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Secondary metabolites from plants include those that function as defence compounds, which often possess antimicrobial activity. These metabolites can be valuable for the food and pharmaceutical industries, as natural food preservatives and leads for new antimicrobial compounds, respectively. The main aim of this research was to explore the antimicrobial potential of defence metabolites from peanut and cereals.Prenylated stilbenoids can be produced by germination of peanuts with simultaneous fungal elicitation. We demonstrated that some of these compounds possess good antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The results also indicated that dimeric prenylated stilbenoids could be even more potent than their monomeric precursors.A systematic approach to the mass spectrometric characterisation of defence metabolites in wheat and oat led to a more complete overview of the chemical diversity in these species. This facilitates more accurate quantification of the total content of benzoxazinoids and avenanthramides in wheat and oat, respectively. Contrary to peanuts and other legumes, fungal elicitation did not enhance the quantity or diversity of metabolites formed in cereals during germination.A literature review revealed that monomeric natural benzoxazinoids lack antimicrobial potency. Synthetic derivatives with a 1,4-benzoxazin-3-one backbone were much more potent than natural analogues. QSAR studies on the antimicrobial activity of 1,4-benzoxazin-3-ones and in silico drug design showed the potential of this backbone for the development of novel antimicrobial drugs.

Contains fulltext : 193251.pdf (publisher's version ) (Open Access), Prenylated flavonoids and isoflavonoids are phytochemicals with remarkable antibacterial activity. In this study, 30 prenylated (iso)flavonoids were tested against Listeria monocytogenes and Escherichia coli (the latter in combination with an efflux pump inhibitor). Minimum inhibitory concentrations of the most active compounds ranged between 6.3-15.0 microg/mL. Quantitative structure-activity relationships (QSAR) analysis was performed and linear regression models were proposed with R(2) between 0.77-0.80, average R(2)m between 0.70-0.75, Q(2)LOO between 0.66-0.69, and relatively low amount of descriptors. Shape descriptors (related to flexibility and globularity), together with hydrophilic/hydrophobic volume and surface area descriptors, were identified as important molecular characteristics related to activity. A 3D pharmacophore model explaining the effect of the prenyl position on the activity of compounds was developed for each bacterium. These models predicted active compounds with an accuracy of 71-88%. With regard to the mode of action, good antibacterial prenylated (iso)flavonoids with low relative hydrophobic surface area caused remarkable membrane permeabilization, whereas those with higher relative hydrophobic surface area did not. Based on the QSAR and membrane permeabilization studies, the mode of action of antibacterial prenylated (iso)flavonoids was putatively rationalized.

Unicellular, photosynthetic microalgae and cyanobacteria are potential new protein sources for both food and feed. Variations between these sources are reflected in chemical variations (e.g. in the chemical composition) and in structural variation (e.g. in cell wall robustness). The aim of this thesis was to understand how chemical and structural variations between unicellular photosynthetic sources affect the application of unicellular protein as techno-functional ingredients and as a fish feed ingredient. To study the applicability of unicellular protein as techno-functional ingredient in food, proteins were extracted and isolated from the cyanobacterium Arthrospira (spirulina) maxima and the microalgae Nannochloropsis gaditana, Tetraselmis impellucida and Scenedesmus dimorphus. Chemical variations observed between photosynthetic unicellular sources were reflected in variations in protein extractability. The isolates (62–77% w/w protein) varied in protein solubility as a function of pH and ionic strength, especially at pH < 4.0. Isolates from N. gaditana, T. impellucida and A. maxima were able to form emulsions (d3,2 0.2–0.3 µm) at pH 8.0. The amount of each isolate needed to form emulsions varied between the isolates (9–74 mg protein / mL oil), but was within the range of proteins from both similar (photosynthetic) sources (algae and sugar beet leaves) and other protein sources (dairy, legume and egg). Minor differences were observed in the pH dependence of flocculation amongst the isolate stabilized emulsions. To study the applicability of microalgae and cyanobacteria as dietary protein sources for fish, Chlorella vulgaris, S. dimorphus, N gaditana, and A. maxima biomass was incorporated in fish feed (30% inclusion) and fed to Nile tilapia and African catfish. The cell walls of these unicellular sources used were quantified to vary in their robustness to mechanical degradation (structural variations). Although protein digestibility varied between the unicellular

The research described in this PhD-thesis aimed to identify new routes to release monosaccharides from hard-to-convert feedstocks by a detailed structural analysis of the recalcitrant carbohydrate and lignin released from grass feedstocks mildly treated with 1) intrinsic chemical catalysts (acetic acid and ammonium); 2) novel enzymes and; 3) combination of both.Loosening of the architecture of cell walls is known to enhance accessibility to enzymes. However, the residual lignin structures are not fully understood and will vary depending on the plant source and the targeted product (i.e. conversion to biochemicals, opening the structures for compost). Hence, this research focused on the variability and the lignin recalcitrant structures of 2 main grasses (corn stover, wheat straw) and how they can be affected by a biological process (rumen digestion or composting process), by an ammonia pretreatment, and by an acetic acid (enzymatically released or added to the treatment) or sulfuric acid pretreatment. The effects and the modifications of lignin were evaluated for to their influence on further enzymatic degradation of polysaccharides. Furthermore, the presence and the use of intrinsic catalysts within the biomass or within the process was investigated to increase the severity of pretreatment and its following degradability.

The experimental maximum degree of hydrolysis (DHmax,exp) was typically lower than the theoretical maximum degree of hydrolysis (DHmax,theo), calculated using the number of cleavage sites (as based on protease specificity) on the protein. The aim of this thesis is, therefore, to develop a method to better define expectations of the DHmax,exp. The developed method is based on the molecular properties of the protein and the protease, and the effects of modifications on protein primary structure and changes in system conditions. Protein primary structure was modified by glycation, the initial stage of the Maillard reaction upon heating proteins in the presence of carbohydrates. The effect of glycation on DHmax,exp for five different enzymes could be predicted based on the protease primary and secondary specificity, and the sensitivity of the enzyme to modifications of the binding site. Secondary specificity was determined by linking the molecular properties of the amino acids on the binding site positions of cleavage sites with the enzyme selectivity. The enzyme selectivity, i.e. the relative hydrolysis rate constants towards cleavage sites in a protein, of bovine, porcine and human trypsins was determined after full quantitative analysis of peptides formed during hydrolysis. The prediction of DHmax,exp for bovine trypsin based on the secondary specificity was 5 times better than the prediction based on the primary specificity (DHmax,theo). The large differences found in DHmax,exp by the three trypsins could be predicted based on enzyme secondary specificity. The substrate concentration, one of the system conditions varying in the in vitro digestion models, also showed large effect on DHmax,exp. This was found to be related to changes in the mechanism of hydrolysis, illustrated by the differences in the percentage of intact protein in the hydrolysates as a function of DH. Due to the changes in mechanism, different amounts of inhibitory peptides for the protease were pr

Contains fulltext : 182924.pdf (publisher's version ) (Open Access), Acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyruvate. The isoflavan equol, followed by the flavone 7,4'-dihydroxyflavone, were the best non-genuine acceptor substrates. B-ring C-prenylation was in general preferred over A-ring C-prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl group of the C-ring was able to make stabilizing interactions with the residue Arg160, which might determine the preference observed for B-ring prenylation. No reaction products were formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can be explained by the essential hydrogen bond needed between a phenolic hydroxyl group and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the C3' position (B-ring), were mainly O3'-prenylated (> 80% of the reaction products). This can be explained by the proximity of the C3' hydroxyl group to the donor substrate at the catalytic site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that the orientation of the B-ring and of the phenolic hydroxyl group at position C7 (A-ring) of flavones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family.

Prenylated phenolic compounds, i.e. those bearing a C5-isoprenoid (prenyl) substituent, are abundant in plants from the Fabaceae (legume) family and are potential natural antibacterial agents against resistant pathogenic bacteria. To understand the antibacterial properties of these compounds, (quantitative) structure-activity relationships and mode of action of these molecules were investigated against Gram positive and negative bacteria. Compounds belonging to the flavonoid, isoflavonoid and stilbenoid classes were studied. Antibacterial activity was modulated by the (sub)class of phenolic compound, as well as by the configuration, position and number of prenyl groups. Prenylated isoflavones were found to be better antibacterials than prenylated pterocarpans and prenylated stilbenoids. It was also shown that chain prenylation increased the antibacterial activity more than pyran-ring prenylation. Diprenylated compounds were among the most active antibacterials with minimum inhibitory concentrations of less than 10 µg/mL against Listeria monocytogenes. The main molecular characteristics defining antibacterial activity were molecular shape (including flexibility and globularity) and hydrophobicity. Regarding the mode of action of these compounds, it was shown that prenylated phenolic compounds can disrupt the integrity of the membrane by permeabilization very quickly. Interestingly, some good antibacterial prenylated (iso)flavonoids showed good permeabilization capacity whereas others not (including diprenylated molecules), highlighting potential differences in their interactions with the bacterial membrane. Likewise, it was shown that Gram negative intrinsic resistance towards prenylated phenolic compounds is primarily due to the activity of efflux pump systems and that it can be overcome by using an efflux pump inhibitor in combination with antibacterial prenylated compounds. Last, in vitro production of prenylated phenolic compounds was performed with microbial pre

Upon extraction of proteins from sugar beet leaves (Beta vulgaris L.) and oarweed (Laminaria digitata) for animal food and feed purposes, endogenous phenolics and proteins can interact with each other, which might affect the protein’s applicability. Sugar beet leaf proteins might become covalently modified by phenolics through polyphenol oxidase (PPO) activity. Oligomeric phenolics from seaweed (so-called phlorotannins (PhT)) might bind non-covalently to protein. The first aim of this thesis was to study factors involved in protein modification by phenolics. The second aim was to investigate the effect of PhT supplementation to feed on in vitro ruminal fermentation. Besides PPO activity and the amount of low molecular weight phenolic substrates present, brown colour formation in sugar beet leaves was dependent on the amount of phenolics, which do not serve as a substrate of PPO. These non-substrate phenolics can engage in browning reactions by oxidative coupling and subsequent coupled oxidation of the products formed. Similar reactions might also be involved in covalent protein modification by phenolics, and therewith protein properties. High molecular weight PhT from L. digitata could potentially modify protein properties by non‑covalent interactions. L. digitata contained PhT with subunits mainly connected via C‑O-C linkages, as determined using NMR spectroscopy. Further mass spectrometric analysis revealed the presence of a wide range of oligomers with degrees of polymerisation between 3 and 27. The interaction between PhT and proteins (b-casein and bovine serum albumin) was studied using model systems with different pH values, representing the various environments throughout the ruminants digestive tract. Phlorotannins bound to protein independent of pH, and broadened the pH range of protein precipitation from 0.5 to ~1.5 pH unit around the protein’s pI. At the pH of the abomasum of 2-3, the proteins re-solubilised again, presumably by increase in their net char