Dietary fibers are at the forefront of nutritional research because they positively contribute to human health. Much of our processed foods contain, however, only small quantities of dietary fiber, because their addition often negatively affects the taste, texture, and mouth feel. There is thus an urge for novel types of dietary fibers that do not cause unwanted sensory effects when applied as ingredient, while still positively contributing to the health of consumers. Here, we report the generation and characterization of a novel type of soluble dietary fiber with prebiotic properties, derived from starch via enzymatic modification,yielding isomalto/malto-polysaccharides (IMMPs), which consist of linear (α1 → 6)-glucan chains attached to the nonreducing ends of starch fragments. The applied Lactobacillus reuteri 121 GTFB 4,6-α-lucanotransferase enzyme synthesizes these molecules by transferring the nonreducing glucose moiety of an (α1 → 4)-glucan chain to the nonreducing end of another (α1 → 4)-α-glucan chain, forming an (α1 → 6)-glycosidic linkage. Once elongated in this way, the molecule becomes a better acceptor substrate and is then further elongated with (α1 → 6)-linked glucose residues in a linear way. Comparison of 30 starches, maltodextrins, and α-glucans of various botanical sources, demonstrated that substrates with long and linear (α1 → 4)- glucan chains deliver products with the highest percentage of (α1 → 6) linkages, up to 92%. In vitro experiments, serving as model of the digestive power of the gastrointestinal tract, revealed that the IMMPs, or more precisely the IMMP fraction rich in (α1 → 6) linkages, will largely pass the small intestine undigested and therefore end up in the large intestine. IMMPs are a novel type of dietary fiber that may have health promoting activity.
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Lignocellulose biorefining is a promising technologyfor the sustainable production of chemicals and biopolymers.Usually, when one component is focused on, the chemical natureand yield of the others are compromised. Thus, one of thebottlenecks in biomass biorefining is harnessing the maximumvalue from all of the lignocellulosic components. Here, we describea mild stepwise process in a flow-through setup leading to separateflow-out streams containing cinnamic acid derivatives, glucose,xylose, and lignin as the main components from differentherbaceous sources. The proposed process shows that minimaldegradation of the individual components and conservation oftheir natural structure are possible. Under optimized conditions,the following fractions are produced from wheat straw based ontheir respective contents in the feed by the ALkaline ACid ENzyme process: (i) 78% ferulic acid from a mild ALkali step, (ii) 51%monomeric xylose free of fermentation inhibitors by mild ACidic treatment, (iii) 82% glucose from ENzymatic degradation ofcellulose, and (iv) 55% native-like lignin. The benefits of using the flow-through setup are demonstrated. The retention of the ligninaryl ether structure was confirmed by HSQC NMR, and this allowed monomers to form from hydrogenolysis. More importantly, thecrude xylose-rich fraction was shown to be suitable for producing polyhydroxybutyrate bioplastics. The direct use of the xylose-richfraction by means of the thermophilic bacteria Schlegelella thermodepolymerans matched 91% of the PHA produced with commercialpure xylose, achieving 138.6 mgPHA/gxylose. Overall, the ALACEN fractionation method allows for a holistic valorization of theprincipal components of herbaceous biomasses.
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Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived beta-lactam nuclei, a conversion that can be used for the industrial synthesis of beta-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues alphaR145 and alphaF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with beta-lactam nucleophile instead of water, mutants alphaR145G, alphaR145S and alphaR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic beta-lactam antibiotics.
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Agricultural by-products, that is primary residue, industrial by-products and animal manure, are an important source of nutrients and carbon for maintaining soil quality and crop production but can also be valorised through treatment pathways such as fermentation, incineration or a combination of these called bio-refinery. Here, we provide an overview of opportunity to reduce environmental impact of valorising agricultural by-products. We estimate the available by-products in Northwestern Europe as a case study and the maximum and realistic greenhouse gas reduction potentials. Availability, collectability, the original use and environmental impact including land use changes, soil carbon sequestration and pollution swapping are discussed as critical factors when valorising agricultural by-products.
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Using either freshly pulped or preserved seaweed biomass for the extraction of protein can have a great effect on the amount of protein that can be extracted. In this study, the effect of four preservation techniques (frozen, freeze-dried, and air-dried at 40 and 70 °C) on the protein extractability, measured as Kjeldahl nitrogen, of four seaweed species, Chondrus crispus (Rhodophyceae), Ascophyllum nodosum, Saccharina latissima (both Phaeophyceae) and Ulva lactuca (Chlorophyceae), was tested and compared with extracting freshly pulped biomass. The effect of preservation is species dependent: in all four seaweed species, a differenttreatment resulted in the highest protein extractability. The pellet (i.e., the non-dissolved biomass after extraction) was also analyzed as in most cases the largest part of the initial protein ended up in the pellet and not in the supernatant. Of the four species tested, freeze-dried A. nodosum yielded the highest overall protein extractability of 59.6% with a significantly increased protein content compared with the sample before extraction. For C. crispus extracting biomass air-dried at 40 °C gave the best results with a protein extractability of 50.4%. Preservation had little effect on the protein extraction for S. latissima; only air-drying at 70 °C decreased the yield significantly. Over 70% of the initial protein ended up in the pellet for all U. lactuca extractions while increasing the protein content significantly. Extracting freshly pulped U. lactuca resulted in a 78% increase in protein content in the pellet while still containing 84.5% of the total initial total protein. These results show the importance of the right choice when selecting a preservation method and seaweed species for protein extraction. Besides the extracted protein fraction, the remainingpellet also has the potential as a source with an increased protein content.
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The results of this study indicate that whole body metabolic and cardiovascular responses to 140 min of either steady state or variable intensity exercise at the same average intensity are similar, despite differences in skeletal muscle carbohydrate metabolism and recruitment
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Saliva diagnostics have become increasingly popular due to their non-invasive nature and patient-friendly collection process. Various collection methods are available, yet these are not always well standardized for either quantitative or qualitative analysis. In line, the objective of this study was to evaluate if measured levels of various biomarkers in the saliva of healthy individuals were affected by three distinct saliva collection methods: 1) unstimulated saliva, 2) chew stimulated saliva, and 3) oral rinse. Saliva samples from 30 healthy individuals were obtained by the three collection methods. Then, the levels of various salivary biomarkers such as proteins and ions were determined. It was found that levels of various biomarkers obtained from unstimulated saliva were comparable to those in chew stimulated saliva. The levels of potassium, sodium, and amylase activity differed significantly among the three collection methods. Levels of all biomarkers measured using the oral rinse method significantly differed from those obtained from unstimulated and chew-stimulated saliva. In conclusion, both unstimulated and chew-stimulated saliva provided comparable levels for a diverse group of biomarkers. However, the results obtained from the oral rinse method significantly differed from those of unstimulated and chew-stimulated saliva, due to the diluted nature of the saliva extract.
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Exercise is one of the external factors associated with impairment of intestinal integrity, possibly leading to increased permeability and altered absorption. Here, we aimed to examine to what extent endurance exercise in the glycogen‐depleted state can affect intestinal permeability toward small molecules and protein‐derived peptides in relation to markers of intestinal function. Eleven well‐trained male volunteers (27 ± 4 years) ingested 40 g of casein protein and a lactulose/rhamnose (L/R) solution after an overnight fast in resting conditions (control) and after completing a dual – glycogen depletion and endurance – exercise protocol (first protocol execution). The entire procedure was repeated 1 week later (second protocol execution). Intestinal permeability was measured as L/R ratio in 5 h urine and 1 h plasma. Five‐hour urine excretion of betacasomorphin‐7 (BCM7), postprandial plasma amino acid levels, plasma fatty acid binding protein 2 (FABP‐2), serum pre‐haptoglobin 2 (preHP2), plasma glucagon‐like peptide 2 (GLP2), serum calprotectin, and dipeptidylpeptidase‐4 (DPP4) activity were studied as markers for excretion, intestinal functioning and recovery, inflammation, and BCM7 breakdown activity, respectively. BCM7 levels in urine were increased following the dual exercise protocol, in the first as well as the second protocol execution, whereas 1 h‐plasma L/R ratio was increased only following the first exercise protocol execution. FABP2, preHP2, and GLP2 were not changed after exercise, whereas calprotectin increased. Plasma citrulline levels following casein ingestion (iAUC) did not increase after exercise, as opposed to resting conditions. Endurance exercise in the glycogen depleted state resulted in a clear increase of BCM7 accumulation in urine, independent of DPP4 activity and intestinal permeability. Therefore, strenuous exercise could have an effect on the amount of food‐derived bioactive peptides crossing the epithelial barrier. The health consequence of increased passage needs more in depth studies.
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In the context of sustainability, the use of biocatalysis in organic synthesis is increasingly observed as an essential tool towards a modern and ‘green’ chemical industry. However, the lack of a diverse set of commercially available enzymes with a broad selectivity toward industrially-relevant substrates keeps hampering the widespread implementation of biocatalysis. Aminoverse B.V. aims to contribute to this challenge by developing enzymatic screening kits and identifying novel enzyme families with significant potential for biocatalysis. One of the most important, yet notoriously challenging reaction in organic synthesis is site-selective functionalization (e.g. hydroxylation) of inert C-H bonds. Interestingly, Fe(II)/α-ketoglutarate-dependent oxygenases (KGOs) have been found to perform C-H hydroxylation, as well as other oxyfunctionalization, spontaneously in nature. However, as KGOs are not commercially available, or even extensively studied in this context, their potential is not readily accessible to the chemical industry. This project aims to demonstrate the potential of KGOs in biocatalysis. In order to achieve this, the following challenges will be addressed: i) establishing an enzymatic screening methodology to study the activity and selectivity of recombinant KGOs towards industrially relevant substrates, ii) establishing analytical methods to characterize KGO-catalyzed substrate conversion and product formation. Eventually, the proof-of-principle demonstrated during this project will allow Aminoverse B.V. to develop a commercial biocatalysis kit comprised of KGO enzymes with a diverse activity profile, allowing their application in the sustainable production of either commodity, fine or speciality chemicals. The project consortium is composed of: i) Aminoverse B.V, a start-up company dedicated to facilitate chemical partners towards implementing biocatalysis in their chemical processes, and ii) Zuyd University, which will link Aminoverse B.V. with students and (bio)chemical professionals in creating a novel collaboration which will not only stimulate the development of (bio)chemical students, but also the translation of academic knowledge on KGOs towards a feasible biocatalytic application.
