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1.  Isolation and Characterization of Brewer's Yeast Variants with Improved Fermentation Performance under High-Gravity Conditions▿  
To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22° Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.
doi:10.1128/AEM.02109-06
PMCID: PMC1800776  PMID: 17158628
2.  The impact of different ale brewer’s yeast strains on the proteome of immature beer 
BMC Microbiology  2013;13:215.
Background
It is well known that brewer’s yeast affects the taste and aroma of beer. However, the influence of brewer’s yeast on the protein composition of beer is currently unknown. In this study, changes of the proteome of immature beer, i.e. beer that has not been matured after fermentation, by ale brewer’s yeast strains with different abilities to degrade fermentable sugars were investigated.
Results
Beers were fermented from standard hopped wort (13° Plato) using two ale brewer’s yeast (Saccharomyces cerevisiae) strains with different attenuation degrees. Both immature beers had the same alcohol and protein concentrations. Immature beer and unfermented wort proteins were analysed by 2-DE and compared in order to determine protein changes arising from fermentation. Distinct protein spots in the beer and wort proteomes were identified using Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MS/MS and revealed common beer proteins, such as lipid transfer proteins (LTP1 and LTP2), protein Z and amylase-protease inhibitors. During fermentation, two protein spots, corresponding to LTP2, disappeared, while three protein spots were exclusively found in beer. These three proteins, all derived from yeast, were identified as cell wall associated proteins, that is Exg1 (an exo-β-1,3-glucanase), Bgl2 (an endo-β-1,2-glucanase), and Uth1 (a cell wall biogenesis protein).
Conclusion
Yeast strain dependent changes in the immature beer proteome were identified, i.e. Bgl2 was present in beer brewed with KVL011, while lacking in WLP001 beer.
doi:10.1186/1471-2180-13-215
PMCID: PMC3849757  PMID: 24079909
2-DE; Beer; Fermentation; Brewer’s yeast; Secreted proteins
3.  Engineering Saccharomyces pastorianus for the co-utilisation of xylose and cellulose from biomass 
Background
Lignocellulosic biomass is a viable source of renewable energy for bioethanol production. For the efficient conversion of biomass into bioethanol, it is essential that sugars from both the cellulose and hemicellulose fractions of lignocellulose be utilised.
Results
We describe the development of a recombinant yeast system for the fermentation of cellulose and xylose, the most abundant pentose sugar in the hemicellulose fraction of biomass. The brewer’s yeast Saccharomyces pastorianus was chosen as a host as significantly higher recombinant enzyme activities are achieved, when compared to the more commonly used S. cerevisiae. When expressed in S. pastorianus, the Trichoderma reesei xylose oxidoreductase pathway was more efficient at alcohol production from xylose than the xylose isomerase pathway. The alcohol yield was influenced by the concentration of xylose in the medium and was significantly improved by the additional expression of a gene encoding for xylulose kinase. The xylose reductase, xylitol dehydrogenase and xylulose kinase genes were co-expressed with genes encoding for the three classes of T. reesei cellulases, namely endoglucanase (EG2), cellobiohydrolysase (CBH2) and β-glucosidase (BGL1). The initial metabolism of xylose by the engineered strains facilitated production of cellulases at fermentation temperatures. The sequential metabolism of xylose and cellulose generated an alcohol yield of 82% from the available sugars. Several different types of biomass, such as the energy crop Miscanthus sinensis and the industrial waste, brewer’s spent grains, were examined as biomass sources for fermentation using the developed yeast strains. Xylose metabolism and cell growth were inhibited in fermentations carried out with acid-treated spent grain liquor, resulting in a 30% reduction in alcohol yield compared to fermentations carried out with mixed sugar substrates.
Conclusions
Reconstitution of complete enzymatic pathways for cellulose hydrolysis and xylose utilisation in S. pastorianus facilitates the co-fermentation of cellulose and xylose without the need for added exogenous cellulases and provides a basis for the development of a consolidated process for co-utilisation of hemicellulose and cellulose sugars.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-015-0242-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12934-015-0242-4
PMCID: PMC4417197  PMID: 25928878
S. pastorianus; Co-utilisation of xylose and cellulose; Biomass; Spent grain fermentations
4.  Evaluation of the fermentation of high gravity thick sugar beet juice worts for efficient bioethanol production 
Background
Sugar beet and intermediates of sugar beet processing are considered to be very attractive feedstock for ethanol production due to their content of fermentable sugars. In particular, the processing of the intermediates into ethanol is considerably facilitated because it does not require pretreatment or enzymatic treatment in contrast to production from starch raw materials. Moreover, the advantage of thick juice is high solid substance and saccharose content which eliminates problems with the storability of this feedstock.
Results
The objective of this study were to investigate bioethanol production from thick juice worts and the effects of their concentration, the type of mineral supplement, as well as the dose of yeast inoculum on fermentation dynamics and ethanol yield.
The obtained results show that to ensure efficient ethanolic fermentation of high gravity thick juice worts, one needs to use a yeast strain with high ethanol tolerance and a large amount of inoculum. The highest ethanol yield (94.9 ± 2.8% of the theoretical yield) and sugars intake of 96.5 ± 2.9% were obtained after the fermentation of wort with an extract content of 250 g/kg supplemented with diammonium hydrogen phosphate (0.3 g/L of wort) and inoculated with 2 g of Ethanol Red dry yeast per L of wort. An increase in extract content in the fermentation medium from 250 g/L to 280 g/kg resulted in decreased efficiency of the process. Also the distillates originating from worts with an extract content of 250 g/kg were characterized by lower acetaldehyde concentration than those obtained from worts with an extract content of 280 g/kg.
Conclusions
Under the favorable conditions determined in our experiments, 38.9 ± 1.2 L of 100% (v/v) ethyl alcohol can be produced from 100 kg of thick juice. The obtained results show that the selection of process conditions and the yeast for the fermentation of worts with a higher sugar content can improve the economic performance of the alcohol-distilling industry due to more efficient ethanol production, reduced consumption of cooling water, and energy for ethanol distillation, as well as a decreased volume of fermentation stillage.
doi:10.1186/1754-6834-6-158
PMCID: PMC3826521  PMID: 24206573
Bioethanol; Fermentation; Thick juice; Sugar beet; High gravity wort; Yeast
5.  Effect of Ethanol, Sulfur Dioxide and Glucose on the Growth of Wine Spoilage Yeasts Using Response Surface Methodology 
PLoS ONE  2015;10(6):e0128702.
Response surface methodology (RSM) was used to study the effect of three factors, sulfur dioxide, ethanol and glucose, on the growth of wine spoilage yeast species, Zygosaccharomyces bailii, Schizosaccharomyces pombe, Saccharomycodes ludwigii and Saccharomyces cerevisiae. Seventeen central composite rotatable design (CCRD) trials were designed for each test yeast using realistic concentrations of the factors (variables) in premium red wine. Polynomial regression equations were fitted to experimental data points, and the growth inhibitory conditions of these three variables were determined. The overall results showed Sa. ludwigii as the most resistant species growing under high ethanol/free sulfur dioxide concentrations, i.e., 15% (v/v)/20 mg L-1, 14% (v/v)/32 mg L-1 and 12.5% (v/v)/40 mg L-1, whereas other yeasts did not survive under the same levels of ethanol/free sulfur dioxide concentrations. The inhibitory effect of ethanol was primarily observed during longer incubation periods, compared with sulfur dioxide, which showed an immediate effect. In some CCRD trials, Sa. ludwigii and S. cerevisiae showed growth recovery after a short death period under the exposure of 20–32 mg L-1 sulfur dioxide in the presence of 11% (v/v) or more ethanol. However, Sc. pombe and Z. bailii did not show such growth recovery under similar conditions. Up to 10 g L-1 of glucose did not prevent cell death under the sulfur dioxide or ethanol stress. This observation demonstrates that the sugar levels commonly used in wine to sweeten the mouthfeel do not increase wine susceptibility to spoilage yeasts, contrary to the anecdotal evidence.
doi:10.1371/journal.pone.0128702
PMCID: PMC4479441  PMID: 26107389
6.  The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732T 
Eukaryotic Cell  2014;13(11):1371-1379.
Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732T) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1Δ, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.
doi:10.1128/EC.00137-14
PMCID: PMC4248702  PMID: 25172765
7.  Technological Development of Brewing in Domestic Refrigerator Using Freeze-Dried Raw Materials 
Food Technology and Biotechnology  2017;55(3):325-332.
Summary
Development of a novel directly marketable beer brewed at low temperature in a domestic refrigerator combined with yeast immobilization technology is presented in this study. Separately, freeze-dried wort and immobilized cells of the cryotolerant yeast strain Saccharomyces cerevisiae AXAZ-1 on tubular cellulose were used in low-temperature fermentation (2, 5 and 7 °C). The positive effect of tubular cellulose during low-temperature brewing was examined, revealing that freeze-dried immobilized yeast cells on tubular cellulose significantly reduced the fermentation rates in contrast to freeze-dried free cells, although they are recommended for home-made beer production. Immobilization also enhanced the yeast resistance at low-temperature fermentation, reducing the minimum brewing temperature value from 5 to 2 °C. In the case of high-quality beer production, the effect of temperature and initial sugar concentration on the fermentation kinetics were assessed. Sensory enrichment of the produced beer was confirmed by the analysis of the final products, revealing a low diacetyl concentration, together with improved polyphenol content, aroma profile and clarity. The proposed process for beer production in a domestic refrigerator can easily be commercialized and applied by dissolving the content of two separate packages in tap water; one package containing dried wort and the other dried immobilized cells on tubular cellulose suspended in tap water.
doi:10.17113/ftb.55.03.17.4907
PMCID: PMC5654428
brewing; low-temperature fermentation; novel technology; immobilized cells; freeze-drying; tubular cellulose
8.  Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects 
A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer.
PMCID: PMC203694  PMID: 16347298
9.  High-Gravity Brewing: Effects of Nutrition on Yeast Composition, Fermentative Ability, and Alcohol Production 
A number of economic and product quality advantages exist in brewing when high-gravity worts of 16 to 18% dissolved solids are fermented. Above this level, production problems such as slow or stuck fermentations and poor yeast viability occur. Ethanol toxicity has been cited as the main cause, as brewers' yeasts are reported to tolerate only 7 to 9% (vol/vol) ethanol. The inhibitory effect of high osmotic pressure has also been implicated. In this report, it is demonstrated that the factor limiting the production of high levels of ethanol by brewing yeasts is actually a nutritional deficiency. When a nitrogen source, ergosterol, and oleic acid are added to worts up to 31% dissolved solids, it is possible to produce beers up to 16.2% (vol/vol) ethanol. Yeast viability remains high, and the yeasts can be repitched at least five times. Supplementation does not increase the fermentative tolerance of the yeasts to ethanol but increases the length and level of new yeast cell mass synthesis over that seen in unsupplemented wort (and therefore the period of more rapid wort attenuation). Glycogen, protein, and sterol levels in yeasts were examined, as was the importance of pitching rate, temperature, and degree of anaerobiosis. The ethanol tolerance of brewers' yeast is suggested to be no different than that of sake or distillers' yeast.
PMCID: PMC241579  PMID: 16346630
10.  Indonesian Tapé Ketan Fermentation 1 
Indonesian tapé ketan is a fermentation in which a mold, Amylomyces rouxii Calmette (Chlamydomucor oryzae Went and Prinsen Geerligs), in combination with one or more yeasts such as Endomycopsis burtonii converts steamed rice to a sweet-sour, slightly alcoholic paste. A study was made to determine the biochemical changes that occur in the substrate during fermentation. It was found that the product was ready for consumption after fermentation at 30°C for 36 to 48 h. A. rouxii used about 30% of the total rice solids, resulting in a crude protein of 12% in 96 h, whereas the combination of the mold with E. burtonii reduced total solids by 50% in 192 h, causing crude protein to increase to 16.5%. Soluble solids increased from 5 to about 67% in 36 h and decreased to 12% at 192 h with A. rouxii alone, whereas soluble solids fell to about 8% at 192 h in the fermentation with both the mold and the yeast. The mold, by itself, reduced the starch content of the rice from 78 to 10% in 48 h and to less than 2% in 144 h. The mold plus yeast reduced the starch content to about 18% in 48 h; however the “starch” content did not fall below 6% even at 192 h, presumably because the yeast was producing glycogen, which was determined along with the residual starch. With both the mold and the mold plus yeast fermentations, reducing sugars increased from less than 1% to approximately 5% in 24 h and reached maximum concentration, 16 to 17%, between 36 and 48 h. A. rouxii by itself produced a maximum of about 5.6% (vol/vol) ethanol at 96 h. The highest concentration of ethanol (8%, vol/vol) was produced by the mold plus E. burtonii at 144 h. The mold by itself reduced the starting pH from 6.3 to about 4.0 in 48 h. The combination of the mold and yeast reduced the pH to 4.1 in 144 h. The mold increased total acidity to approximately 6.2 meq of H+ per 100 ml, and the combination of the mold and yeast increased the total acidity to 7.8 meq of H+ per 100 ml in 192 h. At 48 h there was practically no difference in the volatile acidity (0.20) for the combined fermentation compared with 0.26 meq of H+ per 100 ml for the mold fermentation. The mold and at least one species of yeast were required to develop the rich aroma and flavor of typical Indonesian tapé.
PMCID: PMC170828  PMID: 16345243
11.  Brewery Waste Reuse for Protease Production by Lactic 
Acid Fermentation 
Food Technology and Biotechnology  2017;55(2):218-224.
Summary
This study evaluated the use of three solid brewery wastes: brewer’s spent grain, hot trub and residual brewer’s yeast, as alternative media for the cultivation of lactic acid bacteria to evaluate their potential for proteolytic enzyme production. Initially, a mixture experimental design was used to evaluate the effect of each residue, as well as different mixtures (with the protein content set at 4%) in the enzyme production. At predetermined intervals, the solid and liquid fractions were separated and the extracellular proteolytic activity was determined. After selecting the best experimental conditions, a second experiment, factorial experimental design, was developed in order to evaluate the protein content in the media (1 to 7%) and the addition of fermentable sugar (glucose, 1 to 7%). Among the wastes, residual yeast showed the highest potential for the production of extracellular enzymes, generating a proteolytic extract with 2.6 U/mL in 3 h. However, due to the low content of the fermentable sugars in the medium, the addition of glucose also had a positive effect, increasing the proteolytic activity to 4.9 U/mL. The best experimental conditions of each experimental design were reproduced for comparison, and the enzyme content was separated by ethanol precipitation. The best medium produced a precipitated protein with proteolytic activity of 145.5 U/g.
doi:10.17113/ftb.55.02.17.4378
PMCID: PMC5569352
brewery waste; waste reuse; lactic fermentation; proteolytic enzymes
12.  New lager yeast strains generated by interspecific hybridization 
The interspecific hybrid Saccharomyces pastorianus is the most commonly used yeast in brewery fermentations worldwide. Here, we generated de novo lager yeast hybrids by mating a domesticated and strongly flocculent Saccharomyces cerevisiae ale strain with the Saccharomyces eubayanus type strain. The hybrids were characterized with respect to the parent strains in a wort fermentation performed at temperatures typical for lager brewing (12 °C). The resulting beers were analysed for sugar and aroma compounds, while the yeasts were tested for their flocculation ability and α-glucoside transport capability. These hybrids inherited beneficial properties from both parent strains (cryotolerance, maltotriose utilization and strong flocculation) and showed apparent hybrid vigour, fermenting faster and producing beer with higher alcohol content (5.6 vs 4.5 % ABV) than the parents. Results suggest that interspecific hybridization is suitable for production of novel non-GM lager yeast strains with unique properties and will help in elucidating the evolutionary history of industrial lager yeast.
Electronic supplementary material
The online version of this article (doi:10.1007/s10295-015-1597-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s10295-015-1597-6
PMCID: PMC4412690  PMID: 25682107
Lager yeast; S. eubayanus; Brewing; Hybrid; Mating; Heterosis
13.  Nucleotide sequence and expression of the Enterobacter aerogenes alpha-acetolactate decarboxylase gene in brewer's yeast. 
The nucleotide sequence of a 1.4-kilobase DNA fragment containing the alpha-acetolactate decarboxylase gene of Enterobacter aerogenes was determined. The sequence contains an entire protein-coding region of 780 nucleotides which encodes an alpha-acetolactate decarboxylase of 260 amino acids. The DNA sequence coding for alpha-acetolactate decarboxylase was placed under the control of the alcohol dehydrogenase I promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both S. cerevisiae and Escherichia coli. Brewer's yeast cells transformed by this plasmid showed alpha-acetolactate decarboxylase activity and were used in laboratory-scale fermentation experiments. These experiments revealed that the diacetyl concentration in wort fermented by the plasmid-containing yeast strain was significantly lower than that in wort fermented by the parental strain. These results indicated that the alpha-acetolactate decarboxylase activity produced by brewer's yeast cells degraded alpha-acetolactate and that this degradation caused a decrease in diacetyl production.
PMCID: PMC202393  PMID: 3278689
14.  Yeasts in sustainable bioethanol production: A review 
Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.
Highlights
•Yeast strains applied in bioethanol production are highlighted.•Fermentation in bioethanol production by yeasts are discussed.•Factors that affect bioethanol production are considered.•Immobilization of yeasts in bioethanol production are highlighted.
doi:10.1016/j.bbrep.2017.03.003
PMCID: PMC5637245
Yeasts; Fermentation; Bioethanol; Immobilization
15.  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.
doi:10.3389/fmicb.2017.01690
PMCID: PMC5596070  PMID: 28943864
brewing; Sacchromyces pastorianus; evolutionary engineering; chemostat; maltose; maltotriose consumption rate; transport
16.  Improved Fermentation Performance of a Lager Yeast after Repair of Its AGT1 Maltose and Maltotriose Transporter Genes▿ †  
The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. α-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose. The AGT1 genes of brewer's ale yeast strains encode functional transporters, but the AGT1 genes of the lager strains studied contain a premature stop codon and do not encode functional transporters. In the present work, one or more copies of the AGT1 gene of a lager strain were repaired with DNA sequence from an ale strain and put under the control of a constitutive promoter. Compared to the untransformed strain, the transformants with repaired AGT1 had higher maltose transport activity, especially after growth on glucose (which represses endogenous α-glucoside transporter genes) and higher ratios of maltotriose transport activity to maltose transport activity. They fermented VHG (24° Plato) wort faster and more completely, producing beers containing more ethanol and less residual maltose and maltotriose. The growth and sedimentation behaviors of the transformants were similar to those of the untransformed strain, as were the profiles of yeast-derived volatile aroma compounds in the beers.
doi:10.1128/AEM.01558-08
PMCID: PMC2675207  PMID: 19181838
17.  Selection from Industrial Lager Yeast Strains of Variants with Improved Fermentation Performance in Very-High-Gravity Worts▿  
There are economic and other advantages if the fermentable sugar concentration in industrial brewery fermentations can be increased from that of currently used high-gravity (ca. 14 to 17°P [degrees Plato]) worts into the very-high-gravity (VHG; 18 to 25°P) range. Many industrial strains of brewer's yeast perform poorly in VHG worts, exhibiting decreased growth, slow and incomplete fermentations, and low viability of the yeast cropped for recycling into subsequent fermentations. A new and efficient method for selecting variant cells with improved performance in VHG worts is described. In this new method, mutagenized industrial yeast was put through a VHG wort fermentation and then incubated anaerobically in the resulting beer while maintaining the α-glucoside concentration at about 10 to 20 g·liter−1 by slowly feeding the yeast maltose or maltotriose until most of the cells had died. When survival rates fell to 1 to 10 cells per 106 original cells, a high proportion (up to 30%) of survivors fermented VHG worts 10 to 30% faster and more completely (residual sugars lower by 2 to 8 g·liter−1) than the parent strains, but the sedimentation behavior and profiles of yeast-derived flavor compounds of the survivors were similar to those of the parent strains.
doi:10.1128/AEM.03153-09
PMCID: PMC2832358  PMID: 20081007
18.  Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation 
Background
The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.
Results
The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations.
Conclusion
In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene expression and enzymatic activity of alcohol deydrogenase and pyruvate decarboxilase.
doi:10.1186/1475-2859-11-18
PMCID: PMC3295710  PMID: 22305374
Multistarter fermentation; Saccharomyces cerevisiae; Starmerella bombicola; Immobilization; Real-time RT-PCR
19.  Multiple α-Glucoside Transporter Genes in Brewer’s Yeast 
Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.
PMCID: PMC91046  PMID: 9925567
20.  Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using "-omics" techniques 
Background
Addition of sugar syrups to the basic wort is a popular technique to achieve higher gravity in beer fermentations, but it results in dilution of the free amino nitrogen (FAN) content in the medium. The multicomponent protease enzyme Flavourzyme has beneficial effect on the brewer's yeast fermentation performance during high gravity fermentations as it increases the initial FAN value and results in higher FAN uptake, higher specific growth rate, higher ethanol yield and improved flavour profile.
Results
In the present study, transcriptome and metabolome analysis were used to elucidate the effect on the addition of the multicomponent protease enzyme Flavourzyme and its influence on the metabolism of the brewer's yeast strain Weihenstephan 34/70. The study underlines the importance of sufficient nitrogen availability during the course of beer fermentation. The applied metabolome and transcriptome analysis allowed mapping the effect of the wort sugar composition on the nitrogen uptake.
Conclusion
Both the transcriptome and the metabolome analysis revealed that there is a significantly higher impact of protease addition for maltose syrup supplemented fermentations, while addition of glucose syrup to increase the gravity in the wort resulted in increased glucose repression that lead to inhibition of amino acid uptake and hereby inhibited the effect of the protease addition.
doi:10.1186/1475-2859-10-27
PMCID: PMC3107165  PMID: 21513553
21.  Vegemite Beer: yeast extract spreads as nutrient supplements to promote fermentation 
PeerJ  2016;4:e2271.
Vegemite is an iconic Australian food spread made from spent brewers’ yeast extract, which has been reported to be used as an ingredient in illegal home brewing. In this study, we tested the utility of Vegemite and the similar spread Marmite in promoting fermentation. We could not culture microorganisms from either Vegemite or Marmite, consistent with these food-grade spreads being essentially sterile. To test if the addition of Vegemite or Marmite could assist in fermentation when additional viable yeast was also present, solutions containing glucose and a range of concentrations of either Vegemite or Marmite were inoculated with brewers’ yeast. No fermentation occurred in any condition without addition of extra brewer’s yeast. Fermentation did not occur when yeast was inoculated into solutions containing only glucose, but progressed efficiently with when Vegemite or Marmite was also added. Gas Chromatography confirmed that ethanol was present at ∼3% v/v post-fermentation in all samples which contained glucose, Vegemite or Marmite, and brewers’ yeast. Trace amounts of methanol were also detected. Mass spectrometry proteomics identified abundant intracellular yeast proteins and barley proteins in Vegemite and Marmite, and abundant secreted yeast proteins from actively growing yeast in those samples to which extra brewers’ yeast had been added. We estimate that the real-world cost of home brewed “Vegemite Beer” would be very low. Our results show that Vegemite or other yeast extract spreads could provide cheap and readily available sources of nutrient supplementation to increase the efficiency of fermentation in home brewing or other settings.
doi:10.7717/peerj.2271
PMCID: PMC4991886  PMID: 27602264
Yeast; Vegemite; Fermentation; Ethanol; Yeast extract
22.  Growth characteristics of Saccharomyces rouxii isolated from chocolate syrup. 
We investigated the growth parameters of Saccharomyces rouxii isolated from spoiled chocolate syrup. The optimum pH range for S. rouxii was 3.5 to 5.5, whereas the minimum and maximum pH values that permitted growth were 1.5 and 10.5, respectively. For cells grown in 0 and 60% sucrose the optimum water activity (aw) values were 0.97 and 0.96, respectively. The optimum temperature for S. rouxii increased with a decreasing aw regardless of whether glucose or sucrose was used as the humectant. The optimum temperatures for S. rouxii were 28 degrees C at an aw of greater than 0.995 and 35 degrees C at an aw of 0.96 to 0.90 in 2 X potato dextrose broth with sucrose. Increasing the sorbate concentration (from 0.03 to 0.10%) caused the growth of S. rouxii to become more inhibited between aws of greater than 0.995 and 0.82. S. rouxii did not grow when the sorbate level was 0.12% (wt/vol). At lower sorbate levels, the effect of sorbate on the growth of S. rouxii depended on the aw level. Lowering the aw enhanced the resistance of S. rouxii to increasing concentrations of potassium sorbate. Permeability and polyol production are discussed with respect to sorbate tolerance of S. rouxii at different aw levels.
PMCID: PMC242508  PMID: 6615600
23.  Performance of non‐conventional yeasts in co‐culture with brewers’ yeast for steering ethanol and aroma production 
Microbial Biotechnology  2017;10(6):1591-1602.
Summary
Increasing interest in new beer types has stimulated the search for approaches to extend the metabolic variation of brewers’ yeast. Therefore, we tested two approaches using non‐conventional yeast to create a beer with lower ethanol content and a complex aroma bouquet. First, the mono‐culture performance was monitored of 49 wild yeast isolates of Saccharomyces cerevisiae (16 strains), Cyberlindnera fabianii (9 strains) and Pichia kudriavzevii (24 strains). Interestingly, both C. fabianii and P. kudriavzevii isolates produced relatively more esters compared with S. cerevisiae isolates, despite their limited fermentation capacity. Next, one representative strain of each species (Sc131, Cf65 and Pk129) was applied as co‐culture with brewers’ yeast (ratio 1:1). Co‐cultures with Cf65 and Pk129 resulted in a beer with lower alcohol content (3.5, 3.8 compared with 4.2% v/v) and relatively more esters. At higher inoculum ratios of Cf65 over brewers’ yeast, growth inhibition of brewers’ yeast was observed, most likely caused by competition for oxygen between brewers’ yeast and Cf65 resulting in a reduced level of ethanol and altered aroma profiles. With this study, we demonstrate the feasibility of using non‐conventional yeast species in co‐cultivation with traditional brewers’ yeast to tailor aroma profiles as well as the final ethanol content of beer.
doi:10.1111/1751-7915.12717
PMCID: PMC5658577  PMID: 28834151
24.  Ploidy influences the functional attributes of de novo lager yeast hybrids 
The genomes of hybrid organisms, such as lager yeast (Saccharomyces cerevisiae × Saccharomyces eubayanus), contain orthologous genes, the functionality and effect of which may differ depending on their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification. The hybrids were characterized in fermentations of both high gravity wort (15 °P) and very high gravity wort (25 °P), which were monitored for aroma compound and sugar concentrations. The hybrid strains with higher DNA content performed better during fermentation and produced higher concentrations of flavor-active esters in both worts. The hybrid strains also outperformed both the parent strains. Genome sequencing revealed that several genes related to the formation of flavor-active esters (ATF1, ATF2¸ EHT1, EEB1, and BAT1) were present in higher copy numbers in the higher ploidy hybrid strains. A direct relationship between gene copy number and transcript level was also observed. The measured ester concentrations and transcript levels also suggest that the functionality of the S. cerevisiae- and S. eubayanus-derived gene products differs. The results contribute to our understanding of the complex molecular mechanisms that determine phenotypes in lager yeast hybrids and are expected to facilitate targeted strain development through interspecific hybridization.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-016-7588-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-016-7588-3
PMCID: PMC4947488  PMID: 27183995
Lager yeast; S. eubayanus; Brewing; Hybrid; Rare mating; Heterosis
25.  Population Size Drives Industrial Saccharomyces cerevisiae Alcoholic Fermentation and Is under Genetic Control▿†‡  
Alcoholic fermentation (AF) conducted by Saccharomyces cerevisiae has been exploited for millennia in three important human food processes: beer and wine production and bread leavening. Most of the efforts to understand and improve AF have been made separately for each process, with strains that are supposedly well adapted. In this work, we propose a first comparison of yeast AFs in three synthetic media mimicking the dough/wort/grape must found in baking, brewing, and wine making. The fermentative behaviors of nine food-processing strains were evaluated in these media, at the cellular, populational, and biotechnological levels. A large variation in the measured traits was observed, with medium effects usually being greater than the strain effects. The results suggest that human selection targeted the ability to complete fermentation for wine strains and trehalose content for beer strains. Apart from these features, the food origin of the strains did not significantly affect AF, suggesting that an improvement program for a specific food processing industry could exploit the variability of strains used in other industries. Glucose utilization was analyzed, revealing plastic but also genetic variation in fermentation products and indicating that artificial selection could be used to modify the production of glycerol, acetate, etc. The major result was that the overall maximum CO2 production rate (Vmax) was not related to the maximum CO2 production rate per cell. Instead, a highly significant correlation between Vmax and the maximum population size was observed in all three media, indicating that human selection targeted the efficiency of cellular reproduction rather than metabolic efficiency. This result opens the way to new strategies for yeast improvement.
doi:10.1128/AEM.02547-10
PMCID: PMC3126379  PMID: 21357433

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