Saccharomyces Carlsbergensis Research Articles

Old Yellow Enzyme homologues in Mucor circinelloides: expression profile and biotransformation

The reduction of C=C double bond, a key reaction in organic synthesis, is mostly achieved by traditional chemical methods. Therefore, the search for enzymes capable of performing this reaction is rapidly increasing. Old Yellow Enzymes (OYEs) are flavin-dependent oxidoreductases, initially isolated from Saccharomyces pastorianus. In this study, the presence and activation of putative OYE enzymes was investigated in the filamentous fungus Mucor circinelloides, which was previously found to mediate C=C reduction. Following an in silico approach, using S. pastorianus OYE1 amminoacidic sequence as template, ten putative genes were identified in the genome of M. circinelloides. A phylogenetic analysis revealed a high homology of McOYE1-9 with OYE1-like proteins while McOYE10 showed similarity with thermophilic-like OYEs. The activation of mcoyes was evaluated during the transformation of three different model substrates. Cyclohexenone, α-methylcinnamaldehyde and methyl cinnamate were completely reduced in few hours and the induction of gene expression, assessed by qRT-PCR, was generally fast, suggesting a substrate-dependent activation. Eight genes were activated in the tested conditions suggesting that they may encode for active OYEs. Their expression over time correlated with C=C double bond reduction.

Identification and Characterization of Phospholipids with Very Long Chain Fatty Acids in Brewer's Yeast.

Yeast lipids and fatty acids (FA) were analyzed in Saccharomyces pastorianus from seven breweries and in the dietary yeast supplement Pangamin. GC-MS identified more than 30 FA, half of which were very-long chain fatty acids (VLCFA) with hydrocarbon chain lengths of ≥22 C atoms. Positional isomers ω-9 and ω-7 were identified in FA with C18-C28 even-numbered alkyl chains. The most abundant ω-7 isomer was cis-vaccenic acid. The structure of monounsaturated FA was proved by dimethyl disulfide adducts (position of double bonds and cis geometric configuration) and by GC-MS of pyridyl carbinol esters. Ultra-high performance liquid chromatography-tandem mass spectrometry with negative electrospray ionization identified the phospholipids phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine, with more than 150 molecular species. Wild-type unmutated brewer's yeast strains conventionally used for the manufacture of food supplements were found to contain VLCFA.

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.

Alternative Saccharomyces interspecies hybrid combinations and their potential for low-temperature wort fermentation.

The lager yeast hybrid (Saccharomyces cerevisiae × Saccharomyces eubayanus) possesses two key characteristics that are essential for lager brewing: efficient sugar utilization and cold tolerance. Here we explore the possibility that the lager yeast phenotype can be recreated by hybridizing S. cerevisiae ale yeast with a number of cold‐tolerant Saccharomyces species including Saccharomyces arboricola, Saccharomyces eubayanus, Saccharomyces mikatae and Saccharomyces uvarum. Interspecies hybrids performed better than parental strains in lager brewing conditions (12°C and 12°P wort), with the S. mikatae hybrid performing as well as the S. eubayanus hybrid. Where the S. cerevisiae parent was capable of utilizing maltotriose, this trait was inherited by the hybrids. A greater production of higher alcohols and esters by the hybrids resulted in the production of more aromatic beers relative to the parents. Strong fermentation performance relative to the parents was dependent on ploidy, with polyploid hybrids (3n, 4n) performing better than diploid hybrids. All hybrids produced 4‐vinyl guaiacol, a smoke/clove aroma generally considered an off flavour in lager beer. This characteristic could however be eliminated by isolating spore clones from a fertile hybrid of S. cerevisiae and S. mikatae. The results suggest that S. eubayanus is dispensable when constructing yeast hybrids that express the typical lager yeast phenotype. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

The Importance of a Comparative Characterization of Saccharomyces Cerevisiae and Saccharomyces Pastorianus Strains for Brewing

The volume and market share loss for classical beer types such as pils beer and wheat beer has been declining for several years, but the overall beer market remains almost unchanged as a result of the increasing interest in beer specialties Due to high biodiversity, the diversity of the strains, and the different flavor profiles, reliable and practical information regarding the characteristics of individual brewing strains is required to help brewers to find the right strain for their brewing purposes. This paper presents a comparison of 10 commercially available Technical University of Munich (TUM) brewing yeast strains. The strains were screened for genetic and phenotypic characteristics. After confirming the genetic distinctiveness by using species-specific real-time polymerase chain reaction (RT-PCR) systems and a strain typing method based on PCR-capillary electrophoresis of the partial intergenic spacer 2 (IGS2) fragment (IGS2-314 PCR-capillary electrophoresis), the strains were tested regarding phenotypic characteristics under controlled and identical fermentation conditions in small-scale brewing trials. Besides the fermentation performance, flocculation behavior, sugar metabolism and other phenotypic characteristics, the main focus was on the flavor and aroma profile of each investigated TUM yeast strain.

Recombination sites on hybrid chromosomes in Saccharomyces pastorianus share common sequence motifs and define a complex evolutionary relationship between group I and II lager yeasts.

Saccharomyces pastorianus, referred to as lager yeasts, are hybrids of S. cerevisiae and S. eubayanus. Isolates within the species are divided into two groups (I and II) based on chromosome structure and composition. Following the hybridisation, the parental chromosomes underwent homeologous recombination, generating a set of hybrid chromosomes unique to the species. Here, we assessed the recombination events in seven lager yeast genomes to more clearly define the evolutionary route of lager yeasts. Meta-analysis of the recombination epicentres, as well as a detailed analysis of recombination events at the MAT locus, reveals a more complex evolutionary relationship between the group I and II lager yeasts than previously considered and identifies several divergent routes of evolution leading to the current S. pastorianus strains. We show that recombination epicentres contain sequential runs of pyrimidines, often flanked by purines, on one strand of the DNA, and identify two common sequence motifs present in >80% of the recombination epicentres, indicating that a common mechanism might account for the recombination events. Taken together, the data support a sequential hybridisation model of evolution for the two types of lager yeasts and suggest that the genomes of this newly emerged species are highly dynamic and continually evolving.

Characterization of dimethyl sulphoxide reductase from brewing yeast

Dimethyl sulphoxide (DMSO) reductase is present in all brewing yeast strains tested, but is more readily detectable using activity stains on polyacrylamide gels than through direct spectrophotometric assay of crude extracts. The enzyme is detectable at higher activities in yeast cultivated at lower temperatures and this is because of increased expression of the primary gene that codes for the enzyme in yeast, MXR1. DMSO somewhat increases enzyme expression, notably in lager yeast by up to 70%, whereas methionine suppresses it. Both ale and lager strains produce enzymes of ~100,000 in molecular weight, but ion exchange chromatography divides the activity for both ale and lager yeasts into two fractions with distinct properties (heat tolerance, pH optimum, kinetic parameters). Copyright © 2017 The Institute of Brewing & Distilling

Reduction of toxicologically relevant styrene in wheat beer using specially produced wheat and barley malts

2-Methoxy-4-vinylphenol and 4-vinylphenol are important odorants in wheat beer, which are mainly enzymatically formed by decarboxylation during fermentation with the top fermenting yeast Saccharomyces cerevisiae from their respective precursors, the free phenolic acids ferulic acid and p-coumaric acid. However, also the undesired toxicologically relevant styrene can be formed from cinnamic acid by the same mechanism. Micro-brewing trials with specially produced barley and wheat malts were performed, and the concentrations of the free phenolic acids (precursors) and the corresponding vinyl aromatics were quantitated in the malts and the intermediates of beer production (unboiled wort, cast wort, and green beer) using stable isotopically labelled internal standards. Thereby, 100% wheat or barley malts produced with either high or low malting parameters (steeping degree, germination temperature, and germination time) were used for brewing. Furthermore, the influence of two different yeasts, Saccharomyces cerevisiae strain W68 (top fermenting) and Saccharomyces carlsbergensis strain W34/78 (bottom fermenting), on the amounts of the phenolic acids and the corresponding vinyl aromatics was investigated. Results indicated that modifications in barley malting parameters did not have a mitigating influence on the styrene concentration in beer. However, using wheat malt produced with low malting parameters for brewing revealed a reduced styrene amount by about 30% compared to the brew produced with wheat malt obtained by high malting parameters. In addition, the lowered styrene beer did still fulfil the consumer´s expectation in regard to its overall aroma.

Bactericidal effect of 266 to 279 nm wavelength UVC-LEDs for inactivation of Gram positive and Gram negative foodborne pathogenic bacteria and yeasts

Recently, UVC-LED technology has been validated as an alternative to irradiation with conventional mercury UV lamps. In this study, we sought to determine primary factors affecting reduction trends shown in several microorganisms. Four major foodborne pathogens (Escherichia coli O157:H7, Salmonella spp. Listeria monocytogenes, Staphylococcus aureus) and spoilage yeasts (Saccharomyces pastorianus, Pichia membranaefaciens), important to the brewing industry, were inoculated onto selective and non-selective media in order to investigate reduction tendencies at 4 different peak wavelengths (266 to 279nm). As irradiation dose increased, inactivation levels for every microorganism were enhanced, but there were different UV-sensitivities in Gram positive bacteria (GP), Gram negative bacteria (GN), and yeasts (Y). Loss of membrane integrity measured by propidium iodide (PI) increased as peak wavelength increased for every microorganism studied. Similar results were observed in membrane potential measured by DiBAC4(3). However, there were contrasting results which showed that greater DNA damage occurred at a lower peak wavelength as measured by Hoechst 33,258. The level of DNA damage was strongly related to trends of microbial inactivation. This study showed that even though membrane damage was present in every microorganism studied, DNA damage was the primary factor for inactivating microorganisms through UVC-LED treatment.

Inheritance of brewing-relevant phenotypes in constructed Saccharomyces cerevisiae\xa0\xd7\xa0Saccharomyces eubayanus hybrids

BackgroundInterspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity.ResultsHere we generated a set of five hybrids from three different parent strains, two of which contained DNA from all three parent strains. These hybrids were constructed with fertile allotetraploid intermediates, which were capable of efficient sporulation. We used these eight brewing strains to examine two brewing-relevant phenotypes: stress tolerance and phenolic off-flavour formation. Lipidomics and multivariate analysis revealed links between several lipid species and the ability to ferment in low temperatures and high ethanol concentrations. Unsaturated fatty acids, such as oleic acid, and ergosterol were shown to positively influence growth at high ethanol concentrations. The ability to produce phenolic off-flavours was also successfully removed from one of the hybrids, Hybrid T2, through meiotic segregation. The potential application of these strains in industrial fermentations was demonstrated in wort fermentations, which revealed that the meiotic segregant Hybrid T2 not only didn’t produce any phenolic off-flavours, but also reached the highest ethanol concentration and consumed the most maltotriose.ConclusionsOur study demonstrates the possibility of constructing complex yeast hybrids that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. Yeast lipid composition was also shown to have a central role in determining ethanol and cold tolerance in brewing strains.

Application of Plackett-Burman experimental design for investigating the effect of wort amino acids on flavour-active compounds production during lager yeast fermentation

Aroma-active higher alcohols and esters are produced intracellularly in the cytosol by fermenting lager yeast cells, which are of major industrial interest because they determine aroma and taste characteristics of the fermented beer. Wort amino acid composition and their utilization by yeast during brewer's wort fermentation influence both the yeast fermentation performance and the flavour profile of the finished product. To better understand the relationship between the yeast cell and wort amino acid composition, Plackett–Burman screening design was applied to measure the changes in nitrogen composition associated with yeast amino acids uptake and flavour formation during fermentation. Here, using an industrial lager brewing strain of Saccharomyces pastorianus, we investigated the effect of amino acid composition on the accumulation of higher alcohols and volatile esters. The objective of this study was to identify the significant amino acids involved in the flavour production during beer fermentation. Our results showed that even though different flavour substances were produced with different amino acid composition in the fermentation experiments, the discrepancies were not related to the total amount of amino acids in the synthetic medium. The most significant effect on higher alcohol production was exercised by the content of glutamic acid, aromatic amino acids and branch chain amino acids. Leucine, valine, glutamic acid, phenylalanine, serine and lysine were identified as important determinants for the formation of esters. The future applications of this information could drastically improve the current regime of selecting malt and adjunct or their formula with desired amino acids in wort. Copyright © 2017 The Institute of Brewing & Distilling

Biosorption of Cd(II) and Cs(I) from aqueous solution by live and dead cells of Saccharomyces carlsbergensis PTCC 5051

ABSTRACTThe biosorption characteristics of Cd(II) and Cs(I) using live and dead cells of Saccharomyces carlsbergensis PTCC 5051 as biosorbents have been investigated in the present research. The influence of different experimental parameters such as initial pH (pHi), shaking rate, sorption time and initial metal concentration was evaluated. The optimum pH was obtained as 4 for Cd(II) and 7 for Cs(I). The experimental adsorption data were fitted to the Langmuir linear equation adsorption model. The highest metal uptake values of 0.593 and 0.473 mmol g−1 were calculated for Cd(II) and Cs(I), respectively. The results of Fourier transform infrared analysis suggested the involvement of amine, carboxyl and hydroxyl groups during the biosorption process and also indicated that more functional groups were involved in the biosorption process of live adsorbents, compared with those linked to dead biomass. The results showed that the biomass of S. carlsbergensis PTCC 5051 is a suitable biosorbent for the removal of Cd(II) and Cs(I) from the aqueous solutions.

Characterization of Saccharomyces uvarum (Beijerinck, 1898) and related hybrids: assessment of molecular markers that predict the parent and hybrid genomes and a proposal to name yeast hybrids.

The use of the nuclear DNA reassociation technique has led taxonomists to consider Saccharomyces uvarum a synonym of S. bayanus. The latter, however, is not a species but a hybrid harbouring S. eubayanus (Seu) and S. uvarum (Su) subgenomes with a minor DNA contribution from S. cerevisiae (Sc). To recognize genetically pure lines of S. uvarum and putative interspecies hybrids among so-called S. bayanus strains present in public culture collections, we propose the use of four markers that were defined from the S. bayanus CBS 380T composite genome, namely SeuNTS2 (rDNA), ScMAL31, MTY1 and SuMEL1. Saccharomyces carlsbergensis CBS 1513 was found to be similar to S. bayanus except that it carries the SeuMEL1 allele. Different marker combinations revealed that among 33 strains examined only a few were similar to CBS 380T, but many pure S. uvarum lines and putative Su/Seu-related hybrids occurred. Our results demonstrated that these hybrids were erroneously considered authentic S. bayanus and therefore the varietal state 'Saccharomyces bayanus var. uvarum comb. nov. Naumov' is not valid. Our markers constitute a tool to get insights into the genomic makeup of Saccharomyces interspecies hybrids. We also make a proposal to name those hybrids that may also be applicable to other fungal hybrids.

Effect of Furfural on Saccharomyces carlsbergensis Growth, Physiology and Ethanol Production.

This work described the effect of furfural, a product resulting from the lignocellulosic material pretreatment, on Saccharomyces carlsbergensis growth and ethanol production. Flow cytometry was used to evaluate the yeast membrane potential, membrane integrity, reactive oxygen species production and lipid content. Above 0.3g/L of furfural, a progressive decrease in the maximal specific growth rate was observed, reaching 53% of the value obtained in the absence of toxic when the cells were grown in the presence of 4g/L of furfural. In general, the yeast biomass concentration and yield were less affected by the furfural presence than the specific growth rate, and a maximum reduction of 25% was observed for the assay at 4g/L. The ethanol production was even less affected by the furfural presence than the yeast growth. At 4g/L of furfural, the maximum ethanol concentration was reduced by only 10% relatively to the maximum ethanol concentration observed in the absence of toxic. At 5g/L of furfural, the yeast cells were barely able to keep metabolic functions and produced a final ethanol concentration of 0.87g/L although growth was undetectable. S. carlsbergensis membrane potential was affected by the furfural presence, concomitantly with the ethanol production. However, at 4g/L, most of the yeast cells (90%) displayed the cytoplasmic membrane depolarized. The proportion of cells with increasing reactive oxygen species (ROS) production levels increased for the experiments at 0-4g/L. For the experiment at 4.5g/L of furfural, ROS production was observed for only 11% of the yeast cells. The yeast lipid content was also severely affected by the furfural presence. Both polar and neutral lipids decreased in the presence of furfural, and this reduction was more notorious during the stationary phase.

Saccharomyces species in the Production of Beer

The characteristic flavour and aroma of any beer is, in large part, determined by the yeast strain employed and the wort composition. In addition, properties such as flocculation, wort fermentation ability (including the uptake of wort sugars, amino acids, and peptides), ethanol and osmotic pressure tolerance together with oxygen requirements have a critical impact on fermentation performance. Yeast management between fermentations is also a critical brewing parameter. Brewer’s yeasts are mostly part of the genus Saccharomyces. Ale yeasts belong to the species Saccharomyces cerevisiae and lager yeasts to the species Saccharomyces pastorianus. The latter is an interspecies hybrid between S. cerevisiae and Saccharomyces eubayanus. Brewer’s yeast strains are facultative anaerobes—they are able to grow in the presence or absence of oxygen and this ability supports their property as an important industrial microorganism. This article covers important aspects of Saccharomyces molecular biology, physiology, and metabolism that is involved in wort fermentation and beer production.

Using Flow Cytometry to Evaluate the Stress Physiological Response of the Yeast Saccharomyces carlsbergensis ATCC 6269 to the Presence of 5-Hydroxymethylfurfural During Ethanol Fermentations.

Lignocellulosic materials have been considered low-cost effective substrates for bioethanol production. However, lignocellulosic pretreatment releases toxic compounds such as 5-hydroxymethylfurfural (HMF) that is known to inhibit the yeast growth and ethanol production. In this work, flow cytometry was used to monitor the physiological response of the yeast Saccharomyces carlsbergensis ATCC 6269 in the presence of different initial HMF concentrations within the range of 0-15g/L, in terms of cell membrane integrity, potential, and intracellular lipids. It was observed that the HMF presence affected more significantly the yeast growth than the ethanol production. At 15g/L HMF, the yeast growth and fermentation ability were completely inhibited. The cell membrane integrity and potential decreased as the initial HMF concentration increased. At the end of the fermentation process with 10g/L HMF, the yeast culture contained 45% of cells with depolarized plasma membrane, 52% of cells with permeabilized plasma membrane, and 53% of cells with increasing reactive oxygen species (ROS) levels. Using the Nile Red stain, it was observed that intracellular polar lipids were more affected by the initial HMF concentration than the neutral lipids, probably due to the extensive membrane damage.

Distinct Domestication Trajectories in Top-Fermenting Beer Yeasts and Wine Yeasts

Beer is one of the oldest alcoholic beverages and isproduced by the fermentation of sugars derived from starches present in cereal grains. Contrary to lager beers, made by bottom-fermenting strains of Saccharomyces pastorianus, a hybrid yeast, ale beers are closer to the ancient beer type and are fermented by S.cerevisiae, a top-fermenting yeast. Here, we use population genomics to investigate (1) the closest relatives of top-fermenting beer yeasts; (2) whether top-fermenting yeasts represent an independent domestication event separate from those already described; (3) whether single ormultiple beer yeast domestication events can be inferred; and (4) whether top-fermenting yeasts represent non-recombinant or recombinant lineages. Our results revealed that top-fermenting beer yeasts are polyphyletic, with a main clade composed of at least three subgroups, dominantly represented by the German, British, and wheat beer strains. Other beer strains were phylogenetically close to sake, wine, or bread yeasts. We detected genetic signatures of beer yeast domestication by investigating genes previously linked tobrewing and using genome-wide scans. We propose that the emergence of the main clade of beer yeasts is related with a domestication event distinct from the previously known cases of wine and sake yeast domestication. The nucleotide diversity of the main beer clade more than doubled that of wine yeasts, which might be a consequenceof fundamental differences in the modes of beerand wine yeast domestication. The higher diversity of beer strains could be due to the more intense and different selection regimes associated to brewing.

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.

Quantitative analysis of 3-OH oxylipins in fermentation yeast.

Despite the ubiquitous distribution of oxylipins in plants, animals, and microbes, and the application of numerous analytical techniques to study these molecules, 3-OH oxylipins have never been quantitatively assayed in yeasts. The formation of heptafluorobutyrate methyl ester derivatives and subsequent analysis with gas chromatography - negative chemical ionization - mass spectrometry allowed for the first determination of yeast 3-OH oxylipins. The concentration of 3-OH 10:0 (0.68-4.82 ng/mg dry cell mass) in the SMA strain of Saccharomyces pastorianus grown in laboratory-scale beverage fermentations was elevated relative to oxylipin concentrations in plant tissues and macroalgae. In fermenting yeasts, the onset of 3-OH oxylipin formation has been related to fermentation progression and flocculation initiation. When the SMA strain was grown in laboratory-scale fermentations, the maximal sugar consumption rate preceded the lowest concentration of 3-OH 10:0 by ∼4.5 h and a distinct increase in 3-OH 10:0 concentration by ∼16.5 h.

Intracellular acidification and damage of cellular membrane of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles

The effects of intracellular acidification and damage of cellular membrane on the inactivation of Saccharomyces pastorianus by a two-stage method of low-pressure carbon dioxide microbubbles (two-stage MBCO2) were investigated. Intra/extra cellular pH (pHin and pHex) and propidium iodide (PI) uptake of S. pastorianus cells treated with the two-stage MBCO2 were measured by fluorescence analysis, and were compared with the inactivation efficiency. The pHex and pHin of S. pastorianus cells treated with only mixing vessel at 10 °C of two-stage MBCO2 was lower than those that were untreated. However, the surviving number and the pHin of S. pastorianus cells treated with the two-stage MBCO2 containing the mixing vessel at 1.0 MPa and the heating coil of 40 °C decreased concomitant with the exposure time, and the increase of the PI uptake ratio was confirmed. In addition, when the pressure of the mixing vessel was 1.0 MPa, the surviving number and the pHin of S. pastorianus cells significantly decreased, and the PI uptake ratio increased by increasing the temperature to 45 and 50 °C with the heating coil of the two-stage MBCO2. These results suggested that the inactivation of S. pastorianus by two-stage MBCO2 might be induced by the damage to the cellular membrane at 45 and 50 °C, and by the lowering of the pHin at 40 °C.