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A Gram-stain-positive, catalase-positive and pleomorphic rod organism was isolated from malted barley in Finland, classified initially by partial 16S rRNA gene sequencing and originally deposited in the VTT Culture Collection as a strain of Propionibacterium acidipropionici (currently Acidipropionibacterium acidipropionici). The subsequent comparison of the whole 16S rRNA gene with other representatives of the genus Acidipropionibacterium revealed that the strain belongs to a novel species, most closely related to Acidipropionibacterium microaerophilum and Acidipropionibacterium acidipropionici, with similarity values of 98.46 and 98.31 %, respectively. The whole genome sequencing using PacBio RS II platform allowed further comparison of the genome with all of the other DNA sequences available for the type strains of the Acidipropionibacterium species. Those comparisons revealed the highest similarity of strain JS278T to A. acidipropionici, which was confirmed by the average nucleotide identity analysis. The genome of strain JS278T is intermediate in size compared to the A. acidipropionici and Acidipropionibacterium jensenii at 3 432 872 bp, the G+C content is 68.4 mol%. The strain fermented a wide range of carbon sources, and produced propionic acid as the major fermentation product. Besides its poor ability to grow at 37° C and positive catalase reaction, the observed phenotype was almost indistinguishable from those of A. acidipropionici and A. jensenii. Based on our findings, we conclude that the organism represents a novel member of the genus Acidipropionibacterium, for which we propose the name Acidipropionibacterium virtanenii sp. nov. The type strain is JS278T (=VTT E-113202T =DSM 106790T ).

In 2010, bottles of beer containing viable bacteria of the common beer-spoilage species Lactobacillus backii and Pediococcus damnosus were recovered from a shipwreck near the Åland Islands, Finland. The 170-year quiescent state maintained by the shipwreck bacteria presented a unique opportunity to study lactic acid bacteria (LAB) evolution vis-a-vis growth and survival in the beer environment. Three shipwreck bacteria (one L. backii strain and two P. damnosus strains) and modern-day beer-spoilage isolates of the same two species were genome sequenced, characterized for hop iso-α-acid tolerance, and growth in degassed lager and wheat beer. In addition, plasmid variants of the modern-day P. damnosus strain were analyzed for the effect of plasmid-encoded genes on growth in lager beer. Coding content on two plasmids was identified as essential for LAB growth in modern lager beer. Three chromosomal regions containing genes related to sugar transport and cell wall polysaccharides were shared by pediococci able to grow in beer. Our results show that the three shipwreck bacteria lack the necessary plasmid-located genetic content to grow in modern lager beer, but carry additional genes related to acid tolerance and biofilm formation compared to their modern counterparts.

The food industry, including the meat industry, is currently looking for natural preservatives to prevent the growth of harmful microbes in foods. The potential of plant-derived antimicrobial extracts to increase the shelf life and to delay the microbiological spoilage of marinated broiler chicken cuts in modified atmosphere packages during cold storage was investigated in this study. We evaluated the impact of aqueous ethanolic extracts of Finnish sea buckthorn berries and lingonberries and supercritical CO2-extracted herbal extracts from an antimicrobial blend and oregano leaves on the shelf life of broiler meat. The commercial antimicrobial blend extract and the oregano extract inhibited the growth of lactic acid bacteria (LAB) and Brochothrix thermosphacta in the marinated samples. The antimicrobial blend extract also reduced the growth of psychrotrophic aerobic bacteria, whereas the sea buckthorn and lingonberry extracts did not. Only minor antimicrobial activity against Enterobacteriaceae by all the extracts was observed. Plate count analysis, denaturing gradient gel electrophoresis, and quantitative real-time PCR indicated that LAB, which are the major spoilage group in marinated modified atmosphere–packaged poultry products, were not significantly affected by the berry extracts studied. During this shelf-life study, LAB isolates of Lactobacillus and Leuconostoc were identified in the marinated samples. Antimicrobial blends and oregano leaf extracts can act as antimicrobial agents in marinade blends, although tailoring of the dose is needed because of their strong taste. Further studies for exploiting synergistic effects of plant extracts could contribute to the development of potential and more effective antimicrobial blends. Studies are needed in meat matrices and in product applications to demonstrate the efficacy of these compounds.

Titanium dioxide (TiO2) surfaces doped with molybdenum (Mo) were investigated to determine if their photocatalytic ability could enhance process hygiene in the brewery industry. Doping TiO2 with Mo showed a 5-log reduction in bacterial counts within 4 to 24 h and a 1-log reduction in yeast numbers within 72 h. The presence of a dilute brewery soil on the surface did not interfere with antimicrobial activity. The TiO2–M surface was also active in the dark, showing a 5-log reduction in bacteria within 4 to 24 h and a 1-log reduction in yeast numbers within 72 h, suggesting it could have a novel dual function, being antimicrobial and photocatalytic. The study suggests the TiO2–Mo coating could act as a secondary barrier in helping prevent the build-up of microbial contamination on surfaces within the brewery industry, in particular in between cleaning/disinfection regimes during long production runs.

Due to the presence of moisture and nutrients, brewery filling line surfaces are susceptible to unwanted microbial attachment. Knowledge of the attaching microbes will aid in designing hygienic control of the process. In this study the bacterial diversity present on brewery filling line surfaces was revealed by next generation sequencing. The two filling lines studied maintained their characteristic bacterial community throughout three sampling times (13-163 days). On the glass bottle line, γ-proteobacteria dominated (35-82% of all OTUs), whereas on the canning line α-, β- and γ-proteobacteria and actinobacteria were most common. The most frequently detected genera were Acinetobacter, Propinobacterium and Pseudomonas. The halophilic genus Halomonas was commonly detected, which might be due to its tolerance to alkaline foam cleaners. This study has revealed a detailed overall picture of the bacterial groups present on filling line surfaces. Further effort should be given to determine the efficacy of washing procedures on different bacterial groups.

Microbial attachment and biofilm formation in brewery bottling plants was studied by mounting sterile, stainless steel coupons onto critical sites of the fillers in three breweries. Microbiological...

Bacteria use quorum sensing signalling in various functions, e.g. while forming biofilms, and inhibition of this signalling could be one way to control biofilm formation. The aim of this study was to evaluate the production of signalling molecules and its correlation with the biofilm formation capability of bacteria isolated from brewery filling process. A further aim was to study berry extracts and wood-derived terpenes for their possible quorum sensing inhibitory effects. Out of the twenty bacteria studied, five produced short-chain and five long-chain AHL (acyl homoserine lactone) signalling molecules when tested with the Chromobacterium violaceum CV026 reporter bacterium. Production of AI-2 (autoinducer-2) signalling molecules was detected from nine strains with the Vibrio harveyi BB170 bioassay. Over half of the strains produced biofilm in the microtitre plate assay, but the production of AHL and AI-2 signalling molecules and biofilm formation capability did not directly correlate with each other. Out of the 13 berry extracts and wood-derived terpenes screened, four compounds decreased AHL signalling without effect on growth. These were betulin, raspberry extract and two cloudberry extracts. The effect of these compounds on biofilm formation of the selected six bacterial strains varied. The phenolic extract of freeze-dried cloudberry fruit caused a statistically significant reduction of biofilm formation of Obesumbacterium proteus strain. Further experiments should aim at identifying the active compounds and revealing whether quorum sensing inhibition causes structural changes in the biofilms formed.

The effects of age and repeated cleaning-in-place (CIP) cycles on the hygienic properties of gasket materials used in the food industry were studied. The materials included in the study were EPDM (ethylene propylene diene monomer rubber), NBR (nitrile butyl rubber, also known as Buna-N), PTFE (polytetrafluoroethylene) and Viton (fluoroelastomer). The materials were exposed to prolonged alkali-acid treatments simulating repeated CIP cycles. In addition, materials aged in industrial dairy or brewing processes were examined. Mixed cultures of Bacillus thuringiensis and Pseudomonas fragi or Pantoea agglomerans and Pediococcus inopinatus were used as test organisms and the CIP was performed in an experimental test rig. The susceptibility to biofilm formation and the cleanability in CIP were assessed by the plate count method, epifluorescence microscopy, impedimetry and ATP bioluminescence measurements. Physical deterioration was observed both on experimentally aged materials and on materials aged in industrial processes, causing reduced cleanability. PTFE was most resistant to alkali-acid treatments and repeated CIP cycles. The hygienic properties of EPDM, NBR and Viton degenerated in repeated CIP cycles. The methods used for assessing cleanability are discussed.

Superhydrophobic surface with and without nano-sized silver particles (AgNPs) using a sol–gel technique were prepared for antibacterial and chemical stability tests and electrochemical characterization. Antibacterial tests showed that the surface with AgNPs reduced the number of the bacteria. However, to be used in long-term industrial applications, the surfaces are required to have sufficient chemical stability in the operation environment. This is why the stability of the produced surfaces was studied as a function of pH value of the solution. According the results, the produced surfaces are stable in neutral environments but release silver ions at high rates and dissolve or undergo other structural changes in strongly acidic and alkaline environments during a timeframe of few weeks. Chemical stability of the produced surfaces may be monitored by electrochemical measurements in addition to straight-forward contact angle measurements. Open circuit potential monitoring effectively revealed the presence of silver on both unexposed and exposed coatings, due to the noble character of the element. Electrochemical impedance spectroscopy yielded information on the overall degradation mechanism of the superhydrophobic silver-containing coating, although its use requires calibration by the use of model systems involving different combinations of all included surface layers and relatively complex data modelling.

Two distinct genetic groups (Saaz and Frohberg) exist within the hybrid Saccharomyces pastorianus (S. cerevisiae × S. eubayanus) taxon. However, physiological/technological differences that exist between the two groups are not known. Fermentative capability of the parental S. eubayanus has likewise never been studied. Here, 58 lager strains were screened to determine which hybrid group they belonged to, and selected strains were characterized to determine salient characteristics. In 15 °P all-malt wort fermentations at 22 °C, Frohberg strains showed greater growth and superior fermentation (80% apparent attenuation, 6.5% alcohol by volume in 3-4 days) compared to all other strains and maintained highest viability values (93%). Fermentation with S. eubayanus was poor at the same temperature (33% apparent attenuation, 2.7% alcohol by volume at 6 days and viability reduced to 75%). Saaz strains and S. eubayanus were the least sensitive to cold (10 °C), though this did not translate to greater fermentation performance. Fermentation with S. eubayanus was poor at 10 °C but equal to or greater than that of the Saaz strains. Performance of Saaz yeast/S. eubayanus was limited by an inability to use wort maltotriose. [(14)C]-Maltotriose transport assays also showed negligible activity in these strains (≤0.5 µmol min(-1) g(-1) dry yeast). Beers from Saaz fermentations were characterized by two- to sixfold lower production of the flavour compounds methyl butanol, ethyl acetate and 3-methylbutyl acetate compared to Frohberg strains. Higher alcohol and ester production by S. eubayanus was similar to that of Frohberg strains.

The aim of this study was to find out whether the first bacterial colonizers of brewery process surfaces after washing produce acyl homoserine lactone (AHL) quorum sensing signalling molecules. Microbial attachment and biofilm formation was studied by mounting sterile uncoated and coated stainless steel coupons onto critical places of the filling machines in three breweries. In the present study 26 previously deposited bacterial strains, as well as approximately 2,300 colonies from 76 process surface samples, were screened using reporter bacteria Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTLR4. AHL‐producing bacteria were detected both from process samples and among the previous isolates. From the process samples, up to 15% of the screened colonies produced AHL molecules. Production of long chain AHLs was more common than short chain AHLs. The identified AHL‐producing isolates belonged to the genera Pseudomonas, Serratia, Hafnia, Rahnella, Enterobacter and Aeromonas, which all belong to commonly found primary colonizers of brewery process surfaces. The same microbial genera producing AHL molecules were found from different breweries. Brewery filling equipment is susceptible to microbial attachment and accumulation on surfaces. In the future, inhibition of quorum sensing could be one additional way of controlling biofilm formation.

Beer is generally a hostile environment for most microorganisms rendering it safe in terms of food-borne illnesses. However, species adapted to the brewery environment can cause product spoilage. In this chapter, microorganisms associated with brewing and the production of beer as well as with beer dispensing are briefly described. Keeping the beer free from contaminating organisms is important for image reasons and, additionally, production plant hygiene has both economic and environmental motivation. In the effort of improving brewery hygiene, studies regarding brewery biofilms have lately been carried out. This chapter aims at reviewing the current knowledge of biofilms occurring in the brewery. It also summarises the means available for prevention of biofilm formation with special emphasis on emerging technologies and future trends in biofilm combat and their application in breweries.

Indoor air quality (IAQ) is one of the most important factors for the welfare of the society. Indoor air quality is a complex result of different simultaneous affecting factors. Numerical and experimental simulation studies about the moisture transfer between structures and indoor and outdoor air spaces are relatively well known, but the transfer of volatile organic compounds (VOCs), particles and microbes from or through the building envelope to indoor air have been studied insufficiently. A new laboratory device, the IAQ simulator chamber, has been designed and constructed to study the effect of building materials and structures on the IAQ. The IAQ simulator device can be used to study structure components between controlled 'indoor' and 'outdoor' air spaces, which brings new perspective to the research when compared to the applicability of conventional emission test chambers. The test structure or the outdoor air may contain materials having biological growth that are impurity sources for air. The effect of these sources on the indoor air can be studied under different heat, humidity and air pressure difference conditions. The measurements include evaluation of moisture content of materials, humidity, temperature, air leakage through the building structure, air pressure difference, analyses of indoor air quality (Volatile organic compounds VOC, particles and microbes). This paper presents a short study, where Microbial VOC and new molecular biological techniques were applied to detect and quantify microbes on building materials and in air samples using small conventional emission chambers and the IAQ simulator device. The IAQ simulator device offers a new tool to study the complicated causal connections of building structures, materials and unexpected exposure to water and microbes, especially during the use of the buildings. The results can be used together with a whole building hygrothermal analysis to give ideas to a risk assessment of the building performance under realistic climatic loads. Also renovation concepts needed to protect the indoor air from emissions of polluted building materials can be studied.

Publisher Summary The presence of inhibitors, such as hop compounds, alcohol, carbon dioxide, and sulfur dioxide, as well as the shortage of nutrients and oxygen and the low pH make beer resistant to microbial contamination. Moreover, processes such as filtration, storage at low temperatures and possible pasteurization reduce contamination. The special environment in the brewing process restricts the range of microorganisms likely to be encountered to relatively few species. Although the contaminants found may cause quality defects, pathogens have not to knowledge been reported to grow in standard beer products. Ensuring the well-being of the production, yeast strains is a fundamental part of brewing as in all processes based on fermentation technology. Thus, monitoring yeast quality and quantity is also an important part of the microbiological control carried out in breweries. This chapter presents a summary of the microorganisms likely to be encountered in breweries and the different possibilities to detect and identify them. It also describes the different possibilities to quantify yeast mass and estimate the brewing performance as well as differentiate between yeast strains and discusses both methods currently in use and emerging technologies. The chapter reviews those methods that show most potential for brewery applications till date.

Current cultivation-based methods used by most breweries for microbiological quality control reveal possible contamination only after several days or weeks of delay. Moreover, they do not discriminate between spoilage and nonspoilage microbes or allow the detection of viable but noncultivable cells or tracing of contamination sources. In recent years, several promising molecular biological applications have been described for the detection, characterization, and identification of brewery contaminants. Group-, genus-, and species-specific PCR tests have been designed and evaluated for brewery contaminants, i.e., for lactic and acetic acid bacteria (Lactobacillus, Pediococcus), strictly anaerobic bacteria (Pectinatus, Megasphaera, Selenomonas, Zymophilus), enterobacteria, and wild yeasts. Various PCR detection formats (PCR-ELISA, LightCycler(™) PCR, standard PCR) have been set up for most of these organisms. In order to detect the very low amounts of microbes in brewing samples, an enrichment method has been devised. Practical pre-PCR treatment methods, including collection of cells, DNA extraction, and removal of PCR inhibitors, have been developed for filterable (such as bright beer and process water) and nonfilterable (pitching yeast, wort, fermenting wort) samples. Modular PCR kits for standard PCR and real-time PCR have also become available from several companies. All developed PCR applications are, despite the enrichment step, more rapid and specific than the cultivation methods. Currently, PCR without prior cultivation is less sensitive and more expensive to use than cultivation. Denaturing gradient gel electrophoresis (DGGE) for separation of bacterial or eukaryotic ribosomal DNA amplicons is a valuable tool for characterizing microbial communities in specific environmental niches. This technique has also been applied to study and compare microbial communities during beer, wine, and whisky production. The automated ribotyping system (RiboPrinter® System DuPont Qualicon, U.S.A.) is a rapid molecular biological method for the identification and characterization of bacteria to species or even below species level. A comprehensive identification database has been created for brewery contaminants, such as Lactobacillus spp., Pediococcus spp., Pectinatus spp., M. cerevisiae, and Obesumbacterium proteus, using three different restriction enzymes. In addition to identification and renaming of bacteria, the database has been applied to tracing of contamination sources and detection of troublesome house flora in industrial processes.

Good production hygiene is a single most important factor in preventing microbial deterioration of food. Contaminated raw materials, processing equipment and packaging effectively transmit harmful yeasts to food. Yeasts can proliferate on process surfaces in biofilms, which protect cells from cleaning and disinfectants and serve as contamination sources. At present, information about the effectiveness of various cleaning and disinfecting agents against food-spoilage yeasts is sporadic and restricted to few species and sanitising agents. We evaluated the susceptibility of an extensive panel of food-related yeasts to common and novel sanitising agents using yeast suspensions and biofilms. Suspended cells were generally more susceptible to disinfectants than cells in biofilm. Only the alcohol-based disinfectant was also effective against biofilm cells. The tenside or peracetic based disinfectants or the chlorine-containing disinfectant or foam cleaner were not effective against yeasts in biofilm. The adhesive Candida intermedia, C. parapsilosis and Rhodotorula mucilaginosa strains showed resistance to these disinfectants even in suspension.

Contamination of brewing process by spoilage organisms may lead to production problems and quality defects in final product. Present quality control methods based on cultivation reveal the possible contamination only after several days or weeks of delay. Moreover, they do not discriminate between spoilage and non-spoilage microbes nor allow the detection of fastidious or injured organisms. In recent years, several promising molecular biological applications have been described for the detection, characterization and identification of brewery contaminants. Group, genus and species specific PCR tests have been developed for contaminants present in brewing process, i.e. for lactic acid bacteria (Lactobacillus, Pediococcus), strictly anaerobic bacteria (Pectinatus, Megasphaera, Selenomonas, Zymophilus), enterobacteria, acetic acid bacteria and wild yeasts. Various PCR detection formats (PCR-ELISA, LightCyclerTM PCR, standard PCR) have been set-up for most of the beer spoilage organisms. Pre-PCR treatment methods have been developed for filterable samples, such as bright beer and process water, and for unfilterable samples, such as pitching yeast, fermenting wort and unfilterable beer. Due to the very low amounts of microbes in brewing samples, they need to be enriched in a suitable medium prior to the PCR. Practical DNA extraction methods and removal of PCR inhibitors have been developed for different sample types. For example, a commercial system containing magnetic particles for DNA collection and purification can be used for this purpose. Modular PCR kits for standard PCR and real-time PCR are available from several companies. The developed PCR applications are, despite of the enrichment step, more rapid and specific than conventional methods. Without prior enrichment, sensitivity of PCR methods is lower and they are at the moment expensive to use compared to cultivation methods. An alternative to PCR, a commercial fluorescent in situ hydridization (FISH) method has been developed for beer-spoilage bacteria. It is based on the use of fluorescently labelled gene probes and epifluorescence microscope. The automated ribotyping system (RiboPrinter®System DuPont Qualicon,USA) is rapid molecular biological method for identification and characterization of bacteria to species or even below species level. The comprehensive identification database using three different restriction enzymes has been created to the brewery contaminants including Lactobacillus spp., Pediococcus spp, Pectinatus spp., Megasphaera cerevisiae and Obesumbacterium proteus. In addition to identification purposes and renaming of bacteria, the database has been applied in tracing contamination sources and in detection of troublesome "house flora" in industrial processes.

Immobilized primary fermentation bioreactors were deliberately contaminated with an enterobacterium, Klebsiella terrigena, and changes were monitored in yeast performance and viability and in chemical composition of the green beers. In addition, the formation of biogenic amines, possible carcinogenic substances, was monitored. K. terrigena caused elevated concentrations of dimethyl sulfide in beer but did not cause significant changes in the concentrations of the analyzed higher alcohols or esters. On the other hand, the ratio between diacetyl and pentanedione was increased. The yeast viability was lowered without observed changes in fermentation capacity. The increased concentration of biogenic amines was mainly due to changes in the cadaverine concentration.

Yeasts are spoilage organisms in many food processing industries, where they may grow as biofilms on surfaces of process equipment. Relevant information about the adhesion characteristics of food spoilage yeasts is needed to be able to efficiently prevent the early stages of biofilm formation by control of process parameters. Hydrophobicity has been found to affect attachment of microbial cells to solid surfaces. In this study, the adhesiveness and hydrophobicity of yeast cells isolated from food production processes was investigated. A trend between strong hydrophobicity and high adhesion rate could be observed. The most hydrophobic yeast strains belonged to the species Candida intermedia, Rhodotorula mucilaginosa and Zygosaccharomyces rouxii, whereas the most adhesive strains belonged to the same species and in addition to Trichosporon asahii. However, a clear correlation between adhesion and hydrophobicity could not be shown. Probably other cell surface properties in addition to hydrophobicity also affect the adhesion rate of yeast cells to surfaces.

Present quality control methods do not allow proactive process control, discrimination of spoilage and non-spoilage organisms nor detection of fastidious microbes. BREWPROC (QLKI-CT-2000-01251) is a combined R&D and Demonstration project that is focused on the development and implementation of rapid PCR based methods for microbiological QC in breweries. In this approach, samples are first screened using group specific tests. Species specific identification tests are available for numerous yeast and bacterial species. PCR analysis can be carried out using PCR-ELISA, real-time or end-point PCR. Practical sample treatment protocols, including special enrichment media, have been set-up for filterable and non-filterable samples. All PCR tests will be provided in kits.