in haploid strains and 30 caused slow cellular growth.     

Differential chromosome control of ploidy in the yeast Saccharomyces cerevisiae

In Saccharomyces cerevisiae, aneuploidy is well tolerated and stable. We analysed whether the induced loss of a disomic chromosome favours endo-reduplication of the remaining chromosome or the cells prefer to retain the acquired euploidy. Chromosome VIII disomes and trisomes were tagged with GFP (green fluorescent protein), DsRed (red fluorescent protein) and BFP (blue fluorescent protein) integrated at the thr1 locus, using our newly designed STIK (specific targeted integration of kanamycin resistance-associated, non-selectable DNA) plasmid system. A knockout cassette for centromere 8 was constructed with the hygromycin-B marker, which was transformed into the strains. The transformants lost sensitivity to hygromycin, thereby indicating the event of centromere replacement. Quantitative PCR and Southern analysis were performed for chromosome VIII copy number determination by probing the markers located on both the right (ARG4 and THR1) and left (GUT1) arm whereas, for chromosome V, markers such as HIS1, located on right arm, and URA3, on left arm, were used. The loss of an extranumerary chromosome VIII in a disome and trisome leads to stable euploidy. Furthermore, in a wild-type diploid, deletion of a copy of chromosome VIII, leads to monosomy, and restoration of euploidy after 22 generations, by reduplication of chromosome VIII, and consequent loss of heterozygosis (LOH). However, chromosome V knockouts in chromosome VIII trisome, still showed LOH and duplication of chromosome V, with return to the original aneuploid condition. These results suggest that yeast cells could control the integrity of their genetic complement acting at the individual chromosome level.     

Genetic control of chromosome stability in the yeast Saccharomyces cerevisiae

We have identified four new genetic loci: CHL2 (on chromosome XII), CHL3 (on chromosome XII); CHL4 (on chromosome IV), and CHL5 (on chromosome IX), controlling mitotic transmission of yeast chromosomes. The frequency of loss of chromosomes is 10-100-fold higher in chl5, chl2, chl3 and chl4 mutants than observed in wild-type strains. The mutants also show unstable maintenance of artificial circular minichromosomes with various chromosomal replicators (ARS) and one of the centromeric loci (CEN3, CEN4, CEN5 or CEN6). The instability of minichromosomes in the chl5, chl2, and chl4 mutants is due to the loss of minichromosomes in mitosis (1:0 segregation). In the chl3 mutant the instability of artificial minichromosomes is due to nondisjunction (2:0 segregation). The CHL3 gene therefore appears to affect the segregation of chromosomes during cell division.     

Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae

The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. Copyright © 2014 John Wiley & Sons, Ltd.     

青蛤凝集素CSL对酿酒酵母细胞形态的影响

通过研究青蛤凝集素CSL与酵母细胞壁肽聚糖的相互作用及CSL与酿酒酵母细胞结合后其表面积的变化,分析CSL对酿酒酵母细胞作用的影响。结果表明,固相吸附测试中,CSL可与酵母细胞壁肽聚糖结合,两者的结合具有浓度依赖关系,该结合位点受甘露糖（D-Mannose）及N-乙酰-D-半乳糖胺（N-acetyl-D-galactosamine）的抑制。CSL与酿酒酵母细胞壁相互作用,导致了它的表面积发生了改变,这表明其细胞壁发生了变化。对照组与CSL添加组两组酵母菌内的Ca²⁺含量未发生明显变化。扫描电镜观察表明,对照组酵母细胞表面较圆润光滑,CSL添加组酵母细胞表面有较多赘附凸起。研究结果为CSL促进酵母发酵能力的机理提供基础数据。      

Fate of patulin in the presence of the yeast Saccharomyces cerevisiae

Patulin is known to become analytically non-detectable during the production of cider from contaminated apple juice. The fate of [¹⁴C]-labelled patulin during the alcoholic fermentation of apple juice was studied. Three commercial cider strains of Saccharomyces cerevisiae degraded patulin during active fermentative growth, but not when growing aerobically. The products of patulin degradation were more polar than patulin itself and remained in the clarified fermented cider. Patulin did not appear to bind to yeast cells or apple juice sediment in these model experiments. HPLC analysis of patulin-spiked fermentations showed the appearance of two major metabolites, one of which corresponded by both TLC and HPLC to E-ascladiol prepared by the chemical reduction of patulin using sodium borohydride. Using a diode array detector, both metabolites had a λ_max = 271nm, identical to that of ascladiol. Thenmr spectrum of a crude preparation of these metabolites showed signals corresponding to those of the E-ascladiol prepared chemically and a weaker set of signals corresponding to those reported in the literature for Z-ascladiol.     

Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation

In order to establish an efficient bioethanol production system from rice straw, a new strategy to ferment the mixture of glucose and xylose by a sequential application of Saccharomyces cerevisiae and Pichia stipitis was developed, in which heat inactivation of S. cerevisiae cells before addition of P. stipitis was employed. The results showed that heating at 50°C for 6h was sufficient to give high xylose fermentation efficiency. By application of the inactivation process, 85% of the theoretical yield was achieved in the fermentation of the synthetic medium. At the same time, the xylitol production was reduced by 42.4% of the control process. In the simultaneous saccharification and fermentation of the lime-pretreated and CO(2)-neutralized rice straw, the inactivation of S. cerevisiae cells enabled the full conversion of glucose and xylose within 80 h. Finally, 21.1g/l of ethanol was produced from 10% (w/w) of pretreated rice straw and the ethanol yield of rice straw reached 72.5% of the theoretical yield. This process is expected to be useful for the ethanol production from lignocellulosic materials in the regions where large-scale application of recombinant microorganisms was restricted.