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The present invention relates to fungal enzymes, more in particular of polyphenoloxidases (PPOs), and is based on a newly discovered enzymatic activity of a class of PPOs, i.e. de-methylation of R-substituted mono- or di-methoxyphenolsuch as present in lignin, lignin derived compounds and/or in lignocellulosic biomass. This newly discovered enzymatic activity renders these enzymes highly suitable for a plethora of applications in industry. Provided herein are methods of demethylation, processes to increase the reactivity of lignin or lignin-comprising biomass, processes of conversion lignin or lignin comprising biomass to value added products, processes for degrading and/or modifying (hemi-)cellulose in a hemicellulose-comprising substrate, and expression vectors, host cells and liquids, pastes or solid formulations and compositions for use in the demethylation method of the invention.

Wheat straw based compost is the substrate for commercial growth of Agaricus bisporusmushrooms, but it is unknown to which extent the carbohydrate-lignin matrix changes and howmuch is metabolized during commercial cultivation. In this paper we report yields and remainingstructures of the major compost carbohydrates and lignin. Hereto, a mass balance was conducted ina tunnel-experiment at industrial scale, and total dry matter, cellulose, hemicellulosic xylan andlignin were quantified (Jurak et al., 2015a). Remaining (substituted) xylan structures were extractedand analysed in detail by enzymatic fingerprinting with use of HPAEC and MALDI-TOF MS.Remaining lignin structures were subjected to analytical pyrolysis GC/MS and typical lignin unitswere determined. In addition, to determine why certain fractions remain unused in the compost,activities of water extracted carbohydrate degrading enzymes were analysed for their ability todegrade a range of polysaccharides (Jurak et al., 2015b,c). During growth of A. bisporus,carbohydrates were only slightly consumed and xylan was partially degraded. At the same time,lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was modified, asobserved by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 duringgrowth, while fewer decorations on the phenolic skeleton of both S and G units remained (Jurak etal., 2015a). During mushroom formation, mainly endo-xylanase, endo-glucanase, β-xylosidase andβ-glucosidase activities were determined in the compost extracts. Arabinofuranosidase activity ableto remove arabinosyl residues from doubly substituted xylosyl residues nor α-glucuronidase activitywere detected (Jurak et al., 2015b). The latter correlated with the observed accumulation of xylanfragments substituted with arabinosyl and glucuronic acid substituents in the compost towards theend of the cultivation (Jurak et al., 2015c). Hence, it was concluded that compost grown A. bisporuslacks the ability to degrade and consume highly substituted xylan fragments.

The present invention is in the area of enzymes for (hemi-)cellulose degradation and/or modification, more in particular the degradation and/or modification of xylan. The invention is based on a newly discovered enzymatic activity of a class of lytic polysaccharide monooxygenases (LPMOs), i.e. oxidative cleavage of xylan in addition to oxidative cleavage of cellulose. The present invention therefore relates to a method for degrading and/or modifying xylan in a xylan-comprising substrate, a method for preparing a product from a xylan-comprising substrate, a kit of parts, a liquid, paste or solid formulation, and a xylan-comprising composition, comprising said LPMO. The invention further relates to a use of said LPMO, said kit of parts, said formulation and/or said composition, in a method of the invention.

The increase in hydrolytic activity towards corn silage water unextractable solids by supplementation of acetyl xylan esterase 1 (T. reesei, TrAxe1), belonging to carbohydrate esterase (CE) family 5, to an A. niger / T. emersonii enzyme preparation is presented. TrAxe1 was cloned and expressed in A niger. The hydrolytic activity of TrAxe1 was analysed with a p-nitrophenyl-acetate standard assay and by the measurement of the acetic acid released from neutral and acidic eucalyptus xylo-oligosaccharides. The activity obtained for TrAxe1 was compared to acetyl xylan esterases belonging to CE families 1, 5 and 16 from A. niger. High activities were obtained for the CE5 classified acetyl xylan esterases from T. reesei and A. niger. The data obtained confirm the suggested increase in hydrolytic activity by supplementation of CE5 classified acetyl xylan esterases to the (hemi)cellulolytic A. niger / T. emersonii preparation (Neumüller et al. 2014b).

RATIONALE Mass spectrometry has become a useful technique for elucidating the chemical structures of oligosaccharides. The combined use of chromatography and mass spectrometry for the separation and identification of oligosaccharides has shown much progress in recent years. However, no powerful method has yet been developed to quickly identify isomeric oligosaccharides in complex mixtures. METHODS A rapid travelling-wave ion mobility mass spectrometry (TWIMS-MS) method was developed for the identification of various isomeric oligogalacturonic acids in mixtures and determined their structures, using 3-aminoquinoline (3-AQ) as a labelling agent. RESULTS TWIMS successfully distinguished isomeric oligogalacturonic acids of various degrees of polymerisation (DPs) and levels of methyl-esterification. After derivatisation by 3-AQ, isomeric oligosaccharides of galacturonic acid, with the DP ranging from 2 to 9 and the number of methyl esters ranging from 1 to 5, were identified by 3-AQ-TWIMS-MS. The isomeric oligosaccharides with varying sites of methyl ester substitution were identified by the post-fragmentation mode of TWIMS using 3-AQ labelling to obtain simplified mass spectra. CONCLUSIONS Using the 3-AQ-TWIMS-MS method, the precise distribution of methyl esters within the pectin molecule and isomeric oligogalacturonic acids after enzyme degradation was determined. Simplified product ion mass spectra and precise analysis of the isomers were achieved by labelling 3-AQ at the reducing end of the oligosaccharides. Series of methyl-esterified galacturonic acid oligomers have predictable drift times, depending on the precise position of the methyl ester.

The design, use, and evaluation of a web-based experiment designer, ExperD, are described. ExperD supports students in designing a research strategy for their laboratory class. Next, ExperD supports students in their actual laboratory class work by showing them which experiments they have to carry out, and what the relation is between experiments. The use of ExperD was evaluated in the 2009 and 2011 editions of a Food Chemistry course at Wageningen University in The Netherlands. The evaluations showed that students (n = 60 and 98) find ExperD helpful and that teachers see the ExperD as a valuable addition to the laboratory class. Usage logs show that students used the tool throughout the entire laboratory class. Furthermore, the ExperD proved to be a promising research tool for monitoring both student design activities as well as student actual lab work activities.

Introduction - Sea buckthorn (Hippophaë rhamnoides L.) is known to be rich in many bioactive compounds (such as vitamins, phenolics, carotenoids) important for human health and nutrition. Among the phenolics, berries and leaves contain a wide range of flavonols that are good quality and authenticity biomarkers. Objective - To compare the composition of the main flavonols of Romanian sea buckthorn berry and leaf varieties and to identify the specific biomarkers that contribute to sample differentiation among varieties. Material and methods - Six varieties of cultivated sea buckthorn (ssp. Carpatica) berries and leaves were analysed by UHPLC/PDA–ESI/MS. Results - Berries and leaves contained mainly isorhamnetin (I) glycosides in different ratios. Whereas I-3-neohesperidoside, I-3-glucoside, I-3-rhamnosylglucoside, I-3-sophoroside-7-rhamnoside and free isorhamnetin were predominant for berries (out of 17 compounds identified), I-3-rhamnosylglucoside, I-3-neohesperidoside, I-3-glucoside, quercetin-3-pentoside, kaempferol-3-rutinoside, and quercetin-3-glucoside were predominant in leaves (out of 19 compounds identified). Berries contained, on average, 917¿mg/100¿g DW flavonol glycosides. Leaves had higher content of flavonol glycosides than berries, on average 1118¿mg/100¿g DW. The variation of the quantitative dataset analysed using principal component analysis accounted for 91% of the total variance in the case of berries and 73% in case of leaves, demonstrating a good discrimination among samples. Conclusion - Based on quantitative analysis, by principal component analysis, the flavonol derivatives can be considered as biomarkers to discriminate among varieties and to recognise specifically the berry versus leaf composition

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono- and diesters. For rapid fingerprinting of these esters, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono- and diester palmitate standards in MALDI-TOF/TOF-MS showed that sodium adduct parent masses [M + Na]+ gave much simpler MS2 spectra than radical / protonated [M]+¿ / [M+H]+ parents. [M + Na]+ fragments yielded diagnostic polyene-specific eliminations and fatty acid neutral losses, whereas [M]+¿ / [M+H]+ fragmentation resulted in a multitude of non-diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]+ ionization by addition of sodium acetate, and best signal-to-noise ratios were obtained in the 0.1 to 1.0mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI-TOF/TOF-MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono- and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all-trans esterified esters found in LC were identified with MALDI-TOF/TOF-MS, with the exception of two minor monoesters.

Soybean seedlings and therefrom extractable compositions are described. Such compositions comprise prenylated isoflavones and at least one isoflavonoid, said isoflavonoid being selected from one of the chemical classes of isoflavones, coumestans and pterocarpans. Such compositions usually comprise at least 5 % prenylated isoflavones, in particular prenylated isoflavones selected from prenylated daidzein, prenylated hydroxydaidzein, prenylated glycitein, prenylated hydroxygenistein, and prenylated genistein. Such compositions also comprise pterocarpans, preferably in amounts of at least 20 %, and with a novel ratio of glyceollin I to glyceollin II to glyceollin III to glyceollin IV. Also described is a production method comprising the step of fermenting the soybeans under stress, in particular in the presence of cultures of fungi, preferably in the presence of Rhizopus microsporus Var. oryzae.

Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with a liquid chromatography/mass spectrometry (LC/MS)-based screening method. The glyceollins I–III and glyceollidins I–II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the 1,3AR retro-Diels-Alder (RDA) fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the five pterocarpans in negative-ion (NI) mode were proposed, which involved the cleavage of the C-ring and/or D-ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H2Ox,yRDA fragments, whereas its presence gave predominantly the common RDA fragments.

Intervention studies with procyanidin (PC)-rich extracts and products such as cocoa and wine suggest protective effects of PC against cardiovascular diseases. However, there is no consensus on the absorption and metabolism of PC dimers. Interestingly, nothing is known about the absorption of A-type PC. In this study, the absorption and metabolism of purified PC dimers A1 [epicatechin-(2-O-7, 4–8)-catechin], A2 [epicatechin-(2-O-7, 4–8)-epicatechin], and B2 [epicatechin-(4–8)-epicatechin], A-type trimers, a mixture of A1, B2, and a tetrameric A-type, and monomeric epicatechin were compared by in situ perfusion of the small intestine of rats for 0–30 min. The rats had their bile duct, portal vein, and small intestine cannulated. Unmodified and methylated metabolites were distinguished from their conjugates by differential ß-glucuronidase treatment. A1 and A2 dimers were absorbed from the small intestine of rats and they were better absorbed than dimer B2. Absorption of the A-type dimers was only 5–10% of that of monomeric epicatechin. Dimers were not conjugated or methylated in contrast to epicatechin, which was partly methylated and 100% conjugated. A-type trimers were not absorbed. Furthermore, the presence of tetrameric PC enhanced the absorption of B2 but not that of A1. Epicatechin, methylated epicatechin, and their conjugates were not found as metabolites of the PC tested. In conclusion, dimers A1, A2, and B2 are slightly absorbed but are not conjugated or methylated, thus conserving their biological activity after absorption. Because PC contents of foods are relatively high, dimers may contribute to systemic effects of PC.

This study was aimed at the determination of the pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated Ara h 1, a major allergen from peanuts. Both the oligomeric structure and the trimeric structure of the allergen were investigated. Under the in vitro conditions applied, oligomeric Ara h 1, either unheated or preheated, was hydrolyzed by pepsin at a lower rate than trimeric Ara h 1. Peptides with relatively high molecular masses were shown to be able to bind IgE, whereas peptides with lower molecular masses (

BACKGROUND: Physicochemical properties and digestibility of proteins can be modified by covalent interactions with oxidized phenolic compounds, i.e., quinones. In order to control these interactions in food products, the covalent interactions between quinones from caffeoylquinic acid (CQA) and amino acid side chains were studied with mass spectrometry using N-terminally protected amino acids. RESULTS: The addition of two molecules of CQA, presumably in the form of a pre-formed dimer, was observed for lysine, tyrosine, histidine and tryptophan. A monomer of CQA seemed to be able to react with histidine and tryptophan, whereas no interaction with a CQA monomer was observed for lysine and tyrosine. Serine and threonine showed no covalent interactions with CQA. Cross-linking between CQA and the side chains of two molecules of lysine is likely to occur also in proteins. The results show that protein cross-linking may also be expected to occur via two tyrosine residues in the absence of other phenolic substrates. The side chains of lysine and tyrosine are more reactive than that of histidine and tryptophan. CONCLUSIONS: These results show that covalent protein modification by oxidized phenolics occurs preferentially via an initial dimerization and encompasses not only lysine and cysteine residues.

This paper describes the first stage in the design, development and evaluation of an electronic course book for Food Chemistry. Previous work on the development of digital learning materials for Food Chemistry resulted in several distinct modules for the introductory course `Food Chemistry¿. Evaluations had shown that the students appreciated these modules. However the course as a whole was not appreciated and the effectiveness of the course as a whole was not satisfactory. Students did not recognize the connection between the different digital modules nor did they recognize the connection between the digital and non-digital learning materials. In order to provide the students with a more integrated and more motivating learning experience an electronic course book system is being developed. The paper describes the functional requirements for the electronic course book system and initial experience with the requirements that have been realized.

Colostrum oligosaccharides are known to exhibit prebiotic and immunomodulatory properties. Oligosaccharide composition is species-specific, and equine colostrum has been reported to contain unique oligosaccharides. Therefore, equine oligosaccharides (EMOS) from colostrum from different horse breeds were analyzed by CE-LIF, CE-MSn, HILIC-MSn, and exoglycosidase degradation. Sixteen EMOS were characterized and quantified, of which half were neutral and half were acidic. EMOS showed about 63% structural overlap with human milk oligosaccharides, known for their bioactivity. Seven EMOS were not reported before in equine oligosaccharides literature: neutral Gal(beta 1-4)HexNAc, Gal(beta 1-4)Hex-Hex, beta 4'-galactosyllactose, and lactose-N- hexaose, as well as acidic 6'-Sialyl-Hex-Ac-HexNAc, sialyllacto-N-tetraose-a, and disialylacto-N-tetraose (isomer not further specified). In all colostrum samples, the average oligosaccharide concentration ranged from 2.12 to 4.63 g/L; with beta 6'and 3'- galactosyllactose, 3'-sialyllactose, and disialyllactose as the most abundant of all oligosaccharides (27-59, 16-37, 1-8, and 1-6%, respectively). Differences in presence and in abundance of specific EMOS were evident not only between the four breeds but also within the breed.