Brewery Samples Research Articles

Monitoring the evolution of free and cysteinylated aldehydes from malt to fresh and forced aged beer

During storage, beer staling coincides with a gradual increase in the concentrations of aldehydes resulting in the appearance of undesirable flavours. Cysteinylated aldehydes, also referred to as 2-substituted 1,3-thiazolidine-4-carboxylic acids, have been proposed as potential precursors of this increase. This study aimed to further understand the origin of aldehydes in aged beer, by monitoring both free and cysteinylated aldehydes throughout the brewing process, from the raw materials until the stored product. Quantification of free and cysteinylated aldehydes was performed for two different brews via headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), respectively. All selected marker aldehydes were quantified in malt, wort, and the resulting fresh and aged beer samples. Cysteinylated aldehydes were quantifiable in malt and up to the wort boiling phase. The highest levels of free aldehydes were found in malt, whereas cysteinylated aldehydes showed highest levels at mashing-in pointing to their formation during both malting and subsequent mashing-in. During beer ageing, an increase in all free aldehydes was measured. In particular, a rise in 2-methylpropanal and furfural is most striking. Although the presented experimental data obtained on malt and brewery samples do support the concept of bound-state aldehydes, cysteinylated aldehydes cannot be consider as the cause of increasing levels of staling aldehydes during beer ageing.

Characterization of Hop Acids in Spent Brewer's Yeast from Craft and Multinational Sources

Spent brewer's yeast, a significant byproduct of brewing, presents a disposal issue for craft breweries. To understand value-added opportunities for its use, we measured hop acid profiles of spent yeast from three beer styles produced by a craft brewery and spent yeast from three multinational breweries using high-performance liquid chromatography with photodiode array detection/mass spectrometry. The average α-acid content of craft yeast (2,074 ± 531 μg/g) was 12-fold higher than multinational brewery samples (167 ± 89 μg/g), and the average total hop acid content of craft yeast samples (2,557 ± 622 μg/g) was fivefold higher than the multinational brewery yeast samples (487 ± 136 μg/g). Centrifuged craft yeast slurry from both light and dark ales had 7-to 10-fold, 150- to 158-fold, and >200-fold higher content of iso-α-, α-, and ß-acids, respectively, than that in the corresponding beer, demonstrating a high affinity of brewer's yeast for hop acids. Ongoing research has shown that hop acids have antioxidant, anti-inflammatory, anticancer, and antibacterial activities. The elevated content found in spent craft brewer's yeast suggests consideration for use as a high-value functional food ingredient.

Development of a species-specific PCR assay for identification of the strictly anaerobic bacterium Selenomonas lacticifex found in biofilm-covered surfaces in brewery bottling halls.

In recent years, beer-spoilage cases from strictly anaerobic bacteria have risen in frequency, in connection with the production of non-pasteurized, non-alcohol and low-alcoholic beers and with the lowering of dissolved oxygen in the packaged beer. Selenomonas lacticifex, found in brewer's yeast and in biofilms covering some surfaces in brewery bottling area, is considered to be a beer-spoilage organism. This study aims to develop S.lacticifex-specific PCR assay. The objective of this study was also evaluation of the specificity and reproducibility of the developed PCR assay in real brewery samples. Three primers (one forward and two reverse) were designed for identification of the strictly anaerobic bacterium S.lacticifex on the basis of the species-specific sequences of the 16S rDNA region. The specificity of the primers was tested against 44 brewery-related non-target micro-organisms that could potentially occur in the same brewery specimens. None of the primer pairs amplified DNA from any of the non-S.lacticifex strains tested including genera from the same family (Pectinatus, Megasphaera, Zymophilus) and the closely related species Selenomonas ruminantium, showing thus 100% specificity. The PCR assay developed in this study enables the detection of the strictly anaerobic bacterium S.lacticifex in real brewery samples including pitching yeast. Selenomonas lacticifex-specific PCR assay developed in this study allows for the extension of the spectra of detected beer-spoilage micro-organisms in brewing laboratories and thus lowering the risk of contamination of the final product.

Identification of beer-spoilage bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of beer-spoilage bacteria was examined. To achieve this, an extensive identification database was constructed comprising more than 4200 mass spectra, including biological and technical replicates derived from 273 acetic acid bacteria (AAB) and lactic acid bacteria (LAB), covering a total of 52 species, grown on at least three growth media. Sequence analysis of protein coding genes was used to verify aberrant MALDI-TOF MS identification results and confirmed the earlier misidentification of 34 AAB and LAB strains. In total, 348 isolates were collected from culture media inoculated with 14 spoiled beer and brewery samples. Peak-based numerical analysis of MALDI-TOF MS spectra allowed a straightforward species identification of 327 (94.0%) isolates. The remaining isolates clustered separately and were assigned through sequence analysis of protein coding genes either to species not known as beer-spoilage bacteria, and thus not present in the database, or to novel AAB species. An alternative, classifier-based approach for the identification of spoilage bacteria was evaluated by combining the identification results obtained through peak-based cluster analysis and sequence analysis of protein coding genes as a standard. In total, 263 out of 348 isolates (75.6%) were correctly identified at species level and 24 isolates (6.9%) were misidentified. In addition, the identification results of 50 isolates (14.4%) were considered unreliable, and 11 isolates (3.2%) could not be identified. The present study demonstrated that MALDI-TOF MS is well-suited for the rapid, high-throughput and accurate identification of bacteria isolated from spoiled beer and brewery samples, which makes the technique appropriate for routine microbial quality control in the brewing industry.

Rapid, Simple, and Specific Cultivation-Based Method for Detection of Pectinatus spp. in Brewery Samples

Rapid, Simple, and Specific Cultivation-Based Method for Detection of Pectinatus spp. in Brewery Samples

Rapid, Simple, and Specific Cultivation-Based Method for Detection of Pectinatus spp. in Brewery Samples

A modified de Man–Rogosa–Sharpe (MRS) medium suitable for easy, fast, and specific detection of beer-spoilage bacteria Pectinatus spp. from beer and brewery swab samples was developed. It contains readily commercially available MRS broth, cysteine hydrochloride, and sodium thioglycolate as compounds lowering the redox potential of the medium, Tetra (a mixture of tetrahydroiso-α-acids) and 2-phenylethanol as the specificity-ensuring components, and bacteriological agar, which prevents medium oxidation during sample manipulation and transport. Tests with beer and brewery isolates of Pectinatus frisingensis, P. cerevisiiphilus, P. haikarae, Lactobacillus brevis, L. paracasei, L. plantarum, Lactococcus lactis, Pediococcus damnosus, and brewery strains of Saccharomyces pastorianus and S. cerevisiae showed that the medium does not inhibit the growth of Pectinatus spp. yet suppresses the growth of other beer- and brewery-contaminating microorganisms, which, thus, do not interfere with Pectinatus determination. The characteristic and easy-to-detect snake-like motile cells of Pectinatus spp. appear after a 24-to-48 hr cultivation of samples in MRS medium with Tetra.

Evaluation of Reverse-Transcription PCR Detection of 16S rRNA and Tuf mRNA for Viable/Dead Discrimination of Beer-Spoilage Lactic Acid Bacteria

The aim of this study was to evaluate the feasibility of using 16S rRNA and elongation factor (tuf) mRNA as targets for selective detection of viable beer-spoilage lactic acid bacteria (LAB) by reverse-transcription PCR (RT-PCR). One-step real-time RT-PCR was applied to monitor the target transcripts in Lactobacillus brevis, L. lindneri, and Pediococcus damnosus. Three methods were compared for total RNA extraction. To study the correlation between viability and RNA detection, log-phase cultures were inactivated using brewery disinfectant or heating. RT-PCR, denaturing agarose gel electrophoresis, and cultivation were performed before and at various times after the inactivation treatments. A method combining bead-beating with traditional RNA purification provided good RNA yield from LAB. The 16S rRNA and tuf mRNA targets were detected in inactivated cells by RT-PCR throughout a 21-day storage period. Cultivable cells could not be recovered at any postinactivation time. The 16S rRNA degraded with time, and its degradation appeared to be initially faster after disinfection than after heating. The overall degradation patterns between the strains were similar. Our results indicate that tuf mRNA and 16S rRNA can be stable in LAB cells inactivated under conditions they may encounter during the brewing process. Hence, the RT-PCR analysis of these target transcripts may not be a reliable technique for selective detection of viable LAB in brewery samples.

Amplification Facilitators and Pre-Processing Methods for PCR Detection of Strictly Anaerobic Beer-Spoilage Bacteria of the Class Clostridia in Brewery Samples

The aim of this study was to evaluate easy pre-PCR processing procedures to allow rapid and reliable detection of strictly anaerobic beer-spoilage bacteria throughout the brewing process by end-point and real-time PCR techniques. The efficiencies of the new procedures were evaluated using spiked brewery samples and specific PCRs for the target group bacteria. We found for the first time that the inclusion of 0.25% (w/v) bovine serum albumin (BSA) or 0.5% (w/v) polyvinyl pyrrolidone (PVP) in the end-point PCR mixture reduces the inhibiting effect of brewery sample extracts (3–10%, v/v) on PCR. Membrane filtration with a PVP or a sodium tri-polyphosphate-EDTA wash, and cross-flow filtration were the most promising new methods to reduce inhibitors from beer samples before cell lysis. Together with BSA, they allowed the analysis of 10% (v/v) of crude extracts instead of <3% (v/v). Moreover, we developed a one-hour procedure to prepare target DNA from process samples containing brewer's yeast. It involved removal of inhibitors by a two-step centrifugation followed by physical disruption of cells. The detection limit of the procedure was 101-103 CFU/mL. The developed procedures help to reduce the risk of partial or complete PCR failure due to inhibition and target DNA losses, with minimal sample handling.

Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia

The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism ( KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 10 0–10 3 CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6–7 h and 2–3 h, respectively. Pre-PCR enrichment of beer samples for 1–3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1–2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.

RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples

Recently we showed the applicability and sensitivity of the RNA-based sandwich hybridisation assay (SHA) for detection of Gram-negative cells in environmental samples [Leskelä, T., Tilsala-Timisjärvi, A., Kusnetsov, J., Neubauer, P., Breitenstein, A., 2005. Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay. J. Microbiol. Met. 62, 167–179.]. In this study the aim was to test and optimise this method for the detection of Gram-positive cells from brewery yeast slurries that contain up to 10 9 yeast cells/ml. Eleven new oligonucleotide probes were designed for group-specific detection of different beer-spoiling lactic acid bacteria of the genera Lactobacillus and Pediococcus. Functionality of the designed probes was shown by testing individual and paired probes using in vitro transcribed 16S rRNA and crude cell extracts as samples. Various simple and fast cell disruption methods were evaluated for the efficient disruption of lactobacilli and pediococci. The applicability of the designed oligonucleotide probes and the SHA for detection of brewery contaminants was demonstrated using both artificial and actual yeast slurry samples from brewery fermentation tanks with either fluorimetric readout or an electric biochip analyser.

Megasphaera paucivorans sp. nov., Megasphaera sueciensis sp. nov. and Pectinatus haikarae sp. nov., isolated from brewery samples, and emended description of the genus Pectinatus

Seven unidentified strictly anaerobic, Gram-negative, non-spore-forming bacteria from spoiled beer or the brewery environment were characterized. Based on 16S rRNA gene sequence analyses, all strains were affiliated to the Sporomusa sub-branch of the class 'Clostridia'. Three of the strains were non-motile cocci, on average 1.5 x 1.2 microm or 1.2 x 1.0 microm, occurring mainly singly or in pairs. They shared nearly identical (>99 %) 16S rRNA gene sequences, being most closely related to the species of the Megasphaera-Anaeroglobus group (< or =93.9 % similarity). According to DNA-DNA hybridization results, the coccoid strains represented two genospecies, neither of which was related to any of the recognized Megasphaera species. Several phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The other four novel strains were motile, slightly curved to helical rods, 0.6-0.8 x 3-50 microm or more in size. They shared identical 16S rRNA gene sequences and ribofragment patterns. The highest 16S rRNA gene similarity was found between these isolates and Pectinatus cerevisiiphilus ATCC 29359T (95.6 %) and Pectinatus frisingensis ATCC 33332T (93.6 %). The novel strains also differed from recognized Pectinatus species in their sugar utilization, proteolytic activity, catalase activity, antibiotic resistance and temperature tolerance. The results suggest that the bacteria belong to three novel species, for which the names Megasphaera paucivorans sp. nov. (type strain VTT E-032341T = DSM 16981T), Megasphaera sueciensis sp. nov. (type strain VTT E-97791T = DSM 17042T) and Pectinatus haikarae sp. nov. (type strain VTT E-88329T = DSM 16980T) are proposed.

Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1

To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples. Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells. Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR. Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.

Evaluation of Multinitrogen Source Media for Wild Yeast Detection in Brewing Culture Yeast

A multinitrogen source medium called CLEN (employing cadaverine, lysine, ethylamine, and nitrate as the sole nitrogen sources) was developed based on published data, Theoretically, CLEN would support the growth of a greater number of wild yeast species (415 of 469 or 88%) than would lysine medium (355 or 76%). A comparison of means of detecting wild yeast (growth on CLEN, lysine agar, XMACS [xylose, mannitol, adonitol, cellobiose, and sorbitol] agar, Lin's agar, yeast-maltose agar with cycloheximide, copper sulfate agar, and yeast-maltose agar at 37° C) was made with known pure cultures of a variety of yeast species and with brewery production yeast cultures. CLEN supported the growth of more yeast species and the growth of larger numbers of wild yeast in brewery samples than did lysine medium. Growth on CLEN was more rapid than that on lysine agar. None of the media tested is capable of detecting all wild yeast; several in combination are needed for a thorough examination.

MYGP + COPPER, A MEDIUM THAT DETECTS BOTH SACCHAROMYCES AND NON-SACCHAROMYCES WILD YEAST IN THE PRESENCE OF CULTURE YEAST*

Lin's copper sulphate medium for the detection of non-Saccharomyces wild yeast was modified, mainly by reducing the concentration of copper sulphate, with the aim of producing a medium that could detect a wide range of both non-Saccharomyces and Saccharomyces wild yeast. The performance of the resultant MYGP + Cu medium was compared to that of crystal violet and lysine media using pure cultures of wild yeast, pitching yeast and brewery samples. The growth of the pitching yeasts examined was suppressed more in MYGP + Cu than in crystal violet and the 4 ale yeasts were more susceptible to copper inhibition than the 3 lager yeasts. Neither MYGP + Cu nor crystal violet could detect all the wild Saccharomyces species tested but the proportion detected by each was similar. Twenty-seven out of 28 non-Saccharomyces wild yeasts tested could be detected in MYGP + Cu. When brewery samples were examined, the detection rate by MYGP + Cu compared favourably with that by a combination of crystal violet and lysine. MYGP + Cu is particularly suited to the monitoring of the production of cask-conditioned ales.

DETECTION OFZYMOMONAS

A simple, semi-defined medium has been evaluated and is recommended for detection of Zymomonas in brewery samples.

ENTEROBACTER AGGLOMERANS-A NEW BACTERIAL CONTAMINANT ISOLATED FROM LAGER BEER BREWERIES

From a large number of bacterial strains isolated from all stages of the brewing process in four different beer breweries in South Africa, fifty-five bacterial strains were identified as Enterobacteriaceae by phenotypic analysis. All enterobacterial species previously reported in brewery samples, as well as eighteen strains of Enterobacter agglomerans were found. E. agglomerans strains were isolated from pitching yeast and fermenting wort samples. The occurrence of E. agglomerans in pitching yeast is significant and indicates that these bacteria survive the brewing process. E. agglomerans is regarded as a potential beer spoilage contaminant in lager beer breweries.

Use of Immunofluorescence and Viability Stains in Quality Control

Immunofluorescence has been evaluated as a rapid quality control technique for the detection of low levels of wild yeasts in culture yeast or other brewery samples. Antisera have been prepared agai...

Use of Immunofluorescence and Viability Stains in Quality Control

Immunofluorescence has been evaluated as a rapid quality control technique for the detection of low levels of wild yeasts in culture yeast or other brewery samples. Antisera have been prepared against antigenic groups A to F, pooled and absorbed with culture yeast. Using the indirect staining method and fluorescein isothiocyanate or rhodamine as fluorochrome, levels as low as 10 wild yeasts per million cells can be detected in three hours. Combined immunofluorescence and viability staining allows the differentiation of live and dead, culture and wild yeast cells, on the same slide by alternating the light sources. The preferred method uses fluorescein isothiocyanate, excited by incident blue light, for wild yeast detection, combined with methylene blue, viewed by transmitted light, for viability differentiation. Fluorescein diacetate is a useful viability stain for yeasts although agreement between results comparing it with methylene blue and slide culture viabilities is not exact for heat-stressed cells. Some nonbrewing yeasts require heating for two minutes at 50° C before consistent results are obtained with fluorescein diacetate. An examination of the mechanism of the action of methylene blue as a viability stain has suggested that the action is one of permeability, rather than permeability followed by enzymatic reduction.