Yeast Lysate Research Articles

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a 'hand-over-hand' mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding.

Autolyzed yeast and sodium butyrate supplemented alone to diets promoted improvements in performance, intestinal health and nutrient transporter in weaned piglets

This study investigated the effects of supplemental nucleotides, autolyzed yeast (Saccharomyces cerevisiae), and sodium butyrate in diets for nursery pigs on growth performance, diarrhea incidence, blood profile, intestinal morphology, mRNA expression of nutrient transporters, inflammatory markers, antioxidant profile, and tight junction proteins in the small intestine. One hundred eighty 21-day-old pigs (5.17 ± 0.57 kg) were assigned in a randomized block design to 1 of 4 dietary treatments: (1) CON: control, basal diet, (2) NUC: CON + nucleotides, (3) YSC: CON + lysed yeast S. cerevisiae, (4) ASB: CON + acidifier sodium butyrate. Pigs were fed for 24 days, phase 1 (21–32 days) and 2 (32–45 days). During phase 1, YSC and ASB improved average daily gain (ADG) and feed conversion (FC) compared with CON. At the overall period, ASB improved ADG and YSC improved FC compared with CON. The NUC diet did not affect growth performance. The ASB increased ileal villus height compared to CON. The YSC and ASB reduced the number of Peyer’s patches in the ileum compared with CON. The YSC increased mRNA expression of nutrient transporters (SMCT2, MCT1, and PepT1), tight junction proteins (OCL and ZO-1), antioxidants (GPX), and IL1-β in the jejunum compared with CON. The ASB increased mRNA expression of nutrient transporters (SGLT1 and MCT1), tight junction proteins (OCL and ZO-1), and antioxidants (GPX and SOD) compared with CON. In conclusion, autolyzed yeast and sodium butyrate promoted growth performance by improving the integrity of the intestinal barrier, the mRNA expression of nutrient transporters, and antioxidant enzymes in the jejunum of nursery pigs whereas supplementation of nucleotides did not show such effects.

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

Characterization of an antifungal β-1,3-glucanase from Ficus microcarpa latex and comparison of plant and bacterial β-1,3-glucanases for fungal cell wall β-glucan degradation.

Each β-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of β-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and β-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal β-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall β-glucan, it was compared with β-1,3-glucanase with different substrate specificities. We obtained recombinant β-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I β-1,3-glucanase) and CcGluA (GH64 β-1,3-glucanase).

Fining strategies for quercetin deposit prevention in sangiovese wines

A strategy aimed at decreasing the content of quercetin (Q) and quercetin glycosides (QG) was applied to prevent future quercetin precipitation in wines. Different oenological fining agents were tested: polyvinylpolypyrrolidone (PVPP), a vegetable protein (VP), four yeast lysate (YLs) and a polyfunctional product (MIX) obtained by mixing PVPP, vegetable protein and a yeast lysate selected among the four tested. For each fining agent the adsorption isotherms were obtained; based on Freundlich model, PVPP and MIX showed the best adsorption of Q and QG. The MIX determined also a contemporary lower adsorption of other phenolic compounds. The treatment carried out with MIX on the Sangiovese wines determined a decrease of Q (from −27 to −47% with respect to the initial values) and of QG (−17%) preserving wine anthocyanins.

Enzymatic hydrolysis of spent Saccharomyces cerevisiae derived from sago bioethanol fermentation

Spent Saccharomyces cerevisiae is a by-product of bioethanol fermentation. The spent yeast is abundant in valuable components which can be used for many applications. One of the ways to prepare yeast extract is through enzymatic hydrolysis which is by rupturing the yeast cell walls using exogenous enzymes under certain conditions that promote the leakage of intracellular compounds. To date, enzymatic hydrolysis of spent S. cerevisiae derived from the production of sago bioethanol is yet to be thoroughly explored. In the present study, we examine the feasibility of enzymatic hydrolysis of spent S. cerevisiae generated from sago bioethanol fermentation. The effect of two enzymes namely alcalase and cellulase and their concentrations (0.1-0.5% (v/v) on the release of protein and carbohydrate in the hydrolysate was also investigated. Additionally, the surface morphology of the hydrolysed yeast cells was observed using a Scanning electron microscope (SEM). Our results showed that the optimal concentration of alcalase and cellulase for enzymatic hydrolysis of spent S. cerevisiae was 0.4% (v/v) and 0.5% (v/v) respectively. In addition, cellulase was found to be more superior than alcalase with respect to the protein content in the hydrolysate. The enzymatic hydrolysis of spent yeast by alcalase and cellulase yielded improvements of 1.1 to 1.8-fold and 3.5 to 5.6-fold of protein and total carbohydrate concentration respectively in comparison to that achieved via autolysis. It was evident from the SEM analysis that there was a notable change in the surface morphology of the lysed yeast cells indicating the lysis of the yeast cells throughout the enzymatic hydrolysis. In summary, the current work provides useful insights into the strategies of valorising spent S. cerevisiae generated from sago bioethanol production. This will further help the development of value-added products from the waste, hence promoting a sustainable economy besides reducing the environmental impacts associated with the disposal of spent S. cerevisiae.

Use of a commercial feed supplement based on yeast products and microalgae with or without nucleotide addition in calves

The use of feed additives with antioxidant and immune response modulatory activity could be a useful strategy in suckling calves to reduce morbidity and mortality. This strategy is based on several feed additives tested for these purposes. The aim of the paper is the examination of a commercial feed additive for adult cows for use in calves, with and without nucleotide supplementation. Seventy-five Holstein Friesian male calves were divided in 3 groups, with each calf randomly assigned to a group according to birth order. All calves received 2 L of pooled colostrum within 2 h of birth. The commercial feed supplement group was orally administered with 5 g/head of Decosel (dried brewer's yeast lysate (Saccharomyces cerevisiae), brewer's yeast walls (Saccharomyces cerevisiae), diatoms, spirulina, barley flour, calcium carbonate; Agroteam srl, Torrimpietra, Italy) and the nucleotides + commercial feed supplement group was orally administered with 5 g/head of an additive containing 2.5 g of Decosel and 2.5 g of nucleotides once daily from birth to 25 d. The control group was orally administered 20 mL of fresh water/head once daily. Calves that received the supplement and the nucleotides showed lower rates of protein and metabolizable energy conversion, with longer villi and greater crypt depth in duodenum. Moreover, the commercial feed supplement alone increased antioxidant capacity [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and ferric-reducing antioxidant power] in plasma some activity of antioxidant liver enzymes, and peripheral blood mononuclear cell viability after in vitro concanavalin A and H2O2 stimuli. Dietary supplementation with a commercial feed supplement containing yeast products (yeast cell walls and hydrolyzed yeast) and microalgae enhanced the redox balance and gut morphology in calves, allowing calves to improve their immune response, increasing resistance to stress. Moreover, these beneficial effects were strongly potentiated when dietary nucleotides were added to the supplement.

Mining the nanotube-forming Bacillus amyloliquefaciens MR14M3 genome for determining anti-Candida auris and anti-Candida albicans potential by pathogenicity and comparative genomics analysis

There is a global health concern associated with the emergence of the multidrug-resistant (MDR) fungus Candida auris, which has significant mortality rates. Finding innovative and distinctive anti-Candida compounds is essential for treating infections caused by MDR C. auris. A bacterial strain with anti-Candida activity was isolated and identified using 16 S rRNA gene sequencing. The whole genome was sequenced to identify biosynthesis-related gene clusters. The pathogenicity and cytotoxicity of the isolate were analyzed in Candida and HFF-1 cell lines, respectively. This study set out to show that whole-genome sequencing, cytotoxicity testing, and pathogenicity analysis combined with genome mining and comparative genomics can successfully identify biosynthesis-related gene clusters in native bacterial isolates that encode antifungal natural compounds active against Candida albicans and C. auris. The native isolate MR14M3 has the ability to inhibit C. auris (zone of inhibition 25 mm) and C. albicans (zone of inhibition 25 mm). The 16 S rRNA gene sequence of MR14M3 aligned with Bacillus amyloliquefaciens with similarity (100%). Bacillus amyloliquefaciens MR14M3 establishes bridges of intercellular nanotubes (L 258.56 ± 35.83 nm; W 25.32 ± 6.09 nm) connecting neighboring cells. Candida cell size was reduced significantly, and crushed phenotypes were observed upon treatment with the defused metabolites of B. amyloliquefaciens MR14M3. Furthermore, the pathogenicity of B. amyloliquefaciens MR14M3 on Candida cells was observed through cell membrane disruption and lysed yeast cells. The whole-genome alignment of the MR14M3 genome (3981,643 bp) using 100 genes confirmed its affiliation with Bacillus amyloliquefaciens. Genome mining analysis revealed that MR14M3-coded secondary metabolites are involved in the biosynthesis of polyketides (PKs) and nonribosomal peptide synthases (NRPSs), including 11 biosynthesis-related gene clusters with one hundred percent similarity. Highly conserved biosynthesis-related gene clusters with anti-C. albicans and anti-C. auris potentials and cytotoxic-free activity of B. amyloliquefaciens MR14M3 proposes the utilization of Bacillus amyloliquefaciens MR14M3 as a biofactory for an anti-Candida auris and anti-C. albicans compound synthesizer.

Diverging olfactory sensitivities to yeast volatiles reflect resource partitioning of tephritids and drosophilids

As pests of fruits and vegetables, ovipositing tephritid fruit flies are infamous for their frugivory. Yet, adult tephritids have remained saprophytic in their feeding behavior, as they require decomposing, protein rich media for sexual maturation and oogenesis. Drosophilid fruit flies, in contrast, are saprophytic both during oviposition and feeding. Here we compared the sensory and behavioral responses of two tephritid (Bactrocera dorsalisandCeratitis capitata) and two drosophilid species (Drosophila melanogasterandDrosophila suzukii) to differentially aged cultures of the yeastSaccharomyces cerevisiae. We assessed convergence and divergence in the detection of and behavioral response to these attractive substrates, and how these might be linked to the roles of the substrates for the different taxa. The headspace shifted substantially as broth cultures transitioned from active (1-day) to inactive (8- and 15-days). Interestingly,Drosophilaflies were significantly attracted to actively fermenting 1-day old yeast cultures, whereas the preference shifted to older cultures for the tephritids.Bactrocera dorsalisflies preferred inactive, lysing cultures (8- and 15-days old). We identified compounds from the 1- to 8-days old broth cultures that elicited antennal responses in each species. Synthetic blends composed of antennally active compounds evoked similar behavioral responses as broth cultures. Similarly, the attractiveness of less attractive broth cultures (1- and 8-days old for drosophilids and tephritids, respectively) could be augmented by adding volatiles of the more attractive cultures. The results show that the volatile profiles of fermenting substrates evolve quantitatively and qualitatively, and that fly species key into volatile blends that indicate suitability of the substrates for their purposes. For drosophilids early arrival at fermenting substrates confers a competitive advantage to offspring. In contrast, for tephritid the concentration and availability of protein is facilitated by older, lysed yeast cultures. The data from this comparative study are also instrumental in the development of novel lures for these pests.

DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate

BackgroundQuantification of gene expression such as RNA-Seq is a popular approach to study various biological phenomena. Despite the development of RNA-Seq library preparation methods and sequencing platforms in the last decade, RNA extraction remains the most laborious and costly step in RNA-Seq of tissue samples of various organisms. Thus, it is still difficult to examine gene expression in thousands of samples.ResultsHere, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. The DTT concentration in Direct-RT buffer prevented RNA degradation but not RT in the lysates of several plant tissues, yeast, and zebrafish larvae. Direct reverse transcription on these lysates in Direct-RT buffer produced comparable amounts of cDNA to those synthesized from purified RNA. To maximize the advantage of the Direct-RT buffer, we integrated Direct-RT and targeted RNA-Seq to develop a cost-effective, high-throughput quantification method for the expressions of hundreds of genes: DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq). The DeLTa-Seq method could drastically improve the efficiency and accuracy of gene expression analysis. DeLTa-Seq analysis of 1056 samples revealed the temperature-dependent effects of jasmonic acid and salicylic acid in Arabidopsis thaliana.ConclusionsThe DeLTa-Seq method can realize large-scale studies using thousands of animal, plant, and microorganism samples, such as chemical screening, field experiments, and studies focusing on individual variability. In addition, Direct-RT is also beneficial for gene expression analysis in small tissues from which it is difficult to purify enough RNA for the experiments.

Autolysis of spent baker’s yeast generated from sago bioethanol fermentation: a preliminary study

Spent Baker’s yeast (Saccharomyces cerevisiae) is obtained as a by-product of ethanol fermentation industry and is mainly discarded as waste. The spent yeast can be potentially converted to yeast extract through autolysis process in which the yeast cells are lysed under certain conditions to release valuable and bioactive substances. To date, there are still limited studies on the autolysis of spent S. cerevisiae generated following the production of sago bioethanol. This research aims to investigate the feasibility of autolysis of spent Baker’s yeast generated from sago bioethanol fermentation by focusing on two important parameters namely initial pH and incubation time. The spent Baker’s yeast was autolysed at different initial pH values (3, 5 and 7) and at different incubation times (24, 48, 72 and 96 h). The protein and carbohydrate concentrations of the resulting lysates were analysed using Lowry assay and phenol-sulphuric acid assay respectively. Additionally, the surface morphology of the autolysed yeast cells was observed using Scanning electron microscope. Our results showed that the autolysis conducted at the initial pH of 3 gave the highest concentration of protein and total carbohydrate released from the lysate which were 2.5-fold and 2-fold over that in the control samples respectively. Meanwhile, the optimal incubation time for the autolysis of spent Baker’s yeast for both maximum concentrations of protein and carbohydrate released was found to be 72 h whilst prolonging beyond that did not result in any significant change of both the protein and total carbohydrate concentration. The morphological analysis of the lysed yeast cells clearly showed a significance change of the surface morphology indicative of cell lysis throughout the incubation period. In conclusion, the present work gives significant insights into the feasibility of autolysis of spent Baker’s yeast generated from sago bioethanol fermentation. This will in turn serves as useful fundamental information for developing potential side process streams within an integrated sago biorefinery in the future.

Development of nanocomposite biodegradable film containing iron nanoparticles biosynthesised by Saccharomyces cerevisiae

SummaryThe use of nanoparticles in biodegradable films has been shown to improve their characteristics in food packaging. The study was designed to investigate the effect of iron oxide nanoparticles (IONPs) and lysed yeast cells residue (YSR) on the properties of whey protein concentrate (WPC) films. IONPs (0, 0.5, 1.0 and 1.5 mm) biosynthesised by Saccharomyces cerevisiae and YSR were used to prepare WPC films. The addition of IONPs and YSR increased the tensile strength (TS) of the films by approximately 2.6 MPa compared with the control films (P < 0.05). Percent elongation and water vapour permeability of the films decreased significantly (P < 0.05) by the contribution of IONPs. All the films did not inhibit the growth of tested microorganisms. The incorporation of YSR in the film also did not make a significant change in any properties of the film (P > 0.05). The results indicated the usefulness of IONPs incorporation into WPC films to enhance mechanical but not antimicrobial properties.

Selection of optimal conditions for the lactic acid biosynthesis

Lactic acid is used in various industries: chemical, food, cosmetic, pharmaceutical, agriculture and polymer production. 40% of the domestic market demand for lactic acid is met through imports, while the main methods for producing lactic acid are microbiological or synthetic. The most rational is microbiological synthesis, however, when it is implemented, valuable sugar-containing substrates (crystalline sucrose, molasses, sugar syrup) are used, which significantly affects the cost of the final product. There was an obvious need to search for new technologies for the production of lactic acid. Prospective and cost-effective is the processing of whey into lactic acid using bacterial fermentation of the lactose contained in the whey. The aim of the study is to screen cultures of microorganisms capable of fermenting lactose contained in milk whey and to select the composition of the medium that provides the maximum yield of lactic acid. Strains of lactic acid bacteria were used in the experiment: Leuconostocmesenteroides subsp. mesenteroides 122 (B1699), Lactobacillus brevis B78 (B5728), Lactobacillus plantarum K9 (B5466), Lactobacillus casei C1 (B5726), Lactobacillus acidophilus (B9012), Lactobacillus paracasei BT 24/88 (B6253), Lactobacillus paracasei 139 (B2430), obtained from the Russian National Collection of Industrial Microorganisms (Scientific Center "Kurchatov Institute" -Research Institute for Genetics and Selection of Industrial Microorganisms). The authors determined the optimal composition of the medium for the biosynthesis of lactic acid by selecting the concentrations of yeast autolysate and salt solution that provide the maximum yield of lactic acid. The authors also selected the optimal duration of lactic acid biosynthesis. The study of the acid-forming ability of strains of lactic acid bacteria showed that of the strains studied, Lactobacillus casei C1 (B5726) showed the greatest ability to ferment lactose by synthesizing lactic acid, which correlates with the data on assimilation of lactose by the culture. The optimal cultivation time (132 h) and the concentration of yeast lysate (5%) in a nutrient medium based on curd whey were selected. With optimal parameters, the maximum concentration of lactic acid in the culture liquid of Lactobacillus casei C1 (B5726) was 54.77 g / L, which is comparable with the currently used producers in the industrial production of lactic acid.

Nitroreduction of flutamide by Cunninghamella elegans NADPH: Cytochrome P450 reductase

The microbial model of mammalian drug metabolism, Cunninghamella elegans, has three cytochrome P450 reductase genes in its genome: g1631 (CPR_A), g4301 (CPR_B), and g7609 (CPR_C). The nitroreductase activity of the encoded enzymes was investigated via expression of the genes in the yeast Pichia pastoris X33. Whole cell assays with the recombinant yeast demonstrated that the reductases converted the anticancer drug flutamide to the nitroreduced metabolite that was also produced from the same substrate when incubated with human NADPH: cytochrome P450 reductase. The nitroreductase activity extended to other substrates such as the related drug nilutamide and the environmental contaminants 1-nitronaphthalene and 1,3-dinitronaphthalene. Comparative experiments with cell lysates of recombinant yeast were conducted under aerobic and reduced oxygen conditions and demonstrated that the reductases are oxygen sensitive.

Normalized Ribo-Seq for Quantifying Absolute Global and Specific Changes in Translation.

Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome densities (RD). For library preparation, equal amounts of total RNA are used. Subsequently, all transcript fragments are subjected to linker ligation, cDNA synthesis, and PCR amplification. Importantly, the number of reads obtained for every transcript in input and footprint samples during sequencing depends on sequencing depth and library size, as well as the relative abundance of the transcript in the sample. However, the information pertaining to the absolute amount of input and footprint sequences is lost during sample preparation, hence ruling out any conclusion whether translation is generally suppressed or activated in one condition over the other. Therefore, the RD fold-changes determined for individual genes do not reflect absolute regulation, but have to be interpreted as relative to bulk mRNA translation. Here, we modified the original ribosome profiling protocol that was first established by Ingolia et al. (2009), by adding small amounts of yeast lysate to the mammalian lysates of interest as a spike-in. This allows us to not only detect changes in the RD of specific transcripts relative to each other, but also to simultaneously measure global differences in RD (normalized ribosome density values) between samples. Graphic abstract: Global changes in translation efficiency can be detected with polysome profiling, where the proportion of polysomal ribosomes is interpreted as a proxy for ribosome density (RD) on bulk mRNA. Ribo-Seq measures changes in RD of specific mRNAs relative to bulk mRNA. The addition of a yeast-lysate, as a spike-in for normalization of read counts, allows for an absolute measurement of changes in RD.

Lipid reduction to improve clarification and filterability during primary recovery of intracellular products in yeast lysates using exogenous lipase

AbstractBACKGROUNDThe yeast Pichia pastoris is a popular host organism for production of a range of biological products, several of which are intracellular. The disruption of yeast cells by homogenization also releases large quantities of lipids, which can foul the downstream membranes and chromatography matrices used for purification. This work examines lipid removal from yeast cells following homogenization by enzymatic degradation and its impact on the performance of the subsequent centrifugation and filtration.RESULTSLipase treatment of cell homogenate at 37 °C for 2 h, followed by clarification using a scaled‐down mimic of disc stack centrifugation, resulted in a 6.5‐fold improvement in solids removal when compared to untreated feed material. The lipase‐treated and untreated materials that had undergone initial centrifugation were then tested for filtration performance by passing the material through a 0.45 μm polyethylene sulfone membrane under constant flux. A 50% increase in throughput was observed in comparison to the untreated material.CONCLUSIONThese proof‐of‐concept data suggest enzymatic digestion of lipids, analogous to the widely performed DNA reduction using nucleases, could be a valuable process improvement strategy. © 2021 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Оценка влияния на биологический статус молодняка овец синбиотической кормовой добавки

The article presents the results of studies on the use of synbiotic feed additives based on live spore-forming bacteria of the genus Bacillus, lysates of lactic acid bacteria and yeast to lambs of 10-day-old Volgograd breed in various doses. The use of the feed additive did not have a negative effect on the morphological parameters of the lambs ' blood. Physiological parameters for the entire observation period were within the reference values characteristic of this animal species. The positive effect of synbiotic feed additive was manifested by higher growth rates – an increase in the live weight of the lambs of the experimental groups compared to the control group.

Comparison of protein and peptide fractionation approaches in protein identification and quantification from Saccharomyces cerevisiae

Shotgun proteomics is a high-throughput technology which has been developed with the aim of investigating the maximum number of proteins in cells in a given experiment. However, protein discovery and data generation vary in depth and coverage when different technical strategies are selected. In this study, three different sample preparation approaches, and peptide or protein fractionation methods, were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), filter-aided sample preparation coupled with gas phase fractionation (FASP-GPF), and FASP - high pH reversed phase fractionation (HpH). Fractions were initially analyzed and compared using nanoflow liquid chromatography – tandem mass spectrometry (nanoLC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and analytical replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time. A second set of experiments was performed, comparing FASP-GPF, SDS-PAGE and FASP-HpH using a Q Exactive Orbitrap mass spectrometer. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a substantial increase in protein identifications, and an even greater increase in peptide identifications. This shows that the main advantage of using the higher resolution instrument is in increased proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches, when applied to this type of sample.

Fast and inexpensive whole-genome sequencing library preparation from intact yeast cells.

Through the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limit the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high-quality whole-genome sequencing libraries directly from Saccharomyces cerevisiae cultures in less than 3 h at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting lysed yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow, we expect that our protocol can be adapted to other organisms.

Effect of glutathione on wheat dough properties and bread quality

Reduced glutathione (GSH), released from lysed yeast cells, is well-known for weakening dough structure. However, its influence on bread texture and staling has not yet been completely elucidated. Herein, this study aims at assessing the effects of GSH on dough properties and bread quality, especially bread staling, using Rheofermentometer analysis, texture profile analysis (TPA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and low-field nuclear magnetic resonance (LF-NMR). The results revealed that GSH substantially decreased the dough height and gas-retention capacity during fermentation. The weakened dough structure was attributed to GSH-induced disulfide bond cleavage in gluten proteins and the depolymerization of glutenin polymers. The addition of 0.005%–0.01% GSH resulted in acceptable bread quality. However, bread supplemented with 0.015%–0.03% of GSH exhibited a coarser and more open crumb microstructure, as well as high level of hardness and low resilience during aging. GSH promoted crumb water loss and drove the water shift from the immobilized to the bound state. The DSC and XRD analyses further confirmed that GSH promoted starch retrogradation and recrystallization. These results suggest that apart from the gluten structure, GSH also altered gluten−starch interactions and water redistribution, ultimately decreasing bread quality and accelerating bread staling.