Yeast Saccharomyces Pastorianus Research Articles

Biological recovery of phosphorus (BioP-Rec) from wastewater streams using brewer's yeast on pilot-scale.

Most recent advances for phosphorus (P) recovery using brewery yeast on laboratory scale were used to scale up to a pilot-scale process (BioP-Rec module) and applied in a full-scale wastewater treatment plant (WWTP). A P balance was established for WWTP Markranstädt according to two thresholds: (1) the economic feasibility threshold for P recovery of 0.05kg/m3 of free P, and (2) the German Sewage Sludge Ordinance (GSSO) threshold, which demands that all WWTPs with a P content in dry matter (DM) of biosolids of 20 gP/kgDM or higher in the coming years must perform mandatory P recovery. In terms of defined thresholds, return and excess sludges were identified as the most feasible WWTP process streams for P recovery. In a 1 m3 BioP-Rec module a 3 stage process was established. From the P-rich water-phase of the return sludge produced in stage 1, which contained 0.051kg/m3 of free P, 77.56% was taken up by P-depleted brewer's yeast Saccharomyces pastorianus in 3h in stage 2. In stage 3, the yeast was concentrated in 1h to produce yeast sludge as a fertilizer product. We demonstrated a novel pilot-scale process for the production of bio-based P-rich fertilizer.

Compensatory Genetic and Transcriptional Cytonuclear Coordination in Allopolyploid Lager Yeast (Saccharomyces pastorianus).

Cytonuclear coordination between biparental-nuclear genomes and uniparental-cytoplasmic organellar genomes in plants is often resolved by genetic and transcriptional cytonuclear responses. Whether this mechanism also acts in allopolyploid members of other kingdoms is not clear. Additionally, cytonuclear coordination of interleaved allopolyploid cells/individuals within the same population is underexplored. The yeast Saccharomyces pastorianus provides the opportunity to explore cytonuclear coevolution during different growth stages and from novel dimensions. Using S. pastorianus cells from multiple growth stages in the same environment, we show that nuclear mitochondria-targeted genes have undergone both asymmetric gene conversion and growth stage-specific biased expression favoring genes from the mitochondrial genome donor (Saccharomyces eubayanus). Our results suggest that cytonuclear coordination in allopolyploid lager yeast species entails an orchestrated and compensatory genetic and transcriptional evolutionary regulatory shift. The common as well as unique properties of cytonuclear coordination underlying allopolyploidy between unicellular yeasts and higher plants offers novel insights into mechanisms of cytonuclear evolution associated with allopolyploid speciation.

The Impact of Dry Yeast Rehydrated in Different Plasma Treated Waters (PTWs) on Fermentation Process and Quality of Beer.

Yeast plays a key role in the production of alcoholic beverages. Effective fermentation requires appropriate conditions to ensure the production of high-quality beer. The paper discusses the effect of dry brewing yeast (Saccharomyces cerevisiae and Saccharomyces pastorianus) after rehydration with water exposed to low-temperature, low-pressure glow plasma (PTW) in the atmosphere of air (PTWAir) and nitrogen (PTWN) in the course of the fermentation process, the formation of volatile compounds and other quality parameters of the finished beer. The obtained results show that the lager yeast strain initiated the process of fermentation faster after rehydration in the presence of PTWAir compared to all of the other treatments. It was observed that PTWAir significantly changed the composition of volatile compounds in the finished beer, especially by increasing the number of terpenes, which are compounds that positively shape the aroma of beer. In the case of PTWN samples, lower alcohol content, real extract, apparent extract and amount of biomass were observed in all analyzed strains.

Influence of the time of bilberry (Vaccinium myrtillus L.) addition on the phenolic and protein profile of beer.

In recent years, there has been an increased fruit addition to the brewing process, especially in craft breweries. Fruit addition leads to changes in the organoleptic characteristics and chemical composition of beers. Bilberries are rich in phenolic compounds and possess significant antioxidant capacity. The effects of bilberry addition and brewing process parameters on the changes in the phenolic and protein profile of beer have not been sufficiently studied. The aim of this research was to investigate the changes in the individual phenolic compounds and the protein fractions in beer when bilberries were added at different maturation stages. An infusion mashing method was applied for the purpose of obtaining wort with an original extract of 14°P after boiling. Pilsner malt, bitter and aromatic hops 60/40 (Perle and Cascade, respectively), dry yeast Saccharomyces pastorianus (carlsbergensis) Saflager W 34/70, and bilberries (Vaccinium myrtillus L.) were used. All processes were conducted in a Home Brew 50 all-in-one 50 dm3 brewing system. The fermentation was carried out in a stainless steel cylindroconical fermenter at a temperature of 14°C. The “green beer” was transferred to small stainless-steel fermenters after 60% of the original wort extract had been fermented. The maturation continued for 14 days at 14°C, and the beer lagering for 5 days at 2°C. The bilberries were pasteurised in a water bath for 10 minutes at 70°C. After cooling, they were added to small fermenters at a concentration of 167 g/dm3 at the beginning and on the seventh day of beer maturation. All variants were carried out in duplicate. After lagering, the beer was bottled using a “beer gun”. The beer samples from the experiments were filtered on the day of bottling and frozen until analysis. HPLC/UV-VIS and electrophoresis were used to determine the phenolics and proteins, respectively. The total monomeric anthocyanins were determined by the pH differential method. The original wort extract and alcohol concentration were evaluated, and the sensory analysis was performed according to EBC standard methods. The changes in 10 phenolic acids, 7 flavonoids, and 10 protein fractions in beer with bilberries added at the beginning and on the seventh day of maturation were studied. The addition of bilberries led to an increase in the phenolic acid (3-fold) and flavonoid (6.2-fold) concentrations. The highest enrichment was observed in terms of rutin, chlorogenic, caffeic, and 3,4-dihydrobenzoic acids. Rosmarinic acid and monomeric anthocyanins were only detected in the bilberry beers. Chlorogenic and neochlorogenic acids, rutin, and catechin dominated in the bilberry beers. Neochlorogenic and gallic acids, epicatechin, and catechin dominated in the bilberry-free beers. The addition of bilberries reduced the protein content by 93 to 96%. The number of protein fractions decreased from 10 to 4. The influence of the bilberry addition time on the phenolics and proteins was different, and it affected the individual protein fractions in a different way. More phenolic acids and flavonoids were determined when bilberries were added at the beginning of maturation. The losses of some protein fractions were higher at the beginning of maturation and of others on the seventh day of maturation, whereas the addition time did not matter for some of the fractions. This study provides new information related to the changes in the phenolic and protein profile of beer with bilberries depending on the time of bilberry addition during beer maturation. The protein concentration and number of protein fractions decreased dramatically. In spite of the significant protein losses, the bilberry addition improved the phenolic profile of the beer and its organoleptic characteristics. The presence of more phenolic compounds is related to the antioxidant capacity respective to the biological value of beer. Further research in this direction is needed.

Wine production from ripen pond apple (Annona glabra L.) fruit

Pond apple (Annona glabra L.) trees were widely distributed in swamp regions of Mekong Delta, Vietnam. Pond apple fruits turned from green to yellow when ripening. Ripen pond apple fruits contained numerous phenolic constituents with valuable phytochemical benefits. However, ripen pond apple fruits were not successfully utilized as other commercial fruits. This research examined the possibility of wine production utilized from ripen pond apple fruits. Different various technical variables of fermentation affecting to the quality of pond apple wine were thoroughly examined. Ripen pond apple fruits were naturally collected from Soc Trang province, Vietnam. Ripen pond apple fruits were peeled, blended, deseeded, crushed, enzyme-treated (pectinase 25 mg/L), added with sugar (5-13% w/w), pasteurized (sulphite 30 mg/L), inoculated with yeast Saccharomyces pastorianus ratio (0.1-0.5%), macerated temperature (14-22oC) in different time (6-14 days). Malolactic fermentation was performed in anaerobic condition at 12oC in different durations (4-20 weeks). At the end of malolactic fermentation, wine was racked and clarified with different fining agents (bentonite, polyvinylpyrrolidone, wheat gluten, gelatin, kaolin) at 0.03% (v/v). Results showed that must should be added with 9% sugar and 0.4% yeast inoculation, fermentation temperature of 16oC in 10 days. Malolactic fermentation could be terminated at 12 weeks. Gelatin revealed the best candidate among different clarifying agents to remove turbidity in pond apple wine while retaining the most total phenolic content and antioxidant capacity. Under above technical variable conditions, fermentation gave the high ethanol content (4.26±0.02 % v/v); the total phenolic content (32.79±0.00 mg GAE/100 ml), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging (11.84±0.01 %), overall acceptance (8.34±0.01 score) meanwhile low turbidity (24.41±0.00 NTU) was also noticed. High ethanol content and phytochemical retention contributed to the high sensory score of pond apple wine. These quality parameters were acceptable for an alcoholic drink. Ripen pond apple fruit would be a promising carbohydrate source to convert into a new fruit wine with a pleasant alcoholic flavor and attractive appearance.

Constant temperature mashing at 72 °C for the production of beers with a reduced alcohol content in micro brewing systems

In this paper, we present a constant temperature mashing procedure where grist made of Pilsner malt is mashed-in directly in the temperature regime of alpha-amylase activity, thus omitting all conventional steps, followed by constant temperature mashing at 72 °C. The aim was to investigate an alternative mashing procedure for the production of alcohol-reduced beers. The mashing proceeds with a rapid buildup of sugars and is completed after 120 min at the latest, giving an iodine normal and clear wort. However, the distribution of the different sugars in the worts is strongly altered, in comparison to a more classical mashing procedure. The free amino nitrogen (FAN) concentration is sufficient for vivid fermentation with the bottom fermenting yeast Saccharomyces pastorianus TUM 34/70. The lag phase and initial fermentation performance of this yeast strain are comparable for conventionally and isothermally (72 °C) mashed wort. Under the given conditions the fermentation of the isothermally (72 °C) made wort is finished after 6 days whereas a conventional wort needs 4–5 days more to be completed. The alcohol concentration is remarkably reduced by isothermal mashing leading to roughly 3.4 vol.-% with an original gravity of 11°P whereas with a conventional mashing procedure 4.4 vol.-% are obtained for the same original gravity. In both cases the concentrations of the fermentation by-products are comparable. A preliminary comparison of taste and foam stability did not show striking differences. Constant temperature mashing at 72 °C is a simple way to reduce the alcohol content of beer enriching it at the same time with non-fermentable sugars.

Regulating the ratio of higher alcohols to esters by simultaneously overexpressing ATF1 and deleting BAT2 in brewer's yeast Saccharomyces pastorianus

The quality and aroma of beer are affected by the content of higher alcohols and esters. An appropriate ratio of higher alcohols to esters is essential to the beer quality. The imbalanced ratio can lead to a poor taste of beer. In beer, the production of higher alcohols and esters is closely related to the brewer's yeast metabolism. In Saccharomyces pastorianus, the alcohol acetyltransferase encoding gene (ATF1) is the key gene for producing esters. In this work, the ATF1 gene was overexpressed by different strength promoters (PGK1p(9), PGK1p(12), PGK1p(16), PGK1p(20)) and was constructed recombinant strains S9-M9, S9-M12, S9-M16, S9-M20 by a seamless gene deletion method, respectively. The ethyl acetate content produced by strain S9-M9, S9-M12, S9-M16, S9-M20 was 2.49-, 8.63-, 15.91-, 24.17-fold higher than parental strain S9, respectively. The results illustrated that strain S9-M9 had a favorable ratio of higher alcohols to esters (3.2: 1). The fermentation scale was subsequently expanded to 100-L, while the fermentation performance of strains S9 and S9-M9 remained the same. In this study, the ATF1 gene expression of S. pastorianus by different strength promoters provided an accurate regulation of higher alcohols-esters production ratio in beer. This is the first description of regulating the ratio of higher alcohols to esters using this new strategy, combining different strength promoters and a seamless gene deletion method. This new strategy provided a new perspective for regulating the flavor profile in beer or other beverages.

Improving the Utilization of Isomaltose and Panose by Lager Yeast Saccharomyces pastorianus

Approximately 25% of all carbohydrates in industrial worts are poorly, if at all, fermented by brewing yeast. This includes dextrins, β-glucans, arabinose, xylose, disaccharides such as isomaltose, nigerose, kojibiose, and trisaccharides such as panose and isopanose. As the efficient utilization of carbohydrates during the wort’s fermentation impacts the alcohol yield and the organoleptic traits of the product, developing brewing strains with enhanced abilities to ferment subsets of these sugars is highly desirable. In this study, we developed Saccharomyces pastorianus laboratory yeast strains with a superior capacity to grow on isomaltose and panose. First, we designed a plasmid toolbox for the stable integration of genes into lager strains. Next, we used the toolbox to elevate the levels of the α-glucoside transporter Agt1 and the major isomaltase Ima1. This was achieved by integrating synthetic AGT1 and IMA1 genes under the control of strong constitutive promoters into defined genomic sites. As a result, strains carrying both genes showed a superior capacity to grow on panose and isomaltose, indicating that Ima1 and Agt1 act in synergy to consume these sugars. Our study suggests that non-GMO strategies aiming to develop strains with improved isomaltose and panose utilization could include identifying strains that overexpress AGT1 and IMA1.

Production and characterisation of non-alcoholic beer using special yeast

Non-Saccharomyces yeast strains Saccharomycodes ludwigii, Schizosaccharomyces pombe, Lachancea fermentati and Pichia angusta together with a hybrid yeast strain cross-bred between genetically modified Saccharomyces cerevisiae W303-1A G418R and Saccharomyces eubayanus as well as the parent yeasts of the hybrid were studied for potential use for non-alcoholic beer production. The hybrid yeast, its Saccharomyces cerevisiae W303-1A G418R parent and Saccharomycodes ludwigii were not able to metabolise maltose during Durham tube tests. Schizosaccharomyces pombe, Lachancea fermentati and Pichia angusta metabolised maltose, however, showed limited ethanol production. Parameters, volatile and non-volatile organic compounds of beers produced by the studied yeast were analysed and compared to a beer produced by bottom fermented brewer’s yeast Saccharomyces pastorianus.

Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in Saccharomyces pastorianus.

The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.

Rapid Beer Fermentation: The Effect of Vacuum Pressure on a Pilot Scale Lager Fermentation

Changes to industrial fermentation processes are rare, largely due to the impact on viability for Saccharomyces spp., related to environmental stresses during the process such as: osmotic pressure, ethanol concentration, dissolved CO2. This research studied yeast behavior during a High Gravity (HG) fermentation under partial vacuum pressure during standard brewing operations. This was accomplished by controlling the temperature and pressure of 30 L pilot fermentations. An experimental lager wort recipe with “Diamond” lager yeast (Saccharomyces pastorianus) was used in this study. The modified process conducted at 24.1 kPa of pressure in the headspace was compared to a control process at standard atmospheric pressure (101.3 kPa). This research showed that at 15 °C, lager fermentations (14–15°P) fermented up to 30% faster under vacuum, with an increase in the number of yeast cells in suspension of up to 100%. The rate of sugar consumption under vacuum was greater compared to the control, thus increasing the rate of ethanol production. The removal of volatile CO2 is one hypothesized mechanism for these results. The total ethanol production, pH and yeast viability did not differ significantly between the control and modified process. The application of vacuum during brewing resulted in an increased fermentation rate and number of yeast cells in suspension during fermentation.

Lager-brewing yeasts in the era of modern genetics.

ABSTRACTThe yeast Saccharomyces pastorianus is responsible for the annual worldwide production of almost 200 billion liters of lager-type beer. S. pastorianus is a hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been studied for well over a century. Scientific interest in S. pastorianus intensified upon the discovery, in 2011, of its S. eubayanus ancestor. Moreover, advances in whole-genome sequencing and genome editing now enable deeper exploration of the complex hybrid and aneuploid genome architectures of S. pastorianus strains. These developments not only provide novel insights into the emergence and domestication of S. pastorianus but also generate new opportunities for its industrial application. This review paper combines historical, technical and socioeconomic perspectives to analyze the evolutionary origin and genetics of S. pastorianus. In addition, it provides an overview of available methods for industrial strain improvement and an outlook on future industrial application of lager-brewing yeasts. Particular attention is given to the ongoing debate on whether current S. pastorianus originates from a single or multiple hybridization events and to the potential role of genome editing in developing industrial brewing yeast strains.

Diacetyl control during brewery fermentation via adaptive laboratory engineering of the lager yeast Saccharomyces pastorianus

Diacetyl contributes to the flavor profile of many fermented products. Its typical buttery flavor is considered as an off flavor in lager-style beers, and its removal has a major impact on time and energy expenditure in breweries. Here, we investigated the possibility of lowering beer diacetyl levels through evolutionary engineering of lager yeast for altered synthesis of α-acetolactate, the precursor of diacetyl. Cells were exposed repeatedly to a sub-lethal level of chlorsulfuron, which inhibits the acetohydroxy acid synthase responsible for α-acetolactate production. Initial screening of 7 adapted isolates showed a lower level of diacetyl during wort fermentation and no apparent negative influence on fermentation rate or alcohol yield. Pilot-scale fermentation was carried out with one isolate and results confirmed the positive effect of chlorsulfuron adaptation. Diacetyl levels were over 60% lower at the end of primary fermentation relative to the non-adapted lager yeast and no significant change in fermentation performance or volatile flavor profile was observed due to the adaptation. Whole-genome sequencing revealed a non-synonymous SNP in the ILV2 gene of the adapted isolate. This mutation is known to confer general tolerance to sulfonylurea compounds, and is the most likely cause of the improved tolerance. Adaptive laboratory evolution appears to be a natural, simple and cost-effective strategy for diacetyl control in brewing.

Induced expression of the alcohol acetyltransferase gene ATF1 in industrial yeast Saccharomyces pastorianus TUM 34/70.

Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.

Enantiomeric separation of triacylglycerols containing very long chain fatty acids

Enantiomers of triacylglycerols (TAGs) containing any combination of very long chain fatty acids (VLCFAs) and/or very long chain polyunsaturated fatty acids (VLCPUFAs) with diolein, dilinolein and didocosahexaenoin were synthesized. Gradient non-aqueous reversed-phase high-performance liquid chromatography/high resolution atmospheric pressure chemical ionization-tandem mass spectrometry (NARP-HPLC/HRMS2-APCI) and chiral liquid chromatography were used for the separation and identification of molecular species of these TAGs. Further, NARP-LC and chiral LC were used to separate natural mixtures of TAGs obtained from four natural sources, i.e. ximenia oil (Ximenia americana), green alga (Botryococcus braunii), breweŕs yeast (Saccharomyces pastorianus) and a dinoflagellate (Amphidinium carterae). The ratio of regioisomers and enantiomers in individual samples was determined and a hypothesis has been confirmed on the biosynthetic pathway of natural TAGs, which is based on the preferential representation of VLCFAs and VLCPUFAs in the sn-1 position of the glycerol backbone.

An efficient method for isolating mating-competent cells from bottom-fermenting yeast using mating pheromone-supersensitive mutants.

Crossbreeding is an effective approach to construct novel yeast strains with preferred characteristics; however, it is difficult to crossbreed strains of brewer's yeast, especially the bottom-fermenting yeast Saccharomyces pastorianus, because of the relative inefficiency of the available methods to obtain mating-competent cells (MCCs). Here, we describe a productive method for the isolation of MCCs without artificial genetic modification. We focused on the characteristics of two mating pheromone-supersensitive mutants, Δbar1 and Δsst2, that show a growth defect in the presence of the mating pheromone. When MCCs secreting α-factor and a-factor were spotted on to a lawn of MATa Δbar1 and MATα Δsst2, a halo was observed around the respective MCCs. This plate assay was successful in identifying MCCs from bottom-fermenting yeast strains. Furthermore, by selecting for cells that caused the growth defect in pheromone-supersensitive cells on cultures plates, 40 α/α-type and six a/a-type meiotic segregants of bottom-fermenting yeast strains were successfully isolated and crossed with tester strains to verify their mating type. This method of isolation is expected to be applicable to other industrial yeast strains, including wine, sake and distiller's yeasts, and will enable MCCs without genetic modifications to be obtained. As a result, it will be a useful tool for more convenient and efficient crossbreeding of industrial yeast strains that can be applied to practical brewing. Copyright © 2017 John Wiley & Sons, Ltd.

CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus

BackgroundThe ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae.ResultsThis study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design.ConclusionsThese results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.

Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion (“attenuation”) of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with 14C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.

Antifungal activity of a synthetic human β-defensin 3 and potential applications in cereal-based products

Defensins are cysteine-rich antimicrobial peptides of the innate immune system involved in constitutive and adaptive immunities. The aim of this work was to characterize the antifungal activity of a linear human β-defensin-3 (HBD-3) and test this peptide as a potential food preservative. Antifungal activity of the synthetic HBD-3 was demonstrated against Fusarium culmorum, Penicillium expansum and Aspergillus niger. To determine potential baking applications with the defensin, a parental wild yeast Saccharomyces pastorianus strain, called CMBS-33, was transgenically modified. The novel lager yeast strain, called CM-INT-51, was able to express the gene encoding HBD-3. The use of CM-INT-51 to ferment the dough extends the shelf life of bread by three days in comparison to CMBS-33. Furthermore, synthetic HBD-3 (80 μg·ml−1) delayed fungal growth on bread for > 13 days. These results revealed that both the synthetic peptide and the yeast containing HBD-3 have the potential to increase the shelf-life of baked goods. Fungal spoilage is an important cause of economic losses in the baking industry and might be a source of mycotoxins with detrimental effects on human health. Furthermore, the overuse of preservatives is subject to controversy. We showed that the linear analogue of HBD-3 displays antifungal activity against food contaminants in vitro and can be used to extend the shelf-life of bread. These results have direct applications to the baking and other related industries.

Maltose and maltotriose utilisation by groupI strains of the hybrid lager yeast Saccharomyces pastorianus.

Brewer's wort is a challenging environment for yeast as it contains predominantly α-glucoside sugars. There exist two subgroups of the lager yeast Saccharomyces pastorianus which differ in sugar utilisation. We performed wort fermentations and compared representative strains from both groups with respect to their ability to transport and ferment maltose and maltotriose. Additionally, we mapped the transporters MALx1, AGT1, MPHx and MTT1 by Southern blotting. Contrary to previous observations, group I comprises a diverse set of strains, with varying ability to transport and ferment maltotriose. Of the eight group I strains, three efficiently utilised maltotriose, a property enabled by the presence of transmembrane transporters SeAGT1 and MTT1. A58, a variant of the group I type strain (CBS1513) performed particularly well, taking up maltotriose at a higher rate than maltose and retaining significant transport activity at temperatures as low as 0°C. Analysis of transporter distribution in this strain revealed an increased copy number of the MTT1 gene, which encodes the only permease known with higher affinity for maltotriose than maltose and low temperature dependence for transport. We propose that much of the variation in lager yeast fermentation behaviour is determined by the presence or absence of specific transmembrane transporters.