Johanna Björkroth

Taxonomy and important features of probiotic microorganisms in food and nutrition

Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, eg, morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. Studies based on comparative 16S ribosomal RNA sequencing analysis, however, showed that some taxa generated on the basis of phenotypic features do not correspond with the suggested phylogenetic relations. Thus, some species are not readily distinguishable by phenotypic characteristics. This is especially true for the so-called Lactobacillus acidophilus group, the Lactobacillus casei and Lactobacillus paracasei group, and some bifidobacteria, strains of which have been introduced in many probiotic foods, eg, the novel yogurt-like commodities. Consequently, modern molecular techniques, including polymerase chain reaction-based and other genotyping methods, have become increasingly important for species identification or for the differentiation of probiotic strains. Probiotic strains are selected for potential application on the basis of particular physiologic and functional properties, some of which may be determined in vitro. The classification and identification of a probiotic strain may give a strong indication of its typical habitat and origin. The species, or even genus name, may also indicate the strains safety and technical applicability for use in probiotic products. Molecular typing methods such as pulsed-field gel electrophoresis, repetitive polymerase chain reaction, and restriction fragment length polymorphism are extremely valuable for specific characterization and detection of such strains selected for application as probiotics.

Microbiological ecology of marinated meat products

Marinated meat products are consumed increasingly. In addition to taste, marinating has been considered to increase product safety and shelf life. In Finland, marinades are complex, spiced sauces. They are acidic water-oil emulsions typically containing salt, sugar, sorbate and/or benzoate. Marinated products are usually packaged under modified atmospheres. This results in the growth of psychrotrophic, anaerobic bacteria like lactic acid bacteria (LAB). Marinating did not increase the shelf life of Finnish poultry products and it strongly selected novel spoilage LAB. Surprisingly, it neither had inhibitory effect on Campylobacter. The buffering capability of meat neutralizes the acidic marinade and results in dissociation of the lipophilic acids making their antimicrobial effect nonexistent.

Similar Listeria monocytogenes pulsotypes detected in several foods originating from different sources.

The purpose of the study was to obtain fingerprinting data of Listeria monocytogenes strains isolated in various foods to determine possible associations of strains with product type, producer, country or isolation time. Two hundred and ninety-five L. monocytogenes strains originating from food items of 41 producers of 10 countries were characterized by pulsed-field gel electrophoresis (PFGE) typing. Combination of AscI and ApaI macrorestriction patterns (MRP) yielded 66 different pulsotypes. Ten pulsotypes were common to two or more product types and 17 pulsotypes were detected in foods of more than one producer having no apparent association with each other. Similar pulsotypes of L. monocytogenes were recovered in products of different countries over several years. Some of the pulsotypes were recurrently recovered from the same product of the same producer, suggesting a possible persistence of these strains in the processing plant. However, some of the recurrently isolated L. monocytogenes pulsotypes were repeatedly found in products of several producers, which may indicate that persistent houseflora strains are not always producer-specific. Furthermore, the similarity of macrorestriction patterns expressed as clusters, based on the numerical analysis of macrorestriction patterns, was not found to correlate with product type, country, producer or year of isolation. Our data suggest a wide geographical and temporal distribution of a number of L. monocytogenes strains isolated in food products. The existence of similar L. monocytogenes strains in various food products of several producers should be considered if food strain fingerprint results are used to help trace the vehicles for infections.

rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime

The rRNA gene restriction patterns (ribotypes) of 69 ropy slime producing Lactobacillus sake strains isolated mainly from vacuum-packaged meat products of ten meat plants were determined. Ribotypes were compared to the corresponding patterns of non-ropy L. sake strains, and also to other species of the genus Lactobacillus, Carnobacterium and Weissella associated with meat products. Ropy slime-producing L. sake strains were divided into four characteristic groups corresponding to the phenotypic carbohydrate grouping. No association between certain ribotypes and individual plants was detected. Ribotyping was unable to distinguish slime producing and non-ropy strains of L. sake group sharing the same carbohydrate pattern. Otherwise, ribotyping distinguished the ropy slime producing strains from the non-ropy L. sake reference strains and all L. sake strains from other species of the genus Lactobacillus, Carnobacterium and Weissella. These results suggest that ribotyping is a suitable method for detection and surveillance of the contamination of ropy slime-producing L. sake strains but the patterns alone cannot be used as markers of slime production capability.

Characterization of Lactobacillus sake strains associating with production of ropy slime by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) patterns

Randomly amplified polymorphic DNA (RAPD) patterns and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of ropy slime-producing Lactobacillus sake strains. The two most revealing commercially available primers (OPJ 12 and OPJ 16, Operon Inc. Alameda, USA) and two rare-cutting enzymes (AvrII and SmaI) were chosen from a pretested lot for the typing of 69 ropy slime-producing strains, 7 non-ropy isolates and 4 non-ropy reference strains. Both RAPD and PFGE patterns confirmed the group division established in previous studies and provided new information concerning ropy slime-producing strains. PFGE patterns were found to have the greatest discriminatory power, revealing the genetic variation of the main group of ropy slime-producing L. sake strains and distinguishing all non-ropy strains from slime-producers.

Coagulase-negative staphylococci isolated from bovine extramammary sites and intramammary infections in a single dairy herd

Isolates of various species of coagulase-negative staphylococci (CNS) from extramammary swab samples were compared with isolates of bovine mastitis CNS species. Swab samples were taken from perineum skin and udder skin, teat apices and teat canals of lactating dairy cows of the research dairy herd of the University of Helsinki in 1999 and 2002. In addition, hands of herd staff and liners of teat cups were sampled for CNS. CNS isolates from milk samples of subclinical or clinical mastitis in the same herd were collected during 1998-2002. Species identification was performed using phenotyping (API Staph ID 32 test) and by constructing a 16 and 23S rRNA RFLP library (ribotyping). Based on phenotype, 84% of mastitis isolates and 57% of extramammary isolates were identified at species level with >90% probability. Ribotype patterns formed 24 clusters, and 15 of them included a CNS type strain. If the ribotype clusters contained isolates of both extramammary and mastitis origin, they were further typed using pulsed-field gel electrophoresis (PFGE). The predominant CNS species in mastitis, based both on phenotyping and genotyping, were Staph. chromogenes and Staph. simulans. Phenotyping failed to identify half of the extramammary isolates. Based on phenotyping, Staph. equorum and Staph. sciuri, and based on ribotyping, Staph. succinus and Staph. xylosus, were the predominant CNS species in extramammary samples. The most common species in milk samples, Staph. chromogenes, was also isolated from several extramammary samples, and five out of ten pulsotypes were shared between mastitis and extramammary isolates, indicating that strains from udder skin are highly similar. The second commonest mastitis species, Staph. simulans, was isolated only from three extramammary samples, indicating that Staph. simulans may be more specifically associated with mastitis. Consequently, the origin of CNS mastitis may vary depending on the causing CNS species.

Comparison of microbial communities in marinated and unmarinated broiler meat by metagenomics

Most raw poultry sold in Finland at the retail level is mixed with marinades containing oil, sugar, spices and acetic acid and packaged under modified atmosphere. Premature spoilage of marinated poultry preparations has been observed and associated with high levels of Leuconostoc spp. in meat. In this study we investigated whether marination of broiler fillet strips increased the proportion of Leuconostoc spp. in the microbial communities. To obtain a comprehensive view of the microbiota, we sequenced total DNA and 16S rRNA gene amplicons from the microbial communities. The lactic acid bacterial communities were characterized also by identification of colonies. The results showed that marinade increased the proportions of the spoilage-associated Leuconostoc gasicomitatum in the communities as well as the proportions of Leuconostoc gelidum and Lactobacillus spp. The proportions of Carnobacterium, Vagococcus, Brochothrix thrermosphacta, Clostridium, Enterobacteriaceae and Vibrio were diminished in marinated meat. Analysis of 16S rRNA gene amplicons resulted in 312 and 284 operational taxonomical units (dissimilarity 0.03) in unmarinated and marinated meat, respectively, indicating that the meat communities were more diverse than hitherto shown. Metagenomic analysis revealed a number of bacterial taxa that have not been associated with late shelf-life meat before, including Vagococcus and Vibrio that belonged to the predominating part of the microbial community in unmarinated meat. According to the functional analysis of the metagenomes, the communities in both marinated and unmarinated poultry were characterized by high proportions (15.6% or 17.9%) of genes involved in carbohydrate metabolism.

Characterisation of lactic acid bacteria from spoiled, vacuum-packaged, cold-smoked rainbow trout using ribotyping

A total of 405 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged, salted, sodium nitrite- or potassium nitrate-treated, cold-smoked rainbow trout stored at 4 degrees C or 8 degrees C were characterised and identified using a molecular method. The isolates were initially classified according to their restriction endonuclease profiles using HindIII and EcoRI restriction endonucleases and further characterised by rRNA gene restriction patterns (ribotypes). Numerical analysis of these ribopatterns was performed together with 19 reference LAB strain patterns in order to identify the isolates to species level. The strains were divided with HindIII and EcoRI ribopatterns into ten and nine clusters at the similarity level of 65% and 50%, respectively. The Leuconostoc-clusters and the Lb. sakei/Lb. curvatus-clusters formed the two main groups. Only one isolate was identified as Lactobacillus plantarum and no Carnobacterium strains were discovered. For both enzymes, the 35 isolates possessing six individual ribotypes and forming five clusters could not be identified further with the reference strains used. The relative proportion of Leuconostoc mesenteroides subsp. mesenteroides was higher in all samples stored at 4 degrees C. Most of the Leuconostoc citreum were found in the samples stored at 8 degrees C, and particularly in the nitrite-treated samples.

Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR

Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism. Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions. Ready-To-Go RAPD Analysis Beads and Ready-To-Go PCR Beads were used for PCR reactions with RAPD and rep-PCR, respectively. Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C. botulinum serotypes at the strain level. Group I strains were mainly discriminated at the serotype level. The discriminatory power of rep-PCR was found to be inferior to that of RAPD. The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment. The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results. As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.

The spoilage flora of vacuum-packaged, sodium nitrite or potassium nitrate treated, cold-smoked rainbow trout stored at 4°C or 8°C

The spoilage flora of vacuum-packaged, salted, cold-smoked rainbow trout fillets, with or without the addition of nitrate or nitrite, stored at 4 degrees C and 8 degrees C, was studied. Of 620 isolates, lactic acid bacteria were the major fraction (76%), predominating in all samples of spoiled product. However, the phenotypical tests used were insufficient to identify the lactic acid bacteria to the species level. Gram-positive, catalase-positive cocci, gram-negative, oxidase-negative rods and gram-negative, oxidase-positive rods were found in 6%, 16% and 2% of the samples, respectively. Of 39 gram-positive, catalase-positive cocci, 29 were identified as staphylococci and 10 as micrococci. Eighty-five isolates were found to belong to the family Enterobacteriaceae, with 45 of those being Serratia plymuthica. Eleven isolates from the nitrate treated samples stored at 8 degrees C were identified as Pseudomonas aeruginosa. The occurrence of P. aeruginosa and staphylococci in the nitrate-containing samples, stored at 8 degrees C, may cause problems with respect to the safety of the product. The types of lactic acid and other bacteria in the spoilage flora were generally reduced by the addition of nitrate or nitrite to fillets.