Effects of Sodium Chloride on Tyramine Production in a Fermented Food Model and its Inhibition by Tyrosine-degrading Lactobacillus plantarum JA-1199.

Tyramine is a health-adverse biogenic amine, which can accumulate in fermented foods like cheese by decarboxylation of the free amino acid tyrosine by either starter cultures or resident microbes such as lactic acid bacteria including Enterococcus spp., respectively. Our study aimed to show the effect of sodium chloride concentrations on tyramine production as well as to characterise bacterial strains as anti-tyramine biocontrol agents in a 2 mL micro-cheese fermentation model. The effect of sodium chloride on tyramine production was assayed with tyramine producing strains from eight different species or subspecies. Generally, an increase in sodium chloride concentration enhanced tyramine production, e.g. from 0% to 1.5% of sodium chloride resulted in an increase of tyramine of 870% with a Staphylococcus xylosus strain. In the biocontrol screening among lactic acid bacteria, a Lactobacillus plantarum JA-1199 strain was screened that could consume in successful competition with other resident bacteria tyrosine in the micro-cheese model as a source of energy gain. Thereby tyramine accumulation was reduced between 4% to 99%. The results of this study disclose a feasible strategy for decreasing tyramine concentration and increasing the safety level of fermented food. It is an example of development and application of bacterial isolates as starter or protective cultures in food, a biocontrol topic, which Oreste Ghisalba - in his project evaluation function of SNF and later on CTI - was promoting with great emphasis in our ETH Food Biotechnology research group.

Clustering of Pan- and Core-genome of Lactobacillus provides Novel Evolutionary Insights for Differentiation.

BACKGROUND: Bacterial taxonomy aims to classify bacteria based on true evolutionary events and relies on a polyphasic approach that includes phenotypic, genotypic and chemotaxonomic analyses. Until now, complete genomes are largely ignored in taxonomy. The genus Lactobacillus consists of 173 species and many genomes are available to study taxonomy and evolutionary events. RESULTS: We analyzed and clustered 98 completely sequenced genomes of the genus Lactobacillus and 234 draft genomes of 5 different Lactobacillus species, i.e. L. reuteri, L. delbrueckii, L. plantarum, L. rhamnosus and L. helveticus. The core-genome of the genus Lactobacillus contains 266 genes and the pan-genome 20'800 genes. Clustering of the Lactobacillus pan- and core-genome resulted in two highly similar trees. This shows that evolutionary history is traceable in the core-genome and that clustering of the core-genome is sufficient to explore relationships. Clustering of core- and pan-genomes at species' level resulted in similar trees as well. Detailed analyses of the core-genomes showed that the functional class "genetic information processing" is conserved in the core-genome but that "signaling and cellular processes" is not. The latter class encodes functions that are involved in environmental interactions. Evolution of lactobacilli seems therefore directed by the environment. The type species L. delbrueckii was analyzed in detail and its pan-genome based tree contained two major clades whose members contained different genes yet identical functions. In addition, evidence for horizontal gene transfer between strains of L. delbrueckii, L. plantarum, and L. rhamnosus, and between species of the genus Lactobacillus is presented. Our data provide evidence for evolution of some lactobacilli according to a parapatric-like model for species differentiation. CONCLUSIONS: Core-genome trees are useful to detect evolutionary relationships in lactobacilli and might be useful in taxonomic analyses. Lactobacillus' evolution is directed by the environment and HGT.

A lab-scale model system for cocoa bean fermentation.

Lab-scale systems modelling the spontaneous cocoa bean fermentation process are useful tools to research the influence of process parameters on the fermentation and the final bean quality. In this study in Honduras, a 1-kg lab-scale fermentation (LS-F) was compared to a 300-kg on-farm fermentation (OF-F) in a multiphasic approach, analysing microbial counts, microbial species diversity, physico-chemical parameters, and final dried bean quality. Yeast and total aerobic counts of up to 8 log CFU/g during the LS-F were comparable to the OF-F, while counts for lactic acid bacteria and acetic acid bacteria were up to 3 log CFU/g lower during the LS-F than during the OF-F. While species of the genera Hansenia, Saccharomyces, and Acetobacter dominated most of the fermentation processes, the genera dominating the drying phases were Pichia, Trichosporon, Pediococcus, and Acetobacter. Dried beans resulting from the LS-F, compared to the OF-F, were similar in contents of acetic acid, 6 times lower in lactic acid, up to 4 times higher in residual sugars, and 3-12 times higher in polyphenols. Dried beans processed at LS showed a similar flavour profile in terms of astringency, bitterness, acidity, and brown, fine, and cocoa flavours, but 2 units higher off-flavours than OF processed beans. With 81%, the share of well-fermented beans from the LS-F complied with industrial standards, whereas 7% over-fermented beans were above the threshold. Conclusively, the 5-day model fermentation and subsequent drying successfully mimicked the on-farm process, providing a high-throughput method to screen microbial strains to be used as starter cultures.

Enhancing oxidative stress resistance in Bifidobacterium thermophilum using a novel overexpression vector and transformation protocol.

Bifidobacterium thermophilum is encountered in the GI-tract of pigs and infants. Here we provide a transformation protocol for B. thermophilum and a novel expression vector for this species. The protocol resulted in transformation rates of 1×103 transformed cells per µg DNA. Transformation was shown to be dependent on the presence of fructo-oligosaccharides during growth, polyethylene glycol in the electroporation buffer, and on methylation of the vector. The Escherichia coli - B. thermophilum shuttle vector pLFB1012 for heterologous gene expression was constructed harbouring the glyceraldehyde 3-phosphate dehydrogenase promoter from Bifidobacterium longum (Pgap). Activity of the ß-glucoronidase gene gusA under control of Pgap could be detected at a 20-fold higher rate compared to the wild type, showing activity of the promoter in B. thermophilum. Thereafter, the B. longum gene bl_1404, previously proposed to be involved in oxidative stress resistance, was cloned under control of the Pgap. The wild type cell numbers of B. thermophilum RBL 67 decreased at least 9 log after a 20-mM H2O2 treatment for 60min whereas the mutant strain expressing bl_1404 showed an increased survival of 2 logs compared to the wild type strain. To our knowledge this is the first report on transformation of B. thermophilum. Further, it is shown that pLFB1002 is suitable for engineering B. thermophilum and that bl_1404 from B. longum is involved in peroxide resistance in bifidobacteria.

East and West African milk products are reservoirs for human and livestock-associated Staphylococcus aureus.

Staphylococcus aureus frequently isolated from milk products in sub-Saharan Africa (SSA) is a major pathogen responsible for food intoxication, human and animal diseases. SSA hospital-derived strains are well studied but data on the population structure of foodborne S. aureus required to identify possible staphylococcal food poisoning sources is lacking. Therefore, the aim was to assess the population genetic structure, virulence and antibiotic resistance genes associated with milk-derived S. aureus isolates from Côte d'Ivoire, Kenya and Somalia through spa-typing, MLST, and DNA microarray analysis. Seventy milk S. aureus isolates from the three countries were assigned to 27 spa (7 new) and 23 (12 new) MLST sequence types. Milk-associated S. aureus of the three countries is genetically diverse comprising human and livestock-associated clonal complexes (CCs) predominated by the CC5 (n = 10) and CC30 (n = 9) isolates. Panton-Valentine leukocidin, toxic shock syndrome toxin and enterotoxin encoding genes were predominantly observed among human-associated CCs. Penicillin, fosfomycin and tetracycline, but not methicillin resistance genes were frequently detected. Our findings indicate that milk-associated S. aureus in SSA originates from human and animal sources alike highlighting the need for an overarching One Health approach to reduce S. aureus disease burdens through improving production processes, animal care and hygienic measures.

Phylogenetic, epidemiological and functional analyses of the Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme.

BACKGROUND: The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises seven (sub)species classified as human and animal commensals, emerging opportunistic pathogens and food fermentative organisms. Changing taxonomy, shared habitats, natural competence and evidence for horizontal gene transfer pose difficulties for determining their phylogeny, epidemiology and virulence mechanisms. Thus, novel phylogenetic and functional classifications are required. An SBSEC overarching multi locus sequence type (MLST) scheme targeting 10 housekeeping genes was developed, validated and combined with host-related properties of adhesion to extracellular matrix proteins (ECM), activation of the immune responses via NF-KB and survival in simulated gastric juice (SGJ). RESULTS: Commensal and pathogenic SBSEC strains (n = 74) of human, animal and food origin from Europe, Asia, America and Africa were used in the MLST scheme yielding 66 sequence types and 10 clonal complexes differentiated into distinct habitat-associated and mixed lineages. Adhesion to ECMs collagen I and mucin type II was a common characteristic (23 % of strains) followed by adhesion to fibronectin and fibrinogen (19.7 %). High adhesion abilities were found for East African dairy and human blood isolate branches whereas commensal fecal SBSEC displayed low adhesion. NF-KB activation was observed for a limited number of dairy and blood isolates suggesting the potential of some pathogenic strains for reduced immune activation. Strains from dairy MLST clades displayed the highest relative survival to SGJ independently of dairy adaptation markers lacS/lacZ. CONCLUSION: Combining phylogenetic and functional analyses via SBSEC MLST enabled the clear delineation of strain clades to unravel the complexity of this bacterial group. High adhesion values shared between certain dairy and blood strains as well as the behavior of NF-KB activation are concerning for specific lineages. They highlighted the health risk among shared lineages and establish the basis to elucidate (zoonotic-) transmission, host specificity, virulence mechanisms and enhanced risk assessment as pathobionts in an overarching One Health approach.

Comparative genome analysis of Streptococcus infantarius subsp. infantarius CJ18, an African fermented camel milk isolate with adaptations to dairy environment.

BACKGROUND: Streptococcus infantarius subsp. infantarius (Sii) belongs to the Streptococcus bovis/Streptococcus equinus complex associated with several human and animal infections. Sii is a predominant bacterium in spontaneously fermented milk products in Africa. The genome sequence of Sii strain CJ18 was compared with that of other Streptococcus species to identify dairy adaptations including genome decay such as in Streptococcus thermophilus, traits for its competitiveness in spontaneous milk fermentation and to assess potential health risks for consumers. RESULTS: The genome of Sii CJ18 harbors several unique regions in comparison to Sii ATCC BAA-102T, among others an enlarged exo- and capsular polysaccharide operon; Streptococcus thermophilus-associated genes; a region containing metabolic and hypothetical genes mostly unique to CJ18 and the dairy isolate Streptococcus gallolyticus subsp. macedonicus; and a second oligopeptide transport operon. Dairy adaptations in CJ18 are reflected by a high percentage of pseudogenes (4.9%) representing genome decay which includes the inactivation of the lactose phosphotransferase system (lacIIABC) by multiple transposases integration. The presence of lacS and lacZ genes is the major dairy adaptation affecting lactose metabolism pathways also due to the disruption of lacIIABC.We constructed mutant strains of lacS, lacZ and lacIIABC and analyzed the resulting strains of CJ18 to confirm the redirection of lactose metabolism via LacS and LacZ.Natural competence genes are conserved in both Sii strains, but CJ18 contains a lower number of CRISPR spacers which indicates a reduced defense capability against alien DNA. No classical streptococcal virulence factors were detected in both Sii strains apart from those involved in adhesion which should be considered niche factors. Sii-specific virulence factors are not described. Several Sii-specific regions encoding uncharacterized proteins provide new leads for virulence analyses and investigation of the unclear association of dairy and clinical Sii with human diseases. CONCLUSIONS: The genome of the African dairy isolate Sii CJ18 clearly differs from the human isolate ATCC BAA-102T. CJ18 possesses a high natural competence predisposition likely explaining the enlarged genome. Metabolic adaptations to the dairy environment are evident and especially lactose uptake corresponds to S. thermophilus. Genome decay is not as advanced as in S. thermophilus (10-19%) possibly due to a shorter history in dairy fermentations.

The effect of saturated fatty acids on methanogenesis and cell viability of Methanobrevibacter ruminantium.

Saturated fatty acids (SFAs) are known to suppress ruminal methanogenesis, but the underlying mechanisms are not well known. In the present study, inhibition of methane formation, cell membrane permeability (potassium efflux), and survival rate (LIVE/DEAD staining) of pure ruminal Methanobrevibacter ruminantium (DSM 1093) cell suspensions were tested for a number of SFAs. Methane production rate was not influenced by low concentrations of lauric (C12; 1 µg/mL), myristic (C14; 1 and 5 µg/mL), or palmitic (C16; 3 and 5 µg/mL) acids, while higher concentrations were inhibitory. C12 and C14 were most inhibitory. Stearic acid (C18), tested at 10-80 µg/mL and ineffective at 37°C, decreased methane production rate by half or more at 50°C and ≥50 µg/mL. Potassium efflux was triggered by SFAs (C12 = C14 > C16 > C18 = control), corroborating data on methane inhibition. Moreover, the exposure to C12 and C14 decreased cell viability to close to zero, while 40% of control cells remained alive after 24 h. Generally, tested SFAs inhibited methanogenesis, increased cell membrane permeability, and decreased survival of M. ruminantium in a dose- and time-dependent way. These results give new insights into how the methane suppressing effect of SFAs could be mediated in methanogens.

Tn6198, a novel transposon containing the trimethoprim resistance gene dfrG embedded into a Tn916 element in Listeria monocytogenes.

OBJECTIVES: To characterize Tn6198, a novel conjugative transposon from the clinical Listeria monocytogenes strain TTH-2007, which contains the tetracycline and trimethoprim resistance genes tet(M) and dfrG, respectively, and to assess its transferability in vitro and in situ. METHODS: The complete sequence of Tn6198 was determined using a primer walking strategy. Horizontal gene transfer studies were performed by filter matings, as well as on the surface of smear-ripened cheese and smoked salmon. The presence of Tn916-like circular intermediates was determined by PCR. Antibiotic resistance was determined by the broth microdilution method and microarray hybridization. RESULTS: Sequencing of Tn6198 revealed that a 3.3 kb fragment containing dfrG was integrated between open reading frames 23 and 24 of Tn916. Furthermore, an additional copy of Tn916 was present in L. monocytogenes TTH-2007. Both elements were transferred simultaneously and separately in vitro to recipients L. monocytogenes 10403S and Enterococcus faecalis JH2-2 by conjugation, resulting in either tetracycline- and trimethoprim-resistant or solely tetracycline-resistant transconjugants. On the surface of cheese and salmon, only L. monocytogenes 10403S transconjugants were detected. CONCLUSIONS: This study reports the first Tn916-like element associated with a trimethoprim resistance gene, as well as the first fully characterized transposon conferring multidrug resistance in L. monocytogenes. This is of concern, as trimethoprim is administered to listeriosis patients with ß-lactam allergy and as Tn6198 has a large potential for dissemination, indicated by both intra-species and inter-genus transfer.

pDB2011, a 7.6 kb multidrug resistance plasmid from Listeria innocua replicating in Gram-positive and Gram-negative hosts.

pDB2011, a multidrug resistance plasmid isolated from the foodborne Listeria innocua strain TTS-2011 was sequenced and characterized. Sequence analysis revealed that pDB2011 had a length of 7641 bp and contained seven coding DNA sequences of which two were annotated as replication proteins, one as a recombination/mobilization protein and one as a transposase. Furthermore, pDB2011 harbored the trimethoprim, spectinomycin and macrolide-lincosamide-streptogramin B resistance genes dfrD, spc and erm(A), respectively. However, pDB2011 was only associated with trimethoprim and spectinomycin resistance phenotypes and not with phenotypic resistance to erythromycin. A region of the plasmid encoding the resistance genes spc and erm(A) plus the transposase was highly similar to Staphylococcus aureus transposon Tn554. The dfrD gene was 100% identical to dfrD found in a number of Listeria monocytogenes isolates. Additionally, assessment of the potential host range of pDB2011 revealed that the plasmid was able to replicate in Lactococcus lactis subsp. cremoris MG1363 as well as in Escherichia coli MC1061 and DH5α. This study reports the first multidrug resistance plasmid in L. innocua. A large potential for dissemination of pDB2011 is indicated by its host range of both Gram-positive and Gram-negative bacteria.

Listeria fleischmannii sp. nov., isolated from cheese.

A study was performed on three isolates (LU2006-1(T), LU2006-2 and LU2006-3), which were sampled independently from cheese in western Switzerland in 2006, as well as a fourth isolate (A11-3426), which was detected in 2011, using a polyphasic approach. The isolates could all be assigned to the genus Listeria but not to any known species. Phenotypic and chemotaxonomic data were compatible with the genus Listeria and phylogenetic analysis based on 16S rRNA gene sequences confirmed that the closest relationships were with members of this genus. However, DNA-DNA hybridization demonstrated that the isolates did not belong to any currently described species. Cell-wall-binding domains of Listeria monocytogenes bacteriophage endolysins were able to attach to the isolates, confirming their tight relatedness to the genus Listeria. Although PCR targeting the central portion of the flagellin gene flaA was positive, motility was not observed. The four isolates could not be discriminated by Fourier transform infrared spectroscopy or pulsed-field gel electrophoresis. This suggests that they represent a single species, which seems to be adapted to the environment in a cheese-ripening cellar as it was re-isolated from the same type of Swiss cheese after more than 5 years. Conjugation experiments demonstrated that the isolates harbour a transferable resistance to clindamycin. The isolates did not exhibit haemolysis or show any indication of human pathogenicity or virulence. The four isolates are affiliated with the genus Listeria but can be differentiated from all described members of the genus Listeria and therefore they merit being classified as representatives of a novel species, for which we propose the name Listeria fleischmannii sp. nov.; the type strain is LU2006-1(T) ( = DSM 24998(T)  = LMG 26584(T)).

Complete genome sequence of the African dairy isolate Streptococcus infantarius subsp. infantarius strain CJ18.

Streptococcus infantarius subsp. infantarius, a member of the Streptococcus bovis/Streptococcus equinus complex, is highly prevalent in artisanal dairy fermentations in Africa. Here the complete genome sequence of the dairy-adapted S. infantarius subsp. infantarius CJ18 strain--a strain predominant in traditionally fermented camel milk (suusac) from Kenya--is presented.

CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity.

A gene encoding an O-acetyl-L-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-L-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K(M) for O-acetyl-L-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K(M) for L-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.

Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP.

Pathogenic, spoilage, and technologically important microorganisms were monitored in 21 spontaneously fermented Swiss meat products manufactured with meat from wildlife or animals grown in natural habitat. Thereby, PCR-restriction fragment length polymorphism (RFLP) on rpoB and 16S rRNA gene sequences provided a powerful tool for fast and accurate identification of the main microbial population. Lactobacillus sakei and Lactobacillus curvatus dominated in fermented meat products followed by Staphylococcus species, which constituted 88.2% of all Gram-positive, catalase-positive cocci (GCC(+)) with cell counts varying from 2.6 to 7.0 log cfu/g during maturation. Staphylococcus equorum was prevalent in frequency and cell counts during maturation (18.0%; 5.0-7.3 log cfu/g) and in the end products (28.4%; 1.8-6.2 log cfu/g) implicating a new presumptive starter species for meat fermentation. Nine out of 14 end products indicated safety risks to consumers due to the high incidence of Staphylococcus saprophyticus or Staphylococcus epidermidis combined with cell counts of 7.4 and 4.9 log cfu/g, respectively. This fact was supported by the detection of Staphylococcus aureus and Enterobacteriaceae in ready-to-eat products strongly exceeding the tolerable limit of 2 log cfu/g. Spontaneously fermented meat products produced from wildlife or animals grown in natural habitats not only gave rise to hygienic and safety concerns but also provided new presumptive starter strains.

Novel Streptococcus infantarius subsp. infantarius variants harboring lactose metabolism genes homologous to Streptococcus thermophilus.

Streptococcus infantarius subsp. infantarius belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC) commonly associated with human and animal infections. We elucidated the lactose metabolism of S. infantarius subsp. infantarius predominant in African fermented milk products. S. infantarius subsp. infantarius isolates (n = 192) were identified in 88% of spontaneously fermented camel milk suusac samples (n = 24) from Kenya and Somalia at log10 8.2-8.5 CFU mL⁻¹. African S. infantarius isolates excreted stoichiometric amounts of galactose when grown on lactose, exhibiting a metabolism similar to Streptococcus thermophilus and distinct from their type strain. African S. infantarius subsp. infantarius CJ18 harbors a regular gal operon with 99.7-100% sequence identity to S. infantarius subsp. infantarius ATCC BAA-102(T) and a gal-lac operon with 91.7-97.6% sequence identity to S. thermophilus, absent in all sequenced SBSEC strains analyzed. The expression and functionality of lacZ was demonstrated in a ß-galactosidase assay. The gal-lac operon was identified in 100% of investigated S. infantarius isolates (n = 46) from suusac samples and confirmed in Malian fermented cow milk isolates. The African S. infantarius variant potentially evolved through horizontal gene transfer of an S. thermophilus-homologous lactose pathway. Safety assessments are needed to identify any putative health risks of this novel S. infantarius variant.

High Prevalence of Enterobacterales in the Smear of Surface-Ripened Cheese with Contribution to Organoleptic Properties.

The smear of surface-ripened cheese harbors complex microbiota mainly composed of typical Gram-positive aerobic bacteria and yeast. Gram-negative bacteria are usually classified as un-wanted contaminants. In order to investigate the abundance and impact of Gram-negative bacte-ria naturally occurring in the smear of surface-ripened cheese, we performed a culture-based analysis of smear samples from 15 semi-hard surface-ripened cheese varieties. The quantity, di-versity and species distribution of Proteobacteria in the surface smear of the analyzed cheese vari-eties were unexpectedly high, and comprised a total of 22 different species. Proteus and Morganella predominated most of the analyzed cheese varieties, while Enterobacter, Citrobacter, Hafnia and Serratia were also found frequently. Further physiological characterization of Proteus isolates re-vealed strong proteolytic activity, and the analysis of volatiles in the smear cheese surface head-space suggested that Enterobacterales produce volatile organic flavor compounds that contribute to the organoleptic properties of surface-ripened cheese. Autochthonous members of Enterobac-terales were found in 12 of the 15 smear samples from surface-ripened cheeses, suggesting that they are part of the typical house microbiota that shape the organoleptic properties of the cheese rather than represent unwanted contaminants. However, further investigation on safety issues of the individual species should be performed in order to manage the health risk for consumers.

1,3-Propanediol dehydrogenases in Lactobacillus reuteri: impact on central metabolism and 3-hydroxypropionaldehyde production.

BACKGROUND: Lactobacillus reuteri metabolizes glycerol to 3-hydroxypropionaldehyde (3-HPA) and further to 1,3-propanediol (1,3-PDO), the latter step catalysed by a propanediol dehydrogenase (PDH). The last step in this pathway regenerates NAD+ and enables therefore the energetically more favourable production of acetate over ethanol during growth on glucose. RESULTS: A search throughout the genome of L. reuteri DSM 20016 revealed two putative PDHs encoded by ORFs lr_0030 and lr_1734. ORF lr_1734 is situated in the pdu operon encoding the glycerol conversion machinery and therefore likely involved in 1,3-PDO formation. ORF lr_0030 has not been associated with PDH-activity so far. To elucidate the role of these two PDHs, gene deletion mutant strains were constructed. Growth behaviour on glucose was comparable between the wild type and both mutant strains. However, on glucose + glycerol, the exponential growth rate of Δlr_0030 was lower compared to the wild type and the lr_1734 mutant. Furthermore, glycerol addition resulted in decreased ethanol production in the wild type and Δlr_1734, but not in Δlr_0030. PDH activity measurements using 3-HPA as a substrate revealed lower activity of Δlr_0030 extracts from exponential growing cells compared to wild type and Δlr_1734 extracts.During biotechnological 3-HPA production using non-growing cells, the ratio 3-HPA to 1,3-PDO was approximately 7 in the wild type and Δlr_0030, whereas this ratio was 12.5 in the mutant Δlr_1734. CONCLUSION: The enzyme encoded by lr_0030 plays a pivotal role in 3-HPA conversion in exponential growing L. reuteri cells. The enzyme encoded by lr_1734 is active during 3-HPA production by non-growing cells and this enzyme is a useful target to enhance 3-HPA production and minimize formation of the by-product 1,3-PDO.

Effects of monolaurin on ruminal methanogens and selected bacterial species from cattle, as determined with the rumen simulation technique.

Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal.

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology.

BACKGROUND: Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H2O2 from 0 to 130 ppm. RESULTS: At the beginning of the culture, high cell density of 10(13) CFU per litre of reactor was tested. The continuous culture gradually adapted to increasing H2O2 concentrations. However, after increasing the H2O2 concentration to 130 ppm the OD of the culture decreased to 0. Full wash out was prevented by the immobilization of the cells in gel matrix. Hence after stopping the stress, it was possible to re-grow the cells that survived the highest lethal dose of H2O2 and to select two adapted colonies (HPR1 and HPR2) after plating of the culture effluent. In contrast to HPR1, HPR2 showed stable characteristics over at least 70 generations and exhibited also higher tolerance to O2 than non adapted wild type cells. Preliminary characterization of HPR2 was carried out by global genome expression profile analysis. Two genes coding for a protein with unknown function and possessing trans-membrane domains and an ABC-type transporter protein were overexpressed in HPR2 cells compared to wild type cells. CONCLUSIONS: Our study showed that continuous culture with cell immobilization is a valid approach for selecting cells adapted to hydrogen peroxide. Elucidation of H2O2 adaptation mechanisms in HPR2 could be helpful to develop oxygen resistant bifidobacteria.

Influence of two anti-fungal Lactobacillus fermentum-Saccharomyces cerevisiae co-cultures on cocoa bean fermentation and final bean quality.

The growth of filamentous fungi during the spontaneous cocoa bean fermentation leads to inferior cocoa bean quality and poses a health risk for consumers due to the potential accumulation of mycotoxins. We recently developed anti-fungal cultures with the capacity to inhibit the growth of mycotoxigenic filamentous fungi on cocoa beans. However, it is not clear how these anti-fungal cultures affect the fermentation process and cocoa bean quality. For that, the anti-fungal co-cultures, Lactobacillus fermentum M017-Saccharomyces cerevisiae H290 (A) and Lb. fermentum 223-S. cerevisiae H290 (B), were applied to 180-kg box fermentations in Honduras in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the co-cultures only marginally affected the fermentation process and cocoa bean quality. Microorganisms reached maximal levels of 6.2-7.6 log CFU/g of yeasts and acetic acid bacteria and 7.9-9.5 log CFU/g of lactic acid bacteria during all fermentations and led to maximal metabolite concentrations in bean cotyledons of 4-12 mg/g ethanol, 2-6 mg/g lactic acid and 6-14 mg/g acetic acid. The fermentation and drying processes resulted in 38-90 mg epicatechin equivalents/g in the cotyledons of dried beans. However, the co-cultures led to up to ten times higher mannitol levels in cotyledons of inoculated beans compared to beans during spontaneous fermentation, and caused a slower fermentation process, detectable as up to 8-12 °C lower temperatures in the centre of the fermenting pulp-bean mass and up to 22% lower proportions of well-fermented beans after drying. Co-culture B-with Lb. fermentum 223 -led to improved cocoa bean quality compared to co-culture A-with Lb. fermentum M017 -, i.e. cocoa beans with 0.5-1.9 mg/g less acetic acid, 4-17% higher shares of well-fermented beans and, on a scale from 0 to 10, to 0.2-0.6 units lower astringency, up to 1.1 units lower off-flavours, and 0.2-0.9 units higher cocoa notes. Therefore, the anti-fungal co-culture B is recommended for future applications and its capacity to limit fungal growth and mycotoxin production during industrial-scale cocoa bean fermentation should be investigated in further studies.