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Microbial risk assessment provides a means of estimating consumer risks associated with food products. The methods can also be applied at the plant level. In this study results of microbiological analyses were used to develop a robust single plant level risk assessment. Furthermore, the prevalence and numbers of Listeria monocytogenes in marinated broiler legs in Finland were estimated. These estimates were based on information on the prevalence, numbers and genotypes of L. monocytogenes in 186 marinated broiler legs from 41 retail stores. The products were from three main Finnish producers, which produce 90% of all marinated broiler legs sold in Finland. The prevalence and numbers of L. monocytogenes were estimated by Monte Carlo simulation using WinBUGS®, but the model is applicable to any software featuring standard probability distributions. The estimated mean annual number of L. monocytogenes-positive broiler legs sold in Finland was 7.2 × 106 with a 95% credible interval (CI) 6.7 × 106–7.7 × 106. That would be 34% ± 1% of the marinated broiler legs sold in Finland. The mean number of L. monocytogenes in marinated broiler legs estimated at the sell-by-date was 2 CFU/g, with a 95% CI of 0–14 CFU/g. Producer-specific L. monocytogenes strains were recovered from the products throughout the year, which emphasizes the importance of characterizing the isolates and identifying strains that may cause problems as part of risk assessment studies. As the levels of L. monocytogenes were low, the risk of acquiring listeriosis from these products proved to be insignificant. Consequently there was no need for a thorough national level risk assessment. However, an approach using worst-case and average point estimates was applied to produce an example of single producer level risk assessment based on limited data. This assessment also indicated that the risk from these products was low. The risk-based approach presented in this work can provide estimation of public health risk on which control measures at the plant level can be based.

In the present study, polyphasic analysis [cultivation, combined with the fingerprinting of individual isolates, and denaturing gradient gel electrophoresis (DGGE)] was applied to study whether similar features concerning the diversity and temporal stability of selected bacterial groups could be detected intra-individually in two different niches – the oral cavity and the colon – from ten adult volunteers consuming probiotics. The predominant bacterial microbiota, Clostridium coccoides–Eubacterium rectale group and bifidobacterial populations, were generally stable in salivary and faecal samples, with the greater diversity seen in faeces. Furthermore, different species predominated at the two different sites. Lactobacillus group DGGE profiles were unstable, yet the intra-individual profiles from faecal and salivary samples collected at the same time resembled each other. The ingested probiotic product did not affect the stability of the bacterial groups studied. The culture-based analysis showed that most subjects harboured identical indigenous Lactobacillus genotypes in saliva and faeces (Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus paracasei and Lactobacillus plantarum group). Thus, identical indigenous lactobacilli were able to inhabit both ends of the orogastrointestinal tract, whereas the composition of the other bacterial groups studied varied between the two sites.

Aims: To isolate aerobic mesophilic bacilli and thermophilic bacteria from different paper mill samples and to evaluate their potential harmfulness. Methods and Results: A total of 109 mesophilic and 68 thermophilic isolates were purified and characterized by automated ribotyping and partial 16S rDNA sequencing. The mesophilic isolates belonged to the genera Bacillus (13 taxa), Brevibacillus (three taxa) and Paenibacillus (five taxa). The thermophilic bacteria represented seven taxa of Bacillus, Geobacillus or Paenibacillus, four of proteobacteria and one of actinobacteria. The most frequently occurring bacteria were Bacillus cereus, B. licheniformis, Pseudoxanthomonas taiwanensis and bacteria closely related to Paenibacillus stellifer, P. turicensis or Leptothrix sp. One mill was contaminated throughout with bacteria of a novel mesophilic genus most closely related to Brevibacillus centrosporus and another with bacteria of a novel thermophilic genus most closely related to Hydrogenophilus thermoluteolus. One B. cereus isolate producing haemolytic diarrhoeal enterotoxin was detected and all the tested B. licheniformis isolates produced a metabolite toxic to boar sperm cells. Conclusions: The bacilli and thermophilic bacteria isolated represent species which should not present occupational hazards in paper mill environments. The most harmful bacterium detected was B. licheniformis and potentially also B. cereus. Significance and Impact of the Study: Knowledge of the microbial diversity in a paper mill provides a rational basis for development of an effective controlling programme. A database constructed from the fingerprints generated using automated ribotyping helps to identify and trace the contamination routes of bacteria occurring in paper mills.

Aims: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. Methods and Results: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR–ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. Conclusions: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. Significance and Impact of the Study: Applicability of ribotyping with the automated RiboPrinter® System for identification and genotyping of bifidobacteria was shown in the present study.

The survival of Listeria monocytogenes (inoculation 3 log cfu g−1) in dry sausages fermented by bioprotective experimental meat starter cultures Lactobacillus rhamnosus E-97800, L. rhamnosus LC-705 and Lactobacillus plantarum ALC01 as well as Pediococcus pentosaceus RM2000 (commercial control) was studied. In the first trial both experimental and control sausages (pH 5.0–5.2) were L. monocytogenes negative after 21 days of ripening. However, in the second and third trials the experimental sausages were L. monocytogenes negative after 7 days of fermentation while the control sausages were L. monocytogenes negative only after 28 days of ripening. The pH values were typical for North European type dry sausages (pH 4.7–4.9). It can be concluded that while the control sausage fermented by commercial starter culture was listeria-negative at a late stage of ripening process, L. rhamnosus E-97800, L. rhamnosus LC-705 as well as L. plantarum ALC01 expressed additional antilisterial activity resulting listeria-negative North European type sausages at an early stage of the ripening process.

A total of 564 Listeria monocytogenes isolates were characterized by automated ribotyping. The samples were taken from equipment, personnel and the environment after cleaning procedures and during food processing, as well as from raw materials and products from six meat, two poultry and five seafood processing plants located in the Faroe Islands, Finland, Iceland, Norway and Sweden. Altogether, 25 different ribotypes (RTs) were generated. Two RTs occurred in the samples from all three food sectors--meat, poultry and seafood. Four RTs occurred in meat and poultry plant samples and other four RTs occurred in meat and seafood plant samples. Five RTs occurred only in meat plant samples, five only in poultry plant samples and five only in seafood plant samples. Eight of the thirteen plants had their own in-house L. monocytogenes ribotype. There was geographical differences between the RTs, but no correlation between RTs and food sectors was detected. The discrimination power of automated ribotyping was satisfactory to trace the contamination sources in the food processing plants clearly indicating the sites at which improved cleaning procedures were necessary. In addition, it was possible to screen a large number of isolates with two instruments located at different institutes and to make a reliable combination of the results.

The predominant genera of recycled fibre pulps and process water were Penicillium, Trichoderma, Aspergillus, Mucor, Paecilomyces and Geotrichum. Yeasts were rare in the samples. The drying stages of the paper and boardmaking processes eliminated very effectively all the fungi present.

The bacterial gene, encoding alpha-acetolactate decarboxylase (alpha-ALDC), was expressed in a bottom-fermenting brewer's yeast under the control of a modified Saccharomyces cerevisiae alcohol dehydrogenase (ADH1) promoter which lacks the upstream regions from -800 bp to -1500 bp. In pilot scale brewing conditions, the level of alpha-ALDC produced was high enough to reduce the concentration of diacetyl so that lagering was not required. alpha-ALDC active brewer's yeast strains were also shown to be suitable for high gravity brewing.

During 1998/1999 a total of 36 surveys were carried out in six meat, five seafood and two poultry processing plants. A total of 2522 samples, including processing lines and environment, personnel, raw materials and products (raw or ready-to-eat, RTE), were analysed for the presence of Listeria monocytogenes. The environmental and personnel samples were taken after cleaning and after the process had been running for 2 h. Samples of food products were taken after every important step during processing and samples of poultry carcasses were taken immediately after production and after storage for 21 days. The overall incidence of L. monocytogenes in meat processing plants varied from 0% to 15.1%, in poultry plants from 20.6% to 24.1% and in seafood plants from 5.9% to 22.1%. In raw products the average incidence was 15.6% for meat, 22.2% for poultry and 39.0% for seafood products. The heating steps during the production of RTE products eliminated Listeria. On average, 2.3% of RTE meat and 4.8% of RTE seafood products were recontaminated with L. monocytogenes. In the seafood sector almost all Listeria positive samples also included L. monocytogenes (91.1% of the positive samples), whereas in the meat and poultry sectors other Listeria species (mainly L. innocua) dominated. In most plants, the implemented cleaning procedures were insufficient to eliminate Listeria. The results clearly indicated the problematic sites, which in most plants were conveyer belts and other transporting equipment, floors and drains, but also raw material in the meat industry, cutting boards in the poultry industry and cooking equipment in the seafood industry. In order to solve the problems observed in this study, there is a need for close co-operation between the suppliers of equipment and cleaning agents, the staff of cleaning companies and hygiene specialists from the food industry.

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.

The ability of three probiotic Lactobacillus rhamnosus strains GG, E-97800 and LC-705 and one commercial Pediococcus pentosaceus starter strain (control) to produce dry sausage was studied. During the fermentation process the numbers of inoculated lactic acid bacteria increased from approx. 7 log10 to 8–9 log10 cfu/g and the pH values decreased from 5.6 to 4.9–5.0. The sensory test indicated that the dry sausages fermented by L. rhamnosus LC-705 were inferior to the control sausages. The presence of inoculated experimental strains as predominant organisms in the dry sausages was recognised on the basis of their genetic fingerprints by ribotyping. The concentrations of biogenic amines remained low during the ripening process. These results indicated that the studied Lactobacillus rhamnosus strains, especially strains GG and E-97800, are suitable for use as probiotic starter cultures in fermenting dry sausage.

A bacterial gene encoding a-acetolactate decarboxylase, isolated from Klebsiella ternigena or Enterobacteraerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase(PGKI) or the alcohol dehydrogenase (ADHI) promoter and were integrated by gene replacement byusing cotransformation into the PGKI or ADHI locus, respectively, of a brewer's yeast. The expression levelof the aL-acetolactate decarboxylase gene of the PGKI integrant strains was higher than that of the ADHIintegrants. Under pilot-scale brewing conditions, the a-acetolactate decarboxylase activity of the PGKIintegrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and nolagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, andthe quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.

Four brewer's yeast strains carrying the α- ald gene of Klebsiella terrigena (ex. Aerobacter aerogenes ) or of Enterobacter aerogenes on autonomously replicating plasmids were constructed. The α- ald genes were linked either to the ADC1 promoter or to the PGK1 promoter of yeast Saccharomyces cerevisiae . In pilot scale brewing (50 l) with three of these recombinant yeasts the formation of diacetyl in beer was so low during fermentation that lagering was not required. All other brewing properties of the strains were unaffected and the quality of finished beers was as good as that of finished beer prepared with the control strain. The total process time of beer production could therefore be reduced to 2 weeks, in contrast to about 5 weeks required in the conventional process.

The microbiological and sensory quality of blood pancakes and vacuum-packed cooked ring sausages were studied one day after production, on the sell-by date and one week after the sell-by date. Samples were taken straight from 5 manufacturing plants, stored at 4°C and analyzed by 3 different laboratories using similar methods. Blood pancakes contained high numbers of aerobic organisms and yeasts. The principal sensory defects were visible mould spots, rancid odour and musty taste. A correlation between taste and microbiological counts was observed. Bacillus cereus was observed in some samples and may constitute a potential health hazard when temperature abuse occurs. Cooked ring sausages showed good microbiological and sensory quality after production. Aerobic plate counts and lactobacilli numbers increased during storage. The main defects in sensory analysis were fermented, sour aroma and taste. No correlation between taste and microbiological counts could be determined. The store shelf-lives (sell-by periods) of blood pancakes and cooked ring sausages set by the manufacturers, 7–11 and 20–28 days, respectively, can be considered overly optimistic. Differences between laboratories were observed in the sensory analyses.

Barley β-glucans present in wort reduce beer filtrability and cause hazes and precipitates in the finished beer. The endo-β-1,4-glucanase enzyme, EGI, found in the filamentous fungus Trichoderma reesei, is capable of efficiently hydrolyzing these β-glucans. The cDNA copy of the eg11 gene, which codes for the EGI enzyme, was coupled to yeast regulatory sequences and transferred to a brewer's yeast using the yeast copper chelatin gene CUP1 as a selection marker in the transformation. The eg11 gene was transferred to the yeast both on a multicopy plasmid and on an integrating plasmid. In both cases, highly glycosylated, active EGI enzyme was secreted into the medium. Barley β-glucans present in wort were efficiently hydrolyzed by the recombinant brewer's yeast.