Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis.

Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted ß-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.

Fractional factorial design-based optimisation and application of an extraction and UPLC-MS/MS detection method for the quantification of phytosterols in food, feed and beverages low in phytosterols.

Phytosterols are ubiquitous in plants, as they play an important role in cell membrane stability and as signal transducers. Over the last few decades, scientific interest in phytosterols has significantly increased. Most of the interest has focused on the cholesterol-lowering properties of phytosterols, but they may also interfere with endogenous steroid hormone synthesis. Despite this dual interest in phytosterols, accurate and fully validated methods for the quantification of phytosterols in food and feed samples are scarce. During this study an extraction and detection method for the main free phytosterols (ß-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria, reaching all evaluated performance parameter requirements. The individual recoveries ranged between 95 and 104 %. Good results for repeatability and intralaboratory reproducibility (RSD %) were observed (<10 %). Excellent linearity was proven on the basis of determination coefficient (R 2 > 0.99) and lack-of-fit test (F test, alpha = 0.05). The limits of detection (LODs) and lower limits of quantification (LLOQs) in grain matrices were as low as 0.01-0.03 mg per 100 g and 0.02-0.10 mg per 100 g. This method allowed quantification of all main, free phytosterols in different grains (oats, barley, corn, malt) and it was shown that the method can be used for other solid food and feed samples as well, including new matrices such as straw, hay, mustard seeds, grass and yellow peas. Additionally, the method was shown to perform well in liquid samples low in phytosterols such as concentrate-based juices, soft drinks and beers (<5 µg per 100 mL). Graphical Abstract An extraction and detection method for the main free phytosterols (ß-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria and applied on different matrices including feed and beverages.

The microbial diversity of an industrially produced lambic beer shares members of a traditionally produced one and reveals a core microbiota for lambic beer fermentation.

The microbiota involved in lambic beer fermentations in an industrial brewery in West-Flanders, Belgium, was determined through culture-dependent and culture-independent techniques. More than 1300 bacterial and yeast isolates from 13 samples collected during a one-year fermentation process were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by sequence analysis of rRNA and various protein-encoding genes. The bacterial and yeast communities of the same samples were further analyzed using denaturing gradient gel electrophoresis of PCR-amplified V3 regions of the 16S rRNA genes and D1/D2 regions of the 26S rRNA genes, respectively. In contrast to traditional lambic beer fermentations, there was no Enterobacteriaceae phase and a larger variety of acetic acid bacteria were found in industrial lambic beer fermentations. Like in traditional lambic beer fermentations, Saccharomyces cerevisiae, Saccharomyces pastorianus, Dekkera bruxellensis and Pediococcus damnosus were the microorganisms responsible for the main fermentation and maturation phases. These microorganisms originated most probably from the wood of the casks and were considered as the core microbiota of lambic beer fermentations.

Microbiota and metabolites of aged bottled gueuze beers converge to the same composition.

Gueuze beers are prepared by mixing young and old lambic beers and are bottle-refermented spontaneously for aging. The present study analyzed the microbiota and metabolites present in gueuze beers that were aged between a few months and up to 17 years. Yeasts were cultivated from all beers sampled, but bacteria could not be grown from beers older than 5 years. Lactic acid and ethyl lactate concentrations increased steadily during aging, whereas ethanol concentrations remained constant. The concentrations of isoamyl acetate and ethyl decanoate decreased during the aging process. Hence, ethyl lactate and ethyl decanoate can be considered as positive and negative gueuze beer-aging metabolite biomarkers, respectively. Nevertheless, considerable bottle-to-bottle variation in the metabolite profiles was found, which hindered the generalization of the effects seen during the aging of the gueuze beers examined, but which illustrated the unique character of the lambic beers. The present results further indicate that gueuze beers are preferably aged for less than 10 years.

Effects of growth medium on matrix-assisted laser desorption-ionization time of flight mass spectra: a case study of acetic acid bacteria.

The effect of the growth medium used on the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra generated and its consequences for species and strain level differentiation of acetic acid bacteria (AAB) were determined by using a set of 25 strains. The strains were grown on five different culture media that yielded a total of more than 600 mass spectra, including technical and biological replicates. The results demonstrate that the culture medium can have a profound effect on the mass spectra of AAB as observed in the presence and varying signal intensities of peak classes, in particular when culture media do not sustain optimal growth. The observed growth medium effects do not disturb species level differentiation but strongly affect the potential for strain level differentiation. The data prove that a well-constructed and robust MALDI-TOF mass spectrometry identification database should comprise mass spectra of multiple reference strains per species grown on different culture media to facilitate species and strain level differentiation.

Gluconobacter cerevisiae sp. nov., isolated from the brewery environment.

Three strains, LMG 27748(T), LMG 27749 and LMG 27882 with identical MALDI-TOF mass spectra were isolated from samples taken from the brewery environment. Analysis of the 16S rRNA gene sequence of strain LMG 27748(T) revealed that the taxon it represents was closely related to type strains of the species Gluconobacter albidus (100 % sequence similarity), Gluconobacter kondonii (99.9 %), Gluconobacter sphaericus (99.9 %) and Gluconobacter kanchanaburiensis (99.5 %). DNA-DNA hybridization experiments on the type strains of these species revealed moderate DNA relatedness values (39-65 %). The three strains used d-fructose, d-sorbitol, meso-erythritol, glycerol, l-sorbose, ethanol (weakly), sucrose and raffinose as a sole carbon source for growth (weak growth on the latter two carbon sources was obtained for strains LMG 27748(T) and LMG 27882). The strains were unable to grow on glucose-yeast extract medium at 37 °C. They produced acid from meso-erythritol and sucrose, but not from raffinose. d-Gluconic acid, 2-keto-d-gluconic acid and 5-keto-d-gluconic acid were produced from d-glucose, but not 2,5-diketo-d-gluconic acid. These genotypic and phenotypic characteristics distinguish strains LMG 27748(T), LMG 27749 and LMG 27882 from species of the genus Gluconobacter with validly published names and, therefore, we propose classifying them formally as representatives of a novel species, Gluconobacter cerevisiae sp. nov., with LMG 27748(T) ( = DSM 27644(T)) as the type strain.

Acetobacter lambici sp. nov., isolated from fermenting lambic beer.

An acetic acid bacterium, strain LMG 27439(T), was isolated from fermenting lambic beer. The cells were Gram-stain-negative, motile rods, catalase-positive and oxidase-negative. Analysis of the 16S rRNA gene sequence revealed the strain was closely related to Acetobacter okinawensis (99.7 % 16S rRNA gene sequence similarity with the type strain of this species), A. ghanensis (99.6 %), A. syzygii (99.6 %), A. fabarum (99.4 %) and A. lovaniensis (99.2 %). DNA-DNA hybridization with the type strains of these species revealed moderate DNA-DNA hybridization values (31-45 %). Strain LMG 27439(T) was unable to grow on glycerol or methanol as the sole carbon source, on yeast extract with 10 % ethanol or on glucose-yeast extract medium at 37 °C. It did not produce acid from l-arabinose, d-galactose or d-mannose, nor did it produce 2-keto-d-gluconic acid, 5-keto-d-gluconic acid or 2,5-diketo-d-gluconic acid from d-glucose. It did not grow on ammonium as the sole nitrogen source and ethanol as the sole carbon source. These genotypic and phenotypic data distinguished strain LMG 27439(T) from established species of the genus Acetobacter, and therefore we propose this strain represents a novel species of the genus Acetobacter. The name Acetobacter lambici sp. nov. is proposed, with LMG 27439(T) ( = DSM 27328(T)) as the type strain.

Acetobacter sicerae sp. nov., isolated from cider and kefir, and identification of species of the genus Acetobacter by dnaK, groEL and rpoB sequence analysis.

Five acetic acid bacteria isolates, awK9_3, awK9_4 ( = LMG 27543), awK9_5 ( = LMG 28092), awK9_6 and awK9_9, obtained during a study of micro-organisms present in traditionally produced kefir, were grouped on the basis of their MALDI-TOF MS profile with LMG 1530 and LMG 1531(T), two strains currently classified as members of the genus Acetobacter. Phylogenetic analysis based on nearly complete 16S rRNA gene sequences as well as on concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB indicated that these isolates were representatives of a single novel species together with LMG 1530 and LMG 1531(T) in the genus Acetobacter, with Acetobacter aceti, Acetobacter nitrogenifigens, Acetobacter oeni and Acetobacter estunensis as nearest phylogenetic neighbours. Pairwise similarity of 16S rRNA gene sequences between LMG 1531(T) and the type strains of the above-mentioned species were 99.7%, 99.1%, 98.4% and 98.2%, respectively. DNA-DNA hybridizations confirmed that status, while amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) data indicated that LMG 1531(T), LMG 1530, LMG 27543 and LMG 28092 represent at least two different strains of the novel species. The major fatty acid of LMG 1531(T) and LMG 27543 was C18 : 1ω7c. The major ubiquinone present was Q-9 and the DNA G+C contents of LMG 1531(T) and LMG 27543 were 58.3 and 56.7 mol%, respectively. The strains were able to grow on D-fructose and D-sorbitol as a single carbon source. They were also able to grow on yeast extract with 30% D-glucose and on standard medium with pH 3.6 or containing 1% NaCl. They had a weak ability to produce acid from d-arabinose. These features enabled their differentiation from their nearest phylogenetic neighbours. The name Acetobacter sicerae sp. nov. is proposed with LMG 1531(T) ( = NCIMB 8941(T)) as the type strain.

An rpoD gene sequence based evaluation of cultured Pseudomonas diversity on different growth media.

The last decade has shown an increased interest in the utilization of bacteria for applications ranging from bioremediation to wastewater purification and promotion of plant growth. In order to extend the current number of micro-organism mediated applications, a continued quest for new agents is required. This study focused on the genus Pseudomonas, which is known to harbour strains with a very diverse set of interesting properties. The aim was to identify growth media that allow retrieval of a high Pseudomonas diversity, as such increasing the chance of isolating isolates with beneficial properties. Three cultivation media: trypticase soy agar (TSA), potato dextrose agar (PDA) and Pseudomonas isolation agar (PIA) were evaluated for their abilities to grow Pseudomonas strains. TSA and PDA were found to generate the largest Pseudomonas diversity. However, communities obtained with both media overlapped. Communities obtained with PIA, on the other hand, were unique. This indicated that the largest diversity is obtained by sampling from either PDA or TSA and from PIA in parallel. To evaluate biodiversity of the isolated Pseudomonas members on the media, an appropriate biomarker had to be identified. Hence, an introductory investigation of the taxonomic resolution of the 16S rRNA, rpoD, gyrB and rpoB genes was performed. The rpoD gene sequences not only had a high phylogenetic content and the highest taxonomic resolution amongst the genes investigated, it also had a gene phylogeny that related well with that of the 16S rRNA gene.

Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples.

A polyphasic analysis was undertaken of seven independent isolates of gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7-100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA-DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica. Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria. The name Neisseria oralis sp. nov. (type strain 6332(T)  = DSM 25276(T)  = LMG 26725(T)) is proposed.

Hazenella coriacea gen. nov., sp. nov., isolated from clinical specimens.

A Gram-staining-positive, endospore-forming rod was isolated independently from clinical specimens in New York State, USA, once in 2009 and twice in 2011. The three isolates had identical 16S rRNA gene sequences and, based on their 16S rRNA gene sequence, are most closely related to the type strains of Laceyella sediminis and L. sacchari (94.6 % similarity). The partial 23S rRNA gene sequences of the three strains were also 100 % identical. Maximum-likelihood phylogenetic analysis suggests that the new isolates belong to the family Thermoactinomycetaceae. Additional biochemical and phenotypic characteristics of the strains support the family designation and suggest that the three isolates represent a single species. In each of the strains, the predominant menaquinone is MK-7, the diagnostic diamino acid is meso-diaminopimelic acid and the major cellular fatty acids are iso-C15 : 0, anteiso-C15 : 0 and iso-C13 : 0. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, four unknown phospholipids, four unknown aminophospholipids and an unknown lipid. It is proposed that the novel isolates represent a single novel species within a new genus, for which the name Hazenella coriacea gen. nov., sp. nov. is proposed. The type strain of Hazenella coriacea is strain 23436(T) ( = DSM 45707(T) = LMG 27204(T)).

Pediococcus lolii DSM 19927T and JCM 15055T are strains of Pediococcus acidilactici.

Strain NGRI 0510Q(T), isolated from ryegrass silage, was recently classified as a representative of a novel Pediococcus species, Pediococcus lolii Doi et al. 2009. It was deposited in the DSMZ and JCM culture collections as DSM 19927(T) and JCM 15055(T), respectively. A polyphasic taxonomic study, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, pheS and 16S rRNA gene sequence analysis, fluorescent amplified fragment length polymorphism and DNA-DNA hybridization, was used to prove that both subcultures of the type, and only, strain of this species are strains of Pediococcus acidilactici.

Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly 'thermoglucosidasius'); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov.

Sixty-two strains of thermophilic aerobic endospore-forming bacteria were subjected to polyphasic taxonomic study including 16S rRNA gene sequence analysis, polar lipid and fatty acid analysis, phenotypic characterization, and DNA-DNA hybridization experiments. Distinct clusters of the species Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus toebii and Geobacillus thermoglucosidasius were formed, allowing their descriptions to be emended, and the distinctiveness of the poorly represented species Geobacillus jurassicus, Geobacillus subterraneus and Geobacillus caldoxylosilyticus was confirmed. It is proposed that the name Geobacillus thermoglucosidasius be corrected to Geobacillus thermoglucosidans nom. corrig. Bacillus thermantarcticus clustered between Geobacillus species on the basis of 16S rRNA gene sequence analysis, and its transfer to the genus Geobacillus as Geobacillus thermantarcticus comb. nov. (type strain LMG 23032(T)=DSM 9572(T)=strain M1(T)=R-35644(T)) is proposed. The above-mentioned species, together with Geobacillus thermoleovorans and Geobacillus thermocatenulatus, form a monophyletic cluster representing the genus Geobacillus. The distinctiveness of 'Geobacillus caldoproteolyticus' was confirmed and it is proposed that it be accommodated, along with Geobacillus tepidamans, in the genus Anoxybacillus as Anoxybacillus caldiproteolyticus sp. nov. (type strain DSM 15730(T)=ATCC BAA-818(T)=LMG 26209(T)=R-35652(T)) and Anoxybacillus tepidamans comb. nov. (type strain LMG 26208(T)=ATCC BAA-942(T)=DSM 16325(T)=R-35643(T)), respectively. The type strain of Geobacillus debilis was not closely related to any members of the genera Anoxybacillus and Geobacillus, and it is proposed that this species be placed in the new genus Caldibacillus as Caldibacillus debilis gen. nov. comb. nov. The type strain of the type species, Caldibacillus debilis, is LMG 23386(T) (=DSM 16016(T)=NCIMB 13995(T)=Tf(T)=R-35653(T)).

Lysinibacillus macroides sp. nov., nom. rev.

'Bacillus macroides' ATCC 12905(T) ( = DSM 54(T) = LMG 18474(T)), isolated in 1947 from cow dung, was not included in the Approved Lists of Bacterial Names and so it lost standing in bacteriological nomenclature. Reinvestigation of the strain, including DNA-DNA relatedness experiments, revealed that 'Bacillus macroides' is genomically distinct from its closest relatives Lysinibacillus xylanilyticus, Lysinibacillus boronitolerans and Lysinibacillus fusiformis (as determined by 16S rRNA gene sequence analysis, with pairwise similarity values of 99.2, 98.8 and 98.5 %, respectively, with the type strains of these species). Further analysis showed that 'Bacillus macroides' shares the A4α L-Lys-D-Asp peptidoglycan type with other members of the genus Lysinibacillus and can thus be attributed to this genus. These results, combined with additional phenotypic data, justify the description of strain LMG 18474(T) ( = DSM 54(T) = ATCC 12905(T)) as Lysinibacillus macroides sp. nov., nom. rev.

Sporosarcina newyorkensis sp. nov. from clinical specimens and raw cow's milk.

Twelve independent isolates of a gram-positive, endospore-forming rod were recovered from clinical specimens in New York State, USA, and from raw milk in Flanders, Belgium. The 16S rRNA gene sequences for all isolates were identical. The closest species with a validly published name, based on 16S rRNA gene sequence, is Sporosarcina koreensis (97.13 % similarity). DNA-DNA hybridization studies demonstrate that the new isolates belong to a species distinct from their nearest phylogenetic neighbours. The partial sequences of the 23S rRNA gene for the novel strains and their nearest neighbours also provide support for the novel species designation. Maximum-likelihood phylogenetic analysis of the 16S rRNA gene sequences confirmed that the new isolates are in the genus Sporosarcina. The predominant menaquinone is MK-7, the peptidoglycan has the type A4α L-Lys-Gly-D-Glu, and the polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant fatty acids are iso-C(14 : 0), iso-C(15 : 0) and anteiso-C(15 : 0). In addition, biochemical and morphological analyses support designation of the twelve isolates as representatives of a single new species within the genus Sporosarcina, for which the name Sporosarcina newyorkensis sp. nov. (type strain 6062(T)  = DSM 23544(T)  = CCUG 59649(T)  = LMG 26022(T)) is proposed.

Influence of storage conditions on the growth of Pseudomonas species in refrigerated raw milk.

The refrigerated storage of raw milk throughout the dairy chain prior to heat treatment creates selective conditions for growth of psychrotolerant bacteria. These bacteria, mainly belonging to the genus Pseudomonas, are capable of producing thermoresistant extracellular proteases and lipases, which can cause spoilage and structural defects in pasteurized and ultra-high-temperature-treated milk (products). To map the influence of refrigerated storage on the growth of these pseudomonads, milk samples were taken after the first milking turn and incubated laboratory scale at temperatures simulating optimal and suboptimal preprocessing storage conditions. The outgrowth of Pseudomonas members was monitored over time by means of cultivation-independent denaturing gradient gel electrophoresis (DGGE). Isolates were identified by a polyphasic approach. These incubations revealed that outgrowth of Pseudomonas members occurred from the beginning of the dairy chain (farm tank) under both optimal and suboptimal storage conditions. An even greater risk for outgrowth, as indicated by a vast increase of about 2 log CFU per ml raw milk, existed downstream in the chain, especially when raw milk was stored under suboptimal conditions. This difference in Pseudomonas outgrowth between optimal and suboptimal storage was already statistically significant within the farm tank. The predominant taxa were identified as Pseudomonas gessardii, Pseudomonas gessardii-like, Pseudomonas fluorescens-like, Pseudomonas lundensis, Pseudomonas fragi, and Pseudomonas fragi-like. Those taxa show an important spoilage potential as determined on elective media for proteolysis and lipolysis.

Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus.

Nineteen thermophilic, aerobic, endospore-forming bacterial strains were subjected to 16S rRNA gene sequence analysis. Eight of these strains had been received as cultures of Geobacillus kaustophilus, G. lituanicus, G. stearothermophilus, 'G. thermoleovorans subsp. stromboliensis', G. vulcani, 'Bacillus caldolyticus', 'B. caldotenax' and 'B. caldovelox', but they showed close relationships with the type strain of G. thermoleovorans, as did two other strains received as G. thermoleovorans. All strains underwent further taxonomic analysis by API and other phenotypic tests and fatty acid methyl ester analysis, and selected strains were analysed for their polar lipids and for DNA relatedness. The 11 strains that formed the G. thermoleovorans 16S rRNA cluster also showed some phenotypic similarities, and DNA relatedness data support the reassignment of the strains received as G. kaustophilus, G. lituanicus, 'G. thermoleovorans subsp. stromboliensis', G. vulcani, 'B. caldolyticus', 'B. caldotenax' and 'B. caldovelox', and one of the G. stearothermophilus strains, as members of the species G. thermoleovorans. Four other strains received as G. kaustophilus were misnamed; two were identified as G. stearothermophilus and two appeared to be closely related to Anoxybacillus rupiensis. One strain received as G. stearothermophilus remained unidentified. On the basis of a single strain, Geobacillus thermocatenulatus was shown to represent a distinct species, but study of the type strain of Geobacillus gargensis showed this species to be a later heterotypic synonym of Geobacillus thermocatenulatus. Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus are therefore presented.

Bacillus thermolactis sp. nov., isolated from dairy farms, and emended description of Bacillus thermoamylovorans.

A polyphasic taxonomic study was performed on 22 thermotolerant, aerobic, endospore-forming bacteria from dairy environments. Seventeen isolates were retrieved from raw milk, one from a filter cloth and four from grass, straw or milking equipment. These latter four isolates (R-6546, R-7499, R-7764 and R-7440) were identified as Bacillus thermoamylovorans based on DNA-DNA hybridizations (values above 70 % with Bacillus thermoamylovorans LMG 18084(T)) but showed discrepancies in characteristics with the original species description, so an emended description of this species is given. According to 16S rRNA gene sequence analysis and DNA-DNA hybridization experiments, the remaining 18 isolates (R-6488(T), R-28193, R-6491, R-6492, R-7336, R-33367, R-6486, R-6770, R-31288, R-28160, R-26358, R-7632, R-26955, R-26950, R-33520, R-6484, R-26954 and R-7165) represented one single species, most closely related to Bacillus thermoamylovorans (93.9 % 16S rRNA gene sequence similarity), for which the name Bacillus thermolactis is proposed. Cells were Gram-stain-positive, facultatively anaerobic, endospore-forming rods that grew optimally at 40-50 °C. The cell wall peptidoglycan type of strain R-6488(T), the proposed type strain, was A1γ based on meso-diaminopimelic acid. Major fatty acids of the strains were C(16 : 0) (28.0 %), iso-C(16 : 0) (12.1 %) and iso-C(15 : 0) (12.0 %). MK-7 was the predominant menaquinone, and major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and some unidentified phospholipids. DNA G+C content was 35.0 mol%. Phenotypic properties allowed discrimination from other thermotolerant species of the genus Bacillus and supported the description of the novel species Bacillus thermolactis, with strain R-6488(T) ( = LMG 25569(T)  = DSM 23332(T)) as the proposed type strain.

Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity.

Recent research suggests that gluten-free beers by prolyl-endopeptidase treatment may not be safe for coeliac disease (CD) patients. Therefore, the gluten peptidome of an industrial gluten-free prolyl-endopeptidase treated malt beer (<10 ppm gluten) was compared to its untreated counterpart (58 ppm gluten) as a reference. NanoLC-HRMS analysis revealed the presence of 155 and 158 gluten peptides in the treated and reference beer, respectively. Characterisation of the peptides in treated beer showed that prolyl-endopeptidase activity was not complete with many peptides containing (multiple) internal proline-residues. Yet, prolyl-endopeptidase treatment did eliminate complete CD-immunogenic motifs, however, 18 peptides still contained partial, and potentially unsafe, motifs. In the reference beer respectively 7 and 37 gluten peptides carried (multiple) complete and/or partial CD-immunogenic motifs. Worrying is that many of these partial immunogenic gluten peptides do not contain a recognition epitope for the R5-antibody and would be overlooked in the current ELISA analysis for gluten quantification.

Exploration of isoxanthohumol bioconversion from spent hops into 8-prenylnaringenin using resting cells of Eubacterium limosum.

Hops is an almost unique source of the potent phytoestrogen 8-prenylnaringenin (8-PN). As hops contain only low levels of 8-PN, synthesis may be more attractive than extraction. A strain of the Gram-positive Eubacterium limosum was isolated previously for 8-PN production from more abundant precursor isoxanthohumol (IX) from hops. In this study, spent hops, an industrial side stream from the beer industry, was identified as interesting source of IX. Yet, hop-derived compounds are well-known antibacterial agents and the traces of a large variety of different compounds in spent hops interfered with growth and IX conversion. Critical factors to finally enable bacterial 8-PN production from spent hops, using a food and feed grade medium, were evaluated in this research. The use of bacterial resting cells and complex medium at a pH of 7.8-8 best fulfilled the requirements for 8-PN production and generated a solid basis for development of an economic process.