Hypocholesterolemic Effect of the Lignin-Rich Insoluble Residue of Brewer's Spent Grain in Mice Fed a High-Fat Diet.

Insoluble residue (INS) is a lignin-rich fraction of brewer's spent grain (BSG) that also contains ß-glucan and arabinoxylan, the major constituents of dietary fiber. We investigated the effects of INS in diet-induced obese mice in terms of lipid metabolism and metabolic diseases. Male mice (C57bl6) were fed a high-fat diet (HFD), a HFD + 20% INS, a HFD + 20% cellulose (CEL), a HFD with a combination of 20% INS-CEL (1:1), or a control diet for 14 weeks. Insulin and glucose tolerance tests were performed after 12 weeks. Fasting plasma lipids, bile acid, and fecal bile acid were measured after 14 weeks of feeding, and tissues were collected for gene expression analysis. Body weight gain was significantly reduced with all fibers, but only INS and INS-CEL decreased fasting plasma low-density lipoprotein cholesterol and total cholesterol compared to HFD. CEL and INS-CEL significantly improved insulin resistance. Fecal bile acids were significantly increased by all fibers, but there was no change in plasma bile acid. Clostridium leptum was increased with all fibers, but universal bacterial diversity was only with INS and INS-CEL. In addition, INS significantly increased the abundance of Bacteriodes, while CEL decreased Atopobium and Lactobacillus. INS feeding significantly upregulated various genes of cholesterol and bile acid metabolism, such as Srebp2, Hmgcr, Ldlr, Cyp7a1, Pparα, Fxr, and Pxr, in the liver. INS, INS-CEL, and CEL significantly attenuated liver steatosis. Our results suggest that INS from BSG induced beneficial systemic changes in mice via gut microbiota, bile acids, and gene expression in the liver.

Characterization of sulfhydryl oxidase from Aspergillus tubingensis.

BACKGROUND: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear. RESULTS: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant. CONCLUSIONS: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.

Interactions of Insoluble Residue from Enzymatic Hydrolysis of Brewer's Spent Grain with Intestinal Microbiota in Mice.

Brewer's spent grain (BSG) is the major side-stream from brewing. As BSG is rich in dietary fiber and protein, it could be used in more valuable applications, such as nutritional additives for foods. Our aim was to elucidate whether an insoluble lignin-rich fraction (INS) from BSG is metabolized by mice gut microbiota and how it affects the microbiota. Our results indicated that lignin was partially degraded by the gut microbiota, degradation products were absorbed, and finally excreted in urine. Therefore, they contribute to the phenolic pool circulating in the mammalian body, and may have systemic effects on health. In addition, the effects of the test diets on the microbiota were significant. Most interestingly, diversities of predominant cecal and fecal bacteria were higher after the intervention diet containing INS than after the intervention diet containing cellulose. Since low fecal bacterial diversity has been linked with numerous diseases and disorders, the diversity increasing ability opens very interesting perspectives for the future.

Structure of Brewer's Spent Grain Lignin and Its Interactions with Gut Microbiota in Vitro.

Lignin is part of dietary fiber, but its conversion in the gastrointestinal tract is not well understood. The aim of this work was to obtain structural information on brewer's spent grain (BSG) lignin and to understand the behavior of the polymeric part of lignin exposed to fecal microbiota. The original BSG and different lignin fractions were characterized by pyrolysis-GC/MS with and without methylation. Methylation pyrolysis proved that the ratio between guaiacyl and syringyl units was similar in all lignin samples, but the ratio between p-coumaric and ferulic acids varied by the isolation method. Combined pyrolysis results indicated higher acylation of γ-OH groups in syringyl than in guaiacyl lignin units. The polymeric lignin structure in the alkali-soluble fraction after enzymatic hydrolysis was slightly altered in the in vitro colon fermentation, whereas lignin in the insoluble residue after enzymatic treatments remained intact.

Industrial potential of lipoxygenases.

Lipoxygenases (LOXs) are iron- or manganese-containing oxidative enzymes found in plants, animals, bacteria and fungi. LOXs catalyze the oxidation of polyunsaturated fatty acids to the corresponding highly reactive hydroperoxides. Production of hydroperoxides by LOX can be exploited in different applications such as in bleaching of colored components, modification of lipids originating from different raw materials, production of lipid derived chemicals and production of aroma compounds. Most application research has been carried out using soybean LOX, but currently the use of microbial LOXs has also been reported. Development of LOX composition with high activity by heterologous expression in suitable production hosts would enable full exploitation of the potential of LOX derived reactions in different applications. Here, we review the biological role of LOXs, their heterologous production, as well as potential use in different applications. LOXs may fulfill an important role in the design of processes that are far more environmental friendly than currently used chemical reactions. Difficulties in screening for the optimal enzymes and producing LOX enzymes in sufficient amounts prevent large-scale application so far. With this review, we summarize current knowledge of LOX enzymes and the way in which they can be produced and applied.

Sensitizing potential of enzymatically cross-linked peanut proteins in a mouse model of peanut allergy.

SCOPE: The cross-linking of proteins by enzymes to form high-molecular-weight protein, aggregates can be used to tailor the technological or physiological functionality of food products. Aggregation of dietary proteins by food processing may promote allergic sensitization, but the effects of enzymatic cross-linking of dietary proteins on the allergenic potential of food are not known. In this study, the bioavailability and the sensitizing or tolerizing potential of peanut proteins (PE) cross-linked with microbial tyrosinase from Trichoderma reesei and mushroom tyrosinase from Agaricus bisporus, were investigated. METHODS AND RESULTS: The impact of cross-linking of PE on the in vitro bioavailability of fluorescein isothiocyanate-labeled peanut proteins was tested in a Caco-2 cell monolayer and by competitive ELISA. The in vivo allergenicity or capacity to induce oral tolerance in mice were measured by serum levels of PE-specific antibodies and T cell cytokine production after exposure to PE and cross-linked PE. CONCLUSION: Enzymatic processing of peanut proteins by the two tyrosinases increased the bioavailability of major peanut allergen Ara h 2, but did not significantly change the allergenic or tolerizing properties of peanut. Enzymatic treatment of peanut proteins yielded cross-linked proteins with preserved molecular and immunological features of peanut allergens.

Release of small phenolic compounds from brewer's spent grain and its lignin fractions by human intestinal microbiota in vitro.

Brewer's spent grain (BSG), the major side-stream from brewing, is rich in protein, lignin, and nonstarch polysaccharides. Lignin is a polyphenolic macromolecule considered resilient toward breakdown and utilization by colon microbiota, although some indications of release of small phenolic components from lignin in animals have been shown. The aim of this study was to investigate if the human intestinal microbiota can release lignans and small phenolic compounds from whole BSG, a lignin-enriched insoluble fraction from BSG and a deferuloylated fraction, in a metabolic in vitro colon model. The formation of short-chain fatty acid (SCFA) was also investigated. More lignin-related monomers and dilignols were detected from the lignin-enriched fraction than from BSG or deferuloylated BSG. SCFA formation was not suppressed by any of the fractions. It was shown that small lignin-like compounds were released from these samples in the in vitro colon model, originating most likely from lignin.

Interactions of a lignin-rich fraction from brewer's spent grain with gut microbiota in vitro.

Lignin is a constituent of plant cell walls and thus is classified as part of dietary fiber. However, little is known about the role of lignin in gastrointestinal fermentation. In this work, a lignin-rich fraction was prepared from brewer's spent grain and subjected to an in vitro colon model to study its potential bioconversions and interactions with fecal microbiota. No suppression of microbial conversion by the fraction was observed in the colon model, as measured as short-chain fatty acid production. Furthermore, no inhibition on the growth was observed when the fraction was incubated with strains of lactobacilli and bifidobacteria. In fact, the lignin-rich fraction enabled bifidobacteria to survive longer than with glucose. Several transiently appearing phenolic compounds, very likely originating from lignin, were observed during the fermentation. This would indicate that the gut microbiota was able to partially degrade lignin and metabolize the released compounds.

Pre-hydrolysis with carbohydrases facilitates the release of protein from brewer's spent grain.

Brewer's spent grain (BSG) is the most abundant side-stream from brewing. It is food-grade being rich in dietary fibre and protein and thus having potential as their source for both food and non-food applications. Initial treatment of milled BSG with a carbohydrase cocktail from Humicola insolens significantly enhanced the subsequent solubilisation of protein from the residual biomass. When treated with an alkaline protease, 76% of BSG protein was solubilized, whereas the yields were significantly lower with neutral or acidic proteases. In alkaline conditions significant amount of protein (53%) as predominantly low molecular weight protein was solubilized even without any protease addition. The degree of protein solubilisation was influenced by the time of exposure of modified BSG to the alkaline environment. The non-enzymatic protein solubilisation was, however, only observed when BSG had been initially treated with the carbohydrase, suggesting the protein is surrounded by cell wall polysaccharides restricting its initial release.

Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88.

Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC-MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.

Discovery of novel secreted fungal sulfhydryl oxidases with a plate test screen.

Sulfhydryl oxidases (SOX) are FAD-dependent enzymes capable of oxidising free thiol groups and forming disulphide bonds. Although the quantity of scientific papers and suggested applications for SOX is constantly increasing, only a limited number of microbial SOX have been reported and are commercially available. Hence, the aim of this study was to develop a fast and reliable qualitative plate test for screening novel secreted fungal SOX. The screening was based on the Ellman's reagent, i.e. 5,5'-dithiobis[2-nitrobenzoic acid]. Altogether, 32 fungal strains from an in-house culture collection were screened. A total of 13 SOX-producing strains were found positive in the plate test screen. The novel SOX producers were Aspergillus tubingensis, Chaetomium globusum, Melanocarpus albomyces, Penicillium aurantiogriseum, Penicillium funiculosum, Coniophora puteana and Trametes hirsuta. Six of the discovered SOX were partially characterised by determination of isoelectric point, pH optimum and substrate specificity. A. tubingensis was identified as the most efficient novel SOX producer.

Impact of cell wall-degrading enzymes on water-holding capacity and solubility of dietary fibre in rye and wheat bran.

BACKGROUND: Rye and wheat bran were treated with several xylanases and endoglucanases, and the effects on physicochemical properties such as solubility, viscosity, water-holding capacity and particle size as well as the chemical composition of the soluble and insoluble fractions of the bran were studied. A large number of enzymes with well-defined activities were used. This enabled a comparison between enzymes of different origins and with different activities as well as a comparison between the effects of the enzymes on rye and wheat bran. RESULTS: The xylanases derived from Bacillus subtilis were the most effective in solubilising dietary fibre from wheat and rye bran. There was a tendency for a higher degree of degradation of the soluble or solubilised dietary fibre in rye bran than in wheat bran when treated with most of the enzymes. CONCLUSION: None of the enzymes increased the water-holding capacity of the bran or the viscosity of the aqueous phase. The content of insoluble material decreased as the dietary fibre was solubilised by the enzymes. The amount of material that may form a network to retain water in the system was thereby decreased.

Characterization of lipids and lignans in brewer's spent grain and its enzymatically extracted fraction.

Brewer's spent grain (BSG), the major side stream of brewing, consists of the husks and the residual parts of malts after the mashing process. BSG was enzymatically fractionated by a two-step treatment with carbohydrate- and protein-degrading enzymes, which solubilized 66% of BSG. BSG contained 11% lipids, which were mostly triglycerides, but also a notable amount of free fatty acids was present. Lipids were mostly solubilized due to the alkaline pH applied in the protease treatment. The main fatty acids were linoleic, palmitic, and oleic acids. Several lignans were identified in BSG, syringaresinol and secoisolariciresinol being the most abundant, many associated with the cell wall matrix and released by the alkaline-protease treatment.

Transglutaminase catalyzed cross-linking of sodium caseinate improves oxidative stability of flaxseed oil emulsion.

Sodium caseinate was modified by transglutaminase catalyzed cross-linking reaction prior to the emulsification process in order to study the effect of cross-linking on the oxidative stability of protein stabilized emulsions. The extent of the cross-linking catalyzed by different dosages of transglutaminase was investigated by following the ammonia production during the reaction and using SDS-PAGE gel. O/W emulsions prepared with the cross-linked and non-cross-linked sodium caseinates were stored for 30 days under the same conditions. Peroxide value measurement, oxygen consumption measurement, and headspace gas chromatography analysis were used to study the oxidative stability of the emulsions. The emulsion made of the cross-linked sodium caseinate showed an improved oxidative stability with reduced formation of fatty acid hydroperoxides and volatiles and a longer period of low rate oxygen consumption. The improving effect of transglutaminase catalyzed cross-linking could be most likely attributed to the enhanced physical stability of the interfacial protein layer against competitive adsorption by oil oxidation products.

Effect of a milling pre-treatment on the enzymatic hydrolysis of carbohydrates in brewer's spent grain.

Millions of tonnes of brewer's spent grain (BSG) are annually produced worldwide as a by-product of the brewing industry. BSG has the potential to be a valuable source of food, chemicals and energy if cost-efficient fractionation methods can be developed. A 2-fold improvement in carbohydrate solubilisation could be achieved through the introduction of a milling step prior to enzymatic hydrolysis. Course and fine milled fractions were characterized by particle size distribution and light microscopy. Fine milling decreased particle size down to the micron level and this in turn improved the carbohydrate solubility yield by a multi-enzyme mixture from 23% up to 45%. Carbohydrate solubilisation could be further increased through the supplementation of this enzyme preparation with additional cellulases. The physical degradation caused by the milling also liberated soluble carbohydrates without the requirement of any enzymatic treatment.

Methods for identifying lipoxygenase producing microorganisms on agar plates.

Plate assays for lipoxygenase producing microorganisms on agar plates have been developed. Both potassium iodide-starch and indamine dye formation methods were effective for detecting soybean lipoxygenase activity on agar plates. A positive result was also achieved using the ß-carotene bleaching method, but the sensitivity of this method was lower than the other two methods. The potassium iodide-starch and indamine dye formation methods were also applied for detecting lipoxygenase production by Trichoderma reesei and Pichia pastoris transformants expressing the lipoxygenase gene of the fungus Gaeumannomyces graminis. In both cases lipoxygenase production in the transformants could be identified. For detection of the G. graminis lipoxygenase produced by Aspergillus nidulans the potassium iodide-starch method was successful. When Escherichia coli was grown on agar and soybean lipoxygenase was applied on the culture lipoxygenase activity could clearly be detected by the indamine dye formation method. This suggests that the method has potential for screening of metagenomic libraries in E. coli for lipoxygenase activity.

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus.

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.

One-step method for isolation and purification of native ß-lactoglobulin from bovine whey.

BACKGROUND: The major whey protein ß-lactoglobulin (BLG) has been widely studied for its functional properties. The aim of this study was to develop an efficient, inexpensive and rapid one-step method for the isolation and purification of BLG while preserving its native structure. RESULTS: BLG was purified from defatted whey obtained from raw cow's milk by anion exchange chromatography. Protein purity and identity were determined using reverse phase high-performance liquid chromatography and mass spectrometry. Total BLG yield was 80% with protein purity from 97 to 99%. BLG isoforms A and B were separated into fractions of 91 and 99% purity respectively. The structure and native conformation of the isolated BLG were compared with those of standard commercial BLG by circular dichroism spectrometry, susceptibility to various crosslinking enzymes and enzyme-linked immunosorbent assay inhibition. CONCLUSION: The proposed method is very useful for the rapid preparation of BLG suitable for studying antigenic and molecular characteristics of this protein, as well as the effect of food processing on these properties. The procedure requires only 1 day for the purification of about 300 mg of BLG from a single run using a small column (2.5 cm × 20 cm) of diethylaminoethyl Sephadex and has potential for scaling up.

Charge modifications to improve the emulsifying properties of whey protein isolate.

Whey protein isolate was modified by ethylene diamine in order to shift its isoelectric point to an alkaline pH. The extent of the modification was studied using SDS-PAGE and MALDI-TOF mass spectrometry. The modified whey proteins were used as an emulsifier to stabilize oil-in-water emulsions at acidic and neutral pH ranges, and their emulsifying properties were compared with that of the unmodified whey proteins and with the previously studied ethylene diamine modified sodium caseinate. The emulsifying activity of the modified whey proteins was similar to that of the unmodified ones, but the stability of an emulsion at pH 5 was significantly improved after the modification. Charge and coverage of droplet surface and the displacement of the interfacial proteins by surfactant Tween 20 were further studied as a function of pH. As compared with the unmodified whey proteins, the modified ones were proven to cover the interface more efficiently with extensive surface charge at pH 5, although the interfacial layer was less resistant to the surfactant displacement.

Loosening of globular structure under alkaline pH affects accessibility of ß-lactoglobulin to tyrosinase-induced oxidation and subsequent cross-linking.

Globular proteins such as ß-lactoglobulin (BLG) are poorly accessible to enzymes. We have studied susceptibility of BLG to oxidation by Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) tyrosinases and subsequent intermolecular cross-linking with respect to pH-induced structural changes. We evaluated pH-induced structural changes in BLG using circular dichroism, tryptophan fluorescence and small angle X-ray scattering (SAXS) measurements, where after these results were correlated with the analysis of cross-linking by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Oxygen consumption measurement and changes in radii of gyration determined by SAXS during the enzyme-induced oxidation at the respective reaction conditions were also followed. Intermolecular cross-linking of BLG by TrTyr was found at pH 9 but not at pH 7.5. AbTyr was unable to catalyze cross-linking at pH 7.5 or pH 9. Increased accessibility and cross-linking by TrTyr was addressed to loosening of the three dimensional structure of the protein, increased flexibility of the backbone as well as partial hydrolysis. In addition to basic research of the effect of protein folding on enzymatic cross-linking the research results have significance on the exploitation of TrTyr at alkaline conditions.