Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pH_i) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (μ_max) of 0.58–1.50 h⁻¹. At pH 2.5, slow growth was observed for the isolates from mawè (μ_max of 0.06–0.80 h⁻¹), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pH_i homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.     

The influence of pre‐fermentative practices on the dominance of inoculated yeast starter under industrial conditions

The influence of pre‐fermentative practices on the growth dynamics of a ‘natural’ starter culture with specific phenotype (H₂S^?) concurrently with wild yeast populations was evaluated under winery conditions. Different clarification procedures and added SO₂ strongly influenced species and cell numbers isolable at the pre‐fermentation stage. Independent treatments of must with sulphite addition or vacuum‐filtering clarification caused a 30‐fold reduction in viable cells. Clarification procedures, enhanced by the selective effect of SO₂ addition, induced the appearance of Saccharomyces cerevisiae ‘wild’ yeasts. Correct application of the inoculum generally guarantees the dominance of fermentation by starter cultures. However, inoculated fermentations using unclarified white and red musts exhibited a consistent presence and persistence of non‐Saccharomyces and/or Saccharomyces ‘wild’ yeasts during fermentation. The extent and composition of the initial wild microflora at the start of fermentation may affect the presence and persistence of wild Saccharomyces and non‐Saccharomyces yeasts during guided fermentations under commercial conditions. The above findings confirm the results of previous works carried out at laboratory‐ or pilot‐scale level. Furthermore, they suggest a clear correlation between the modality of pre‐fermentative practices and the presence and persistence of ‘wild’ yeasts during fermentation. © 2002 Society of Chemical Industry     

Characterization of the recombinant succinic semi‐aldehyde dehydrogenase from Saccharomyces cerevisiae

The yeast succinic semi‐aldehyde dehydrogenase gene (SSADH; EC 1.2.1.16) was cloned and overexpressed in Escherichia coli. Based on SDS–PAGE, the molecular mass of the subunit was around 54 kDa, and the purified recombinant enzyme had a tetrameric molecular mass of ca. 200 kDa. The specific activity of the recombinant enzyme was 1.90 µm NADH formed/min/mg, and showed maximal activity at pH 8.4. The recombinant protein was highly specific for succinate semi‐aldehyde (K_m = 15.48 ± 0.14 µm ) and could use both NAD⁺ and NADP⁺ as co‐factors, with K_m values of 579.06 ± 30.1 µm and 1.017 ± 0.46 mm, respectively. Initial velocity studies showed that NADH was a competitive inhibitor with respect to NAD⁺ (K_i = 129.5 µm ) but a non‐competitive inhibitor with respect to succinate semi‐aldehyde. Adenine nucleotides of AMP, ADP and ATP inhibited yeast SSADH activity with K_i = 1.13–10.2 mm, and showed competitive inhibition with respect to NAD⁺ and mixed‐competitive, non‐competitive and non‐competitive inhibition, respectively, with respect to succinate semi‐aldehyde. The kinetic data suggest a 'ping‐pong' mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

Random and targeted gene integrations through the control of non‐homologous end joining in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus DMKU3‐1042 is a thermotolerant yeast strain suitable for high‐temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 × 10⁶ transformants/µg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3‐1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non‐homologous end‐joining (NHEJ) pathway. In yeast lacking KU70, high‐frequency non‐homologous gene integration was abolished and the Kmku70 mutants showed 82–95% homologous gene targeting efficiencies using homologous sequences of 40–1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid‐free gene manipulations in K. marxianus. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluating potential applications of indigenous yeasts and their β‐glucosidases

The potential applications of wild yeast strains with β‐glucosidase activity were investigated by assaying their enzymatic production under simulated oenological conditions, coupled with the exploration of the potential applications of the β‐glucosidases by studying the enzymatic activity and stability under similar oenological conditions. The assay of enzymatic locations revealed that the β‐glucosidase activities from these wild strains occurred in the extracellular fraction, and in whole and permeabilized cells. The effects of different oenological factors on β‐glucosidase production indicated that the F6 Trichosporon asahii strain had higher β‐glucosidase production than the other strains under low pH conditions. However, the F35 Hanseniaspora uvarum strain and the F30 Saccharomyces cerevisiae strain showed higher β‐glucosidase production under high‐sugar conditions. Furthermore, the influence of oenological factors on the activity and stability of the β‐glucosidases revealed that the enzyme from the F6 T. asahii strain had a stronger low‐pH‐value resistance than the other yeast β‐glucosidases. These results suggest that the F35 H. uvarum, F30 S. cerevisiae and the F6 T. asahii β‐glucosidases may have some potentially applicable values in the fermentation industry. Copyright © 2015 The Institute of Brewing & Distilling     

Tryptophan accumulation in Saccharomyces cerevisiae under the influence of an artificial yeast TRP gene cluster

Plasmid pME559, carrying all five yeast TRP genes, was constructed. This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 μm-DNA sequences derived from plasmid pJDB207. We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants. These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2^fbr mutant allele. For stable maintenance of the plasmids in yeast a selection was necessary. Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found. In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.     

A simple colony‐formation assay in liquid medium,termed ‘tadpoling’, provides a sensitive measure of Saccharomyces cerevisiae culture viability

Here we describe the first high‐throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high‐throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10⁸ dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high‐throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space‐saving method, amenable to high‐throughput screens, for accurately measuring yeast cell viability. Copyright © 2013 John Wiley & Sons, Ltd.     

Measurement of urine pH and net acid excretion and their association with urine calcium excretion in periparturient dairy cows

Urine pH (U_pH) and net acid excretion (NAE) are used to monitor the degree of systemic acidification and predict the magnitude of resultant hypercalciuria when feeding an acidogenic ration to control periparturient hypocalcemia in dairy cattle. The objectives of this study were to evaluate the diagnostic performance of urine dipstick and pH paper for measuring U_pH, and to characterize the U_pH–NAE relationship and the association of urine Ca concentration ([Ca]) with U_pH and NAE. Urine samples (n = 1,116) were collected daily from 106 periparturient Holstein-Friesian cows fed an acidogenic ration during late gestation. Net acid excretion was measured by titration, and U_pH was measured by a glass-electrode pH meter (reference method), Multistix-SG urine dipsticks (Siemens Medical Solutions Inc., Ann Arbor, MI), and Hydrion pH paper (Micro Essential Laboratory Inc., Brooklyn, NY). Diagnostic performance was evaluated using Spearman correlation coefficient (r_s), Bland–Altman plots, and logistic regression. Urine pH measured by urine dipstick (r_s = 0.94) and pH paper (r_s = 0.96) were strongly associated with U_pH. Method-comparison studies indicated that the urine dipstick measured an average of 0.28 pH units higher, and pH paper 0.10 pH units lower, than U_pH. Urine [Ca] was more strongly associated with U_pH (r_s = −0.65) than NAE (r_s = 0.52). Goals for controlling periparturient hypocalcemia under the study conditions were U_pH <6.22 and <6.11, based on achieving urine [Ca] ≥5 mmol/L and estimated urinary Ca excretion ≥4 g/d, respectively. Urine pH was as accurate at predicting urine [Ca] as NAE when U_pH >6.11. We conclude that pH paper is an accurate, practical, and low-cost cow-side test for measuring U_pH and provides a clinically useful estimate of urine [Ca].     

MPR1 as a novel selection marker in Saccharomyces cerevisiae

L ‐Azetidine‐2‐carboxylic acid (AZC) is a toxic four‐membered ring analogue of L ‐proline that is transported into cells by proline transporters. AZC and L ‐proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Σ1278b as a multicopy suppressor of AZC‐induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N‐acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C‐derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose‐inducible MPR1 and ppr1⁺, the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC‐resistant colonies were also successfully selected. We concluded that our MPR1–AZC system provides a powerful tool for yeast transformation. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of Intracellular pH in <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 to Determine the Effect of Chlorine Dioxide Decontamination

The aim of this study was to investigate the response of Escherichia coli O157:H7 when exposed to different concentrations of sanitation agent chlorine dioxide (ClO₂) by determining intracellular pH (pH_i). For this purpose, fluorescence ratio imaging microscopy was used together with pH-sensitive, ratiometric green fluorescent protein that was introduced in E. coli O157:H7 cells. Along with pH_i, colony counts were determined during the treatment with ClO₂. Results revealed several post-treatment subpopulations with different physiological states, as judged by their pH_i. The fraction of cells with no pH gradient increased, and the colony count decreased as the concentration of ClO₂ increased.     

Production of fungal biomass from cormel process waste‐water of cocoyam (Xanthosoma sagittifolium (L) Schott) using Aspergillus oryzae obtained from cormel flour

Aspergillus oryzae obtained from spoilt cormel flour was subjected to mutation treatments using X‐rays, solar radiation and bleach. Exposure of A oryzae spores to X‐radiation of 50 kV at 20 mA s and other treatments induced both mutation and lethality in the organism. Following selection and screening of viable colonies on a medium containing cormel process waste‐water as the only carbon source, two strains, A oryzae No 15 and A oryzae No 8, which significantly (P ≤ 0.05) produced more biomass at a higher growth rate than the wild parent, were chosen for single‐cell protein production. Nutrient content of the single‐cell protein produced by the mutants was comparable to that of the wild type. Addition of 3.0 g each of (NH₄)₂SO₄, NH₄NO₃, NH₄Cl, and urea in 1 l of Xanthosoma process waste‐water increased the growth rate of mutants, with the highest increase observed with urea. Medium amended with urea also produced fungal biomass with the highest protein level of 7.97 g l^?1 for A oryzae No 8 strain compared with a protein yield of 3.97 g l^?1 obtained in the control. Total biomass produced after 54 h was 22.47 g l^?1 for A oryzae No 15 strain when urea was added, whereas only 15.20 g l^?1 was produced when no nitrogen source was added. The optimal temperature for single‐cell protein production was found to be 35 °C and the optimal pH was 3.50. A speed of 100 rpm gave the largest quantity of fungal biomass for the mutants tested. Copyright © 2003 Society of Chemical Industry     

Growth of a tropical marine yeast Yarrowia lipolytica NCIM 3589 on bromoalkanes: relevance of cell size and cell surface properties

Yarrowia lipolytica 3589, a tropical marine yeast, grew aerobically on a broad range of bromoalkanes varying in carbon chain length and differing in degree and position of bromide group. Amongst the bromoalkanes studied, viz. 2‐bromopropane (2‐BP), 1‐bromobutane (1‐BB), 1,5‐dibromopentane (1,5‐DBP) and 1‐bromodecane (1‐BD), the best utilized was 1‐BD, with a maximal growth rate (μ_max) of 0.055 h^?1 and an affinity ratio (μ_max/K_s) of 0.022. Utilization of these bromoalkanes as growth substrates was associated with a concomitant release of bromide (8202.9 µm ) and cell mass (36 × 10⁹ cells/ml), occurring maximally on 1‐BD. Adherence of yeast cells to these hydrophobic bromoalkanes was observed microscopically, with an increase in cell size and surface hydrophobicity. The maximal cell diameter was for 1‐BD (4.66 µm), resulting in an increase in the calculated cell surface area (68.19 µm²) and sedimentation velocity (1.31 µm/s). Cell surface hydrophobicity values by microbial adhesion to solvents (MATS) analysis for yeasts grown on bromoalkanes and glucose were significantly high, i.e. >80%. Similarly, water contact angles also indicate that the cell surface of yeast cells grown in glucose possess a relatively more hydrophilic cell surface (θ = 49.1°), whereas cells grown in 1‐BD possess a more hydrophobic cell surface (θ = 90.7°). No significant change in emulsification activity or surface tension was detected in the cell‐free supernatant. Thus adherence to the bromoalkane droplets by an increase in cell size and surface hydrophobicity leading to debromination of the substrate might be the strategy employed in bromoalkane utilization and growth by Y. lipolytica 3589. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of weak acids and external pH on the intracellular pH of Zygosaccharomyces bailii,and its implications in weak-acid resistance

Weak acids and hydrogen ions in different concentration combinations affect the intracellular pH value (pH_i) of Zygosaccharomyces bailii. The lowest pH_i value measured was not at the most extreme, but at intermediate conditions of inhibition. Proton and organic-acid ejection, on a cell volume basis, is greater in cells grown under inhibitory conditions and is stimulated by weak acids, whilst in cells not grown under inhibitory conditions acid efflux is lower and is depressed by weak acids; this may be important in the maintenance of tolerable pH_i values in the presence of weak acids. The concentration of benzoic acid measured internally is identical to the value expected from its pK, external pH and pH_i. Addition of fructose to starved cells causes both a decreased pH_i and a concomitant efflux of previously loaded benzoic acid, quantitatively in accord with the shift in equilibrium of the freely permeable undissociated acid. There is no evidence that weak acids are actively extruded. Protoplast volume also varies with hydrogen-ion and weak-acid concentration and this too may play a role in intracellular pH maintenace.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Near infrared spectroscopy as an on-line method to quantitatively determine glycogen and predict ultimate pH in pre rigor bovine M. longissimus dorsi

The potential of near infrared (NIR) spectroscopy as an on-line method to quantify glycogen and predict ultimate pH (pH_u) of pre rigor beef M. longissimus dorsi (LD) was assessed. NIR spectra (538 to 1677 nm) of pre rigor LD from steers, cows and bulls were collected early post mortem and measurements were made for pre rigor glycogen concentration and pH_u. Spectral and measured data were combined to develop models to quantify glycogen and predict the pH_u of pre rigor LD. NIR spectra and pre rigor predicted values obtained from quantitative models were shown to be poorly correlated against glycogen and pH_u (r² = 0.23 and 0.20, respectively). Qualitative models developed to categorise each muscle according to their pH_u were able to correctly categorise 42% of high pH_u samples. Optimum qualitative and quantitative models derived from NIR spectra found low correlation between predicted values and reference measurements.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Latent polyphenol oxidase from quince fruit pulp (Cydonia oblonga): purification,activation and some properties

Quince fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning in Triton X‐114 and PEG 8000/phosphate with a final ammonium sulfate fractionation between 30% and 75%, to avoid the deep browning of the enzyme due to the high amount of oxidizing substances present in the quince pulp. The clean and stable enzyme was partially purified in a latent form and could be optimally activated by the presence of 0.5 g dm^?3 sodium dodecyl sulfate (SDS) with an optimum pH of 5.0. In the absence of SDS, the enzyme showed maximum activity at acid pH. The apparent kinetic parameters of the latent enzyme were determined at pH 5.0, the V_m value being 15 times higher in the presence of SDS than in its absence, whereas the K_M was the same in both cases, with a value of 1.2 mmol L^?1. The effect of several inhibitors was studied, tropolone being the most active with a K_i value of 4.7 µmol L^?1. In addition, the effect of cyclodextrins was studied, and the complexation constant (K_c) between 4‐tert‐butylcatechol and cyclodextrins was calculated using an enzymatic method. The value obtained for K_c was 15 310 mol L^?1. Copyright © 2006 Society of Chemical Industry     

Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter

A 3950 bp genomic fragment from Candida glycerinogenes, WL2002‐5, containing the CgGAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The CgGAP protein consisted of an N‐terminal NAD⁺‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the CgGAP gene. The influence of CgGAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with CgGAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S. cerevisiae using the green fluorescent protein (gfp) gene as a reporter showed that the GAP promoter (P_CgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of beer‐spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer‐spoilage yeasts

There have been many beer‐spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer‐spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer‐spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer‐spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer‐spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer‐spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling