Contribution of S. xylosus and L. sakei ssp. carnosus Fermentation to the Aroma of Lupin Protein Isolates.

Aroma-active compounds of lupin protein isolate and lupin protein isolate fermented with Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus were investigated. The changes in aroma-active compounds were determined by application of aroma extract dilution analysis in combination with gas chromatography-mass spectrometry/olfactometry for identification, and by stable isotope dilution assays for quantification. A total of 30 aroma-active compounds for non-fermented and fermented samples were identified. The aroma profile of LPI fermented with Lactobacillus sakei ssp. carnosus was characterized as roasty and popcorn-like. Staphylococcus xylosus generated cheesy impressions, being in line with the fact that the main aroma compounds acetic acid, butanoic acid, and 2/3-methylbutanoic acid could be identified. Quantification of butanoic acid further confirmed these findings with the highest concentration of 140 mg/kg for LPI fermented with Staphylococcus xylosus. Our study provides insights into how fermentation utilizing different fermentative microbial strains, namely Staphylococcus xylosus and Lactobacillus sakei ssp. carnosus alters the aroma profile of lupin protein isolates. This demonstrates the potential of shaping fermented protein-based foods via targeted microbiological refinement.

Fermentation of Lupin Protein Hydrolysates-Effects on Their Functional Properties, Sensory Profile and the Allergenic Potential of the Major Lupin Allergen Lup an 1.

Lupin protein isolate was treated using the combination of enzymatic hydrolysis (Papain, Alcalase 2.4 L and Pepsin) and lactic acid fermentation (Lactobacillus sakei ssp. carnosus, Lactobacillus amylolyticus and Lactobacillus helveticus) to investigate the effect on functional properties, sensory profile and protein integrity. The results showed increased foaming activity (2466-3481%) and solubility at pH 4.0 (19.7-36.7%) of all fermented hydrolysates compared to the untreated lupin protein isolate with 1613% of foaming activity and a solubility of 7.3 (pH 4.0). Results of the SDS-PAGE and Bead-Assay showed that the combination of enzymatic hydrolysis and fermentation of LPI was effective in reducing L. angustifolius major allergen Lup an 1 to a residual level of <0.5%. The combination of enzymatic hydrolysis and fermentation enables the production of food ingredients with good functional properties in terms of protein solubility and foam formation, with a balanced aroma and taste profile.

Field and mesocosm methods to test biodegradable plastic film under marine conditions.

The pollution of the natural environment, especially the world's oceans, with conventional plastic is of major concern. Biodegradable plastics are an emerging market bringing along potential chances and risks. The fate of these materials in the environment and their possible effects on organisms and ecosystems has rarely been studied systematically and is not well understood. For the marine environment, reliable field test methods and standards for assessing and certifying biodegradation to bridge laboratory respirometric data are lacking. In this work we present newly developed field tests to assess the performance of (biodegradable) plastics under natural marine conditions. These methods were successfully applied and validated in three coastal habitats (eulittoral, benthic and pelagic) and two climate zones (Mediterranean Sea and tropical Southeast Asia). Additionally, a stand-alone mesocosm test system which integrated all three habitats in one technical system at 400-L scale independent from running seawater is presented as a methodological bridge. Films of polyhydroxyalkanoate copolymer (PHA) and low density polyethylene (LD-PE) were used to validate the tests. While LD-PE remained intact, PHA disintegrated to a varying degree depending on the habitat and the climate zone. Together with the existing laboratory standard test methods, the field and mesocosm test systems presented in this work provide a 3-tier testing scheme for the reliable assessment of the biodegradation of (biodegradable) plastic in the marine environment. This toolset of tests can be adapted to other aquatic ecosystems.

Effect of enzyme-assisted hydrolysis on protein pattern, technofunctional, and sensory properties of lupin protein isolates using enzyme combinations.

The modification of lupin protein isolates (LPI) by means of enzymatic hydrolysis (Lupinus angustifolius cultivar Boregine) was performed with four enzyme preparations (Alcalase 2.4 L, Papain, Corolase 7089, and Neutrase 0.8 L) in a one- and two-step process to determine the efficacy for the destruction of major IgE-reactive polypeptides and the evaluation of the technofunctional and sensory properties of lupin protein hydrolysates. Combinations of Alcalase 2.4 L and Papain were most effective in the degradation of polypeptides in L. angustifolius as measured by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The enzymatic hydrolysis of the LPI increased their technofunctional properties such as protein solubility, foam activity, and emulsifying capacity almost independently of the enzyme preparation used. The sensory results showed a significant increase in bitterness from 1.9 for LPI to 5.7 for the combination of Alcalase 2.4 L and Papain in one-step process. The aroma attributes of the hydrolysates were very similar to untreated LPI. The results of this study show the possibility of enzymatic hydrolysis of LPI to destroy the major IgE-reactive polypeptides that increase the technofunctional properties of the isolates and thus their use in human nutrition as food ingredients.

Myco-DES: Enabling Remote Extraction of Mycotoxins for Robust and Reliable Quantification by Stable Isotope Dilution LC-MS/MS.

Mycotoxins remain a global threat to human and animal health, especially in countries lacking effective measures to detect and control contaminated commodities. As the quantification of mycotoxins usually relies on complex and expensive techniques, the availability of suitable instrumentation is often a bottleneck in reliable mycotoxin detection. As part of our research toward strategies offering widespread access to mycotoxin analysis while cutting down on costs, we present a new extraction and quantification protocol combining materials originally designed for dried blood spot analysis with stable isotope dilution analysis. Its key benefits are that extraction of mycotoxins can be carried out at remote sites and by minimally trained personnel, while quantification will take place in specialized central laboratories simply connected by regular, paper-based mail. As a proof of concept, aflatoxins, ochratoxin A, and deoxynivalenol were extracted from cereal-based foodstuffs, fixed on paper cards for transport, and successfully quantified after re-extraction by stable isotope dilution LC-MS/MS analysis. Several materials (cellulose/polyethylene terephthalate/glass fiber, nontreated/chemically treated) as well as possible transport and storage conditions (temperature, humidity) were evaluated. The final myco-DES (dried extract spots) protocol allows quantification of mycotoxin levels currently recognized as safe (aflatoxin B1: 2 µg/kg, ochratoxin A: 3 µg/kg, deoxynivalenol: 500 µg/kg) after a storage of up to 4 weeks under tropical climate conditions (40 °C, 75% relative humidity).

Technofunctional and Sensory Properties of Fermented Lupin Protein Isolates.

Lupin protein isolate was fermented with eight different microorganisms to evaluate the influence on sensory profile, techno-functional properties and protein integrity. All investigated microorganisms were able to grow in lupin protein isolate. The results showed that the foaming activity in the range of 1646-1703% and the emulsifying capacity in the range of 347-595 mL of the fermented lupin protein isolates were similar to those of the unfermented ones. Protein solubility at pH 4 showed no significant changes compared to unfermented lupin protein isolate, whereas the solubility at pH 7 decreased significantly from 63.59% for lupin protein isolate to solubilities lower than 42.35% for fermented lupin protein isolate. Fermentation with all microorganisms showed the tendency to decrease bitterness from 2.3 for lupin protein isolate (LPI) to 1.0-2.0 for the fermented ones. The most promising microorganisms for the improvement of the sensory properties of lupin protein isolates were Lactobacillus brevis as it reduced the intensity of characteristic aroma impression (pea-like, green bell pepper-like) from 4.5 to 1.0. The SDS-PAGE results showed the fermentation treatment appeared not to be sufficiently effective to destruct the protein integrity and thus, deplete the allergen potential of lupin proteins. Fermentation allows the development of food ingredients with good functional properties in foam formation and emulsifying capacity, with a well-balanced aroma and taste profile.

Enzymatic hydrolysis of lupin protein isolates-Changes in the molecular weight distribution, technofunctional characteristics, and sensory attributes.

Enzymatic hydrolysis of lupin protein isolates (LPI; Lupinus angustifolius L.) was performed with nine different protease preparations to investigate their effect on technofunctionality, sensory properties, and the integrity of the proteins to estimate the reduction of the immunoreactivity. Alcalase 2.4 L, papain, and pepsin were most effective in the degradation of the α- and ß-conglutin examined by SDS-PAGE analysis, although the degree of hydrolysis only slightly increased. The technofunctional properties of LPI-solubility, emulsifying, and foaming activity-were improved by most of the proteolytic enzymes with the most impressive increase from 980% foam activity for LPI up to 3,614% foam activity for pepsin hydrolysate. The formation of bitterness, most likely linked to generation of bitter peptides, was pronounced in the Alcalase hydrolysate, while the other hydrolysates did not show an extensive increase in bitterness compared to the LPI. Other sensory attributes of the hydrolysates-with the exception of Alcalase treatment-were also very similar to the LPI. The results of this study show the potential of enzymatic degradation of LPI to modify the IgE-reacting polypeptides and to improve the technofunctionality of the isolates and therefore their use as food ingredients.

An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/ß-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

Evolution of Na(+) and H(+) bioenergetics in methanogenic archaea.

Methanogenic archaea live at the thermodynamic limit of life and use sophisticated mechanisms for ATP synthesis and energy coupling. The group of methanogens without cytochromes use an Na(+) current across the membrane for ATP synthesis, whereas the cytochrome-containing methanogens have additional coupling sites that also translocate protons. The ATP synthase in this group is promiscuous and uses Na(+) and H(+) simultaneously.

Electron transport during aceticlastic methanogenesis by Methanosarcina acetivorans involves a sodium-translocating Rnf complex.

The anaerobic methanogenic archaeon Methanosarcina acetivorans lives under extreme energy limitation. Methanogenesis from acetate as carried out by M. acetivorans involves an anaerobic electron transport chain with ferredoxin as electron donor and heterodisulfide as electron acceptor, and so far only the heterodisulfide reductase has been shown to translocate H(+) . Here, we describe a second Na(+) -translocating coupling site in this electron transport chain. Inside-out membrane vesicles of M. acetivorans catalyzed Na(+) transport coupled to an electron transport catalyzed by the ferredoxin:heterodisulfide oxidoreductase activity. Ionophore studies revealed that Na(+) transport was primary and electrogenic. A ∆rnf mutant was unable to grow on acetate and the ferredoxin:heterodisulfide oxidoreductase-coupled Na(+) transport was abolished. These data are consistent with the hypothesis that the Rnf complex of M. acetivorans is an Na(+) -translocating coupling site and the entry point of electrons derived from reduced ferredoxin into the electron transport chain leading to the heterodisulfide.

Promiscuous archaeal ATP synthase concurrently coupled to Na+ and H+ translocation.

ATP synthases are the primary source of ATP in all living cells. To catalyze ATP synthesis, these membrane-associated complexes use a rotary mechanism powered by the transmembrane diffusion of ions down a concentration gradient. ATP synthases are assumed to be driven either by H(+) or Na(+), reflecting distinct structural motifs in their membrane domains, and distinct metabolisms of the host organisms. Here, we study the methanogenic archaeon Methanosarcina acetivorans using assays of ATP hydrolysis and ion transport in inverted membrane vesicles, and experimentally demonstrate that the rotary mechanism of its ATP synthase is coupled to the concurrent translocation of both H(+) and Na(+) across the membrane under physiological conditions. Using free-energy molecular simulations, we explain this unprecedented observation in terms of the ion selectivity of the binding sites in the membrane rotor, which appears to have been tuned via amino acid substitutions so that ATP synthesis in M. acetivorans can be driven by the H(+) and Na(+) gradients resulting from methanogenesis. We propose that this promiscuity is a molecular mechanism of adaptation to life at the thermodynamic limit.

Sodium ion translocation and ATP synthesis in methanogens.

Methanogens are the only significant biological producers of methane. A limited number of C(1) substrates, such as methanol, methylamines, methyl sulfate, formate, H(2)+CO(2) or CO, and acetate, serve as carbon and energy source. During degradation of these compounds, a primary proton as well as a primary sodium ion gradient is established, which is a unique feature of methanogens. This raises the question about the coupling ion for ATP synthesis by the unique A(1)A(o) ATP synthase. Here, we describe how to analyze and determine the Na(+) dependence of two model methanogens, the hydrogenotrophic Methanothermobacter thermautotrophicus and the methylotrophic Methanosarcina barkeri. Furthermore, the determination of important bioenergetic parameters like the ΔpH, ΔΨ, or the intracellular volume in M. barkeri is described. For the analyses of the A(1)A(O) ATP synthase, methods for measurement of ATP synthesis as well as ATP hydrolysis in Methanosarcina mazei Gö1 are described.

[X-ray and CT morphology of atlas variants in the dog]. / Röntgen- und CT-Morphologie von Atlas- varianten beim Hund.

To investigate variations in atlas shape in dogs the image archives of the Department for Small Animals and Horses, University of Vienna, and The Royal Veterinary College, University of London were searched for radiographs and CT images of the canine atlas. 32 radiographic and 78 CT studies, including 51 toy and 54 large breed dogs, were retrieved and analysed. Five dogs had both radiographs and CT scans. Patients with pathologic alterations, such as fractures and incomplete ossification of the atlas, as well as oblique projections were excluded. Compared to large breed dogs (body weight more than 30 kg) the atlas of toy breed dogs (body weight less than 10 kg) had a reduced craniocaudal and an increased dorsoventral diameter. The vertebral longitudinal axis appeared to be dorsorotated, resulting in a steeper rise of the atlas wings and a more dorsally located lateral vertebral foramen. When comparing the atlas of toy and large breed dogs, the distribution of three morphologic parameters was statistically different: Ratio length to height (toy breeds > 1, large breeds < or = 1), shape of the vertebral chanal (toy breeds: vertically oval, large breeds: round resp. cross-oval, or vertically oval) and trabecular bone of the dorsal arch (toy breeds: absent, large breeds: present). There was no significant difference in the morphology of the ventral arch. Differences in atlas morphology could contribute to the predisposition of toy breed dogs to atlanto-axial instability; however, further studies are necessary to test this hypothesis.

Genetic and epidemiological risk factors in the development of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the chronic lung disease of preterm infants and still represents a major burden of prematurity. Several clinical risk factors for the onset of the disease are already known. In addition, some candidate genes have recently been identified. We set out to determine clinical as well as genetic risk factors for the development of BPD in the German population. 155 infants born with a gestational age < or = 28 at the tertiary neonatal Centre, Freiburg, were recruited. Clinical data were recorded from hospital charts. 47 children developed moderate or severe BPD. For genetic analyses, 37 polymorphisms within sixteen genes were genotyped on all children. The strongest epidemiological risk factor for BPD was birth weight, followed by low gestational age. Genetic association was detected with single polymorphisms within Tumour necrosis factor alpha, Toll like receptor 10 and vascular endothelial growth factor. The former two genes showed also association with BPD in haplotype analyses. In conclusion, association of BPD was far more convincingly found with a few clinical factors than with genetic polymorphisms. This underscores the genetic complexity of the disease. Furthermore, the identification of predisposing genetic polymorphisms might be hampered by the complex interaction between clinical and genetic factors.

The F1FO ATP synthase genes in Methanosarcina acetivorans are dispensable for growth and ATP synthesis.

There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A(1)A(O) together with the bacterial F(1)F(O) ATP synthase. To address a potential role of the bacterial F(1)F(O) ATP synthase, we have exchanged the F(1)F(O) ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F(1)F(O) ATP synthase is dispensable for the growth of M. acetivorans.