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ispartof: Lwt-Food Science And Technology vol:165 status: published

Purification is a critical step to obtain hydrophobin HFBII for use in positive applications. In this study, hydrophobin HFBII was produced by Trichoderma reesei via submerged fermentation. Using the CO2-foam fractionation method yielded a fourfold increase in protein concentration. The foamate (αL-HFBII) was dried using a nano spray-dryer under optimal temperature. The gushing activity of the dried foamate (αS-HFBII) decreased. Addition of Tween 80 to the foamate before the drying process partially prevented the deactivation of hydrophobin HFBII. The purity of the powder was enhanced based on the theory of CO2-nanobubbles in a CO2-rich environment. The collected CO2-nanobubbles were added to an apolar–polar system and the interface of these two phases was collected. After evaporation of the apolar phase, the purity of the hydrophobins assembled on the surface of the liquid was significantly improved.

Gushing has been recognized as a disastrous phenomenon for contaminated carbonated beverages. Recent advances highlighted the ability of the class II hydrophobin films to interact via the hydrophob...

Fungal hydrophobin is a family of low molecular weight proteins consisting of four disulfide bridges and an extraordinary hydrophobic patch. The hydrophobic patch of hydrophobins and the molecules of gaseous CO2 may interact together and form the stable CO2-nanobubbles covered by an elastic membrane in carbonated beverages. The nanobubbles provide the required energy to provoke primary gushing. Due to the hydrophobicity of hydrophobin, this protein is used as a biosurfactant, foaming agent or encapsulating agent in food products and medicine formulations. Increasing demands for using of hydrophobins led to a challenge regarding production and purification of this product. However, the main issue to use hydrophobin in the industry is the regulatory affairs: yet there is no approved legislation for using hydrophobin in food and beverages. To comply with the legislation, establishing a consistent method for obtaining pure hydrophobins is necessary. Currently, few research teams in Europe are focusing on different aspects of hydrophobins. In this paper, an up-to-date collection of highlights from those special groups about the bio-chemical and physicochemical characteristics of hydrophobins have been studied. The recent advances of those groups concerning the production and purification, positive applications and negative function of hydrophobin are also summarised.

Hydrophobins have exceptional surface activity with potential applications for food and pharmaceutical industry. In this study, the effect of Class II hydrophobin HFBII on the retention of a volatile compound, ocimene in a water phase was investigated. We observed a negative gushing when 200 μ gp ure HFBII (κ-grade) and 40 μg ocimene (assay ≥ 90%) was added to 1 L sparkling water with CO2 concentration of 7 g/L. The droplet size analysis of κ-HFBII, ocimene and the mixture of them showed that the ocimene was emulsified in a solution containing κ-HFBII. The stability of this emulsion was reduced in function of time and this effect was intensified by increasing the temperature and the presence of CO2. Additionally, the retention of ocimene in a HFBII rich solution was measured by SPME-GC-MS. When 0.26±0.03 mg HFBII/mL is added to ocimene solution, 43% of the original ocimene concentration (i.e. 5.9 × 10 � 6 mg/mL) remained in the solution after 3 days, and 32% after 7 days. In contrast, original ocimene concentration in solutions without HFBII was only retained 20% and 14%, respectively. It showed the potential of HFBII to incor- porate insoluble flavour compounds in beverages and control their release. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher' sw eb site

Hydrophobins are surface active molecules that cause gushing of beer. Gushing is vigorous overfoaming of carbonated beverages without any shaking. A hop extract was used to decrease gushing of wort induced by hydrophobin HFBI. The influence of a magnetic field on dispersion of the hop extract was used to decrease gushing by HFBI. The results indicate that when a magnetic field exerted on hop extract, this compound is dispersed more and smaller particles are formed. Therefore, the specific surface areas of the particles are increased and interact with larger numbers of hydrophobins. This resulted in less gushing by HFBI. When hydrophobins and hop extract together were submitted to magnetic field more gushing was obtained than in the absence of magnet. This is due to the extensive dispersion of the combination by the magnet and can be limited by using less amount of hop extract.

Production and purification of hydrophobin HFBII has recently been the subject of intensive research, but the yield of production needs to be further improved for a generic use of this molecule at industrial scale. In a first step, the influence of different carbon sources on the growth of Trichoderma reesei and the production of HFBII was investigated. The optimum productivity was obtained by using 40 g/L lactose. Carbon starvation and excretion of extracellular enzyme were determined as two main conditions for the production of HFBII. In the second phase, and according to the physiological mechanisms observed during the screening phase, a bioreactor set up has been designed and two modes of cultures have been investigated, i.e. the classical submerged fermentation and a fungal biofilm reactor. In this last set-up, the broth is continuously recirculated on a metal packing exhibiting a high specific surface. In this case, the fungal biomass was mainly attached to the metal packing, leading to a simplification of downstream processing scheme. More importantly, the HFBII concentration increased up to 48.6 ± 6.2 mg/L which was 1.8 times higher in this reactor configuration and faster than the submerged culture. X-ray tomography analysis shows that the biofilm overgrowth occurs when successive cultures are performed on the same packing. However, this phenomenon has no significant influence on the yield of HFBII, suggesting that this process could be operated in continuous mode. Protein hydrolysis during stationary phase was observed by MALDI-TOF analysis according to the removal of the last amino acid from the structure of HFBII after 48 h from the beginning of fermentation in biofilm reactor. Hopefully this modification does not lead to alternation of the main physicochemical properties of HFBII.

Food industries are interested in finding highly stable foaming agents for product texture improvements. Hydrophobins have been mentioned many times in that context. For the production and purification of hydrophobin HFBII from Trichoderma reesei we found that foam fractionation of the fungal fermentation media is an interesting first step. After this step, the biophysical properties of the obtained foamate were studied. The foam generated by CO2 as the only sparging gas into a HFBII enriched solution in a closed environment was more stable than the foam generated by sparging with air. The equilibrium surface tension of a foamate containing 100 mg/L HFBII was measured by CAMTEL CDCA-100 tensiometry as equal to 40 mJ/m2, and was confirmed by the maximum bubble pressure tensiometer. Besides, it was found that for a long term foam preservation, the foam created by the HFBII-SDS combination is more stable than that of SDS.

The presence of Class II hydrophobins produced by fungi on barley results in primary gushing of beer. Gushing is the spontaneous overfoaming of carbonated beverages by opening of bottles. Solving gushing problems caused by brewing raw materials has received much scientific attention. Lipophilic extract of hops are introduced to brewers as foam suppressor in fermenters. We studied the effects of hop extract on gushing and found that lipophilic hop extract could reduce gushing. The effects are different when hop extract is added before mashing than when it is added after mashing. Hop extract contains fats and waxes and the effects on gushing are explained by a change in the physical state of its components during mashing which are due to temperature effects. Especially the effect of saturated fatty acids and waxes becomes apparent which are known as gushing inducers. This indicates that with respect to gushing potential of the hop extract's components, it is better to be added to cold wort (after mashing and filtration). Our study also showed an important effect of the filtration step on the amount of gushing.

Hydrophobins are exceptional proteins produced by fungi. Research over the last decade has led to a better understanding of their role in spontaneous self-assembly at hydrophobic/hydrophilic interfaces. This has resulted in many proposals for using hydrophobins in many important scientific and technological applications. Hydrophobins may become attractive as special biosurfactants, as foaming agents and for protein immobilization in the food industries and in biosensors. Moreover, they can be interesting as stabilizers for flavors, and as, encapsulating agents of trace ingredients in beverages. The use of hydrophobins in pharmaceutical formulations and in medicine is another interesting application as they cause an increased stabilization of drugs. The study of hydrophobins must also lead to a better understanding of the gushing phenomenon in beverages like beers, wines and ciders, which causes great economic losses in those fields. To recognize the positive and the negative aspects of hydrophobins these proteins should be commercially available at large scale which however is not the case. An overview of existing possibilities for applications may help to understand their behavior in different environmental conditions and to stimulate finding improved methods for isolation and purification, and possibly other unexpected applications. ispartof: Cerevisia: Belgian Journal of Brewing and Biotechnology vol:37 issue:1 pages:3-9 status: published

When weather conditions are damp, barley crops can be infected by fungi that produce amphiphilic proteins called hydrophobins. We postulated that these hydrophobins contaminate CO2 bubbles during b...

Since their discovery in 1997, work on proteinaceous wheat xylanase inhibitors (XIs) led to the identification and thorough biochemical and structural characterization of three classes of XIs, namely TAXI-type (Triticum aestivum xylanase inhibitor), XIP-type (xylanase inhibiting protein), and TLXI-type (thaumatin-like xylanase inhibitor) XIs. Already early on, a role in plant defense has been put forward for these proteins, mainly based on the observation that these XIs are only active against xylanases of microbial origin and can not inhibit the plant endogenous xylanases known so far. Considerable effort has been devoted to substantiate this plant defense hypothesis. Data at the genomic, transcriptomic and proteomic levels increasingly provide evidence that XIs, occurring as large polymorphic families, do indeed participate in plant defense. This review summarizes the current knowledge on XIs and the accumulated evidence on their role in plant defense. TLXI and XIP can be classified as pathogenesis-rela...

The effects of germination time and temperature on the quality of rye malt and worts derived thereof were investigated using Response Surface Methodology. Amylolytic and proteolytic enzyme activities were increased by long germination periods, while β-glucanase activity was not influenced. Total and Soluble Nitrogen content were also not significantly affected by the variations in germination conditions. Free Amino Nitrogen (FAN) was found in higher amounts in worts prepared from rye malts with long germination times. Extract contents were higher in rye malt than in the control barley malt and could be increased by a favourable germination regime, while no such impact on wort fermentability was found. High wort viscosities could be significantly reduced by a long germination period at low temperatures, but were still unacceptably high. The same conditions favoured the development of endoxylanase activity. Arabinoxylan (AX) accumulated during the germination process and their extractability increased. The results suggest that longer germination periods resulted in an increased number of AX molecules with lower molecular mass. Optimal rye malt qualities within the limits of this study were found for a germination time of 144 h at 10 °C, which resulted in an acceptable FAN content and the lowest measured viscosity.

Within the HEALTHGRAIN diversity screen, the variability of the contents of dietary fiber (DF) and components thereof was studied in wheat. Furthermore, the contribution of genotype and environment to this variability was estimated. The levels of total DF (TDF), total nonstarch polysaccharide (TOTNSP), water-extractable nonstarch polysaccharide (WENSP), total arabinoxylan (TOTAX), lignin, and beta-glucan in whole meal, flour, and/or bran varied approximately 1.8-fold. The highest variability was observed for the water-extractable arabinoxylan (WEAX) level in flour and bran (approximately 3.7-fold). Genotype and environment contributed to a similar extent to the variability in TDF, TOTNSP, and TOTAX content in wheat. The observed relatively high impact of genotype-environment interaction suggests that the levels of these constituents are weak breeding parameters. The WENSP level is a more stable parameter as the effect of the interaction term was much less than the impact of genotype. For TOTAX and WEAX in flour, WEAX in bran, beta-glucan in whole meal, and extract viscosity, wheat genotype determined approximately 50% or higher of the variation observed, whereas the impact of the genotype-environment interaction was relatively low. These findings suggest that the health-related and technological functionality of wheat can be directed to a certain extent by selection of appropriate wheat varieties.

Endo-1,4-β- d -xylanase (EC 3.2.1.8, β- d -xylanase) activity, and arabinoxylan (AX) level and extractability were monitored for the first time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to 24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3–4 DPI). This increase was not accompanied by the discernible presence of homologues of the barley X-I β- d -xylanase as established by immunodetection. This suggests that other, yet unidentified β- d -xylanases operate in this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of unprocessed 67 kDa X-I like β- d -xylanase, which was much more abundant in the presence of GA. The occurrence of higher levels of the unprocessed enzyme was related with higher β- d -xylanase activities and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa β- d -xylanase. During the late stage (up to 24 DPI) gradual processing of the 67 kDa β- d -xylanase occurred and was associated with a drastic increase in β- d -xylanase activity. Up to 120-fold higher activity was recorded at 24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during the late stage, suggesting that the β- d -xylanase is processed into more active forms to achieve extensive AX breakdown.

The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH's and temperatures were determined. All mutations increased the pH optimum by 0.5-1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a T m maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications.

Xylanases (EC 3.2.1.8) hydrolyse the backbone of cereal cell wall arabinoxylans and often have a significant impact on cereal-based processes and end-products. The use of microbial xylanases as processing aids in this respect is well established and has been extensively studied. Much less research has focused on inherently present cereal-associated xylanases and their possible impact. Cereals produce xylanases for re-modeling and expansion of cereal cell walls during normal cell growth and for more drastic cell wall degradation during seed germination. Besides these endogenous xylanases, cereals also contain microbial xylanases from micro-organisms populating the outer grain kernels layers. Unfortunately, these microbial xylanases are often inhibited by wheat proteinaceous xylanase inhibitors and they hence escape standard xylanase activity measurements. It is more correct to refer to these activity levels as ‘apparent’ xylanase activity levels. As a result, the occurrence of cereal-associated xylanases might have been largely underestimated in the past and hence unjustly been disregarded. The levels and the types of cereal-associated xylanases differ strongly between grain species, varieties, and tissues, and are largely affected by grain growing conditions. These variations in the levels of grain-associated xylanase activity affect several cereal-based food and feed applications. This paper provides an overview of the occurrence and variability of cereal-associated xylanases and of their potential impact on bread making, shelf life of refrigerated doughs, brewing, animal feed efficiency, pasta production, and wheat gluten–starch separation.

The prolamins in seven Algerian Sahara sorghum cultivars of varying seed shape and color were investigated. Protein contents ranged from 12 to 16%. Prolamins were the major protein fraction. They could be separated according to degree of disulfide cross-linking. Kafirin monomers and low molecular weight polymers could be extracted with 70% ethanol, whereas highly cross-linked kafirins additionally needed a reducing agent to become extractable. Kafirin monomers of α-, β- and γ-type were purified and N-terminally sequenced. For the first time, δ-kafirin was identified at the protein level. The study clearly revealed intercultivar differences between protein levels. The joint use of SDS-PAGE, SE-HPLC, and RP-HPLC allowed discriminating among cultivars based on the differences in prolamin levels and composition.

Wheat contains high levels of the three classes of xylanase inhibitors (XIs), Triticum aestivum xylanase inhibitor (TAXI), xylanase-inhibiting protein (XIP) and thaumatin-like xylanase inhibitor (TLXI). These proteins have been linked to plant defense. In this study, expression of XIs during wheat ear development and germination was examined using immunoblotting. The three types of XIs accumulated at high levels between the milky and the soft dough stages of ear development, and reached the highest levels at the hard kernel stage. From the hard kernel stage to harvest ripeness, a slight drop in inhibitor levels was observed, which was more marked for TAXI and TLXI than for XIP. During germination, the levels of the three types of XIs initially decreased, but XIs accumulated again after 1-2d, reaching maximum levels between 5 and 9d after imbibition. The levels of TAXI, XIP and TLXI in the seedlings then gradually and continuously declined as a function of time. 1D- and 2D-immunoblotting indicated that the three types of XIs occur in a wide variety of forms. This polymorphism is maintained throughout ear development and germination, although the proportions of the different (iso)forms vary with time. A differential temporal profile was observed for the unprocessed and processed forms of TAXI-type proteins. Finally, the occurrence of TAXI and XIP, but not TLXI, in roots and shoots of young seedlings was demonstrated. No XIs were detected in roots, leaves or stems at later stages of ear development. Overall, the three classes of XIs show remarkable similarities in their temporal distribution, indicating a related function within the wheat plant.

An XIP (xylanase inhibiting protein) type xylanase inhibitor was purified from Algerian pearl millet ( Pennisetum glaucum L.) grains and characterized for the first time. Cation exchange and affinity chromatography with immobilized Trichoderma longibrachiatum glycoside hydrolase (GH) family 11 xylanase resulted in electrophoretically pure protein with a molecular mass of 27-29 kDa and a pI value of 6.7. The experimentally determined N-terminal amino acid sequence of the purified XIP protein is 87.5%, identical to that of sorghum ( Sorghum bicolor L.) XIP and 79.2% identical to that of wheat ( Triticum aestivum L.) XIP-I. The biochemical properties of pearl millet XIP are comparable to those described earlier for sorghum XIP, except for the higher specific activity toward a T. longibrachiatum GH family 11 xylanase. On the basis of immunoblot neither TAXI nor TLXI type xylanase inhibitors were detected in pearl millet grains.

Within the HEALTHGRAIN diversity screening program, the variation in the content of dietary fiber and components thereof in different types of wheat was studied. The wheat types were winter (131 varieties) and spring (20 varieties) wheats (both Triticum aestivum L., also referred to as common wheats), durum wheat (Triticum durum Desf., 10 varieties), spelt wheat (Triticum spelta L., 5 varieties), einkorn wheat (T. monococcum L., 5 varieties), and emmer wheat (Triticum dicoccum Schubler, 5 varieties). Common wheats contained, on average, the highest level of dietary fiber [11.5-18.3% of dry matter (dm)], whereas einkorn and emmer wheats contained the lowest level (7.2-12.8% of dm). Intermediate levels were measured in durum and spelt wheats (10.7-15.5% of dm). Also, on the basis of the arabinoxylan levels in bran, the different wheat types could be divided this way, with ranges of 12.7-22.1% of dm for common wheats, 6.1-14.4% of dm for einkorn and emmer wheats, and 10.9-13.9% of dm for durum and spelt wheats. On average, bran arabinoxylan made up ca. 29% of the total dietary fiber content of wheat. In contrast to what was the case for bran, the arabinoxylan levels in flour were comparable between the different types of wheat. For wheat, in general, they varied between 1.35 and 2.75% of dm. Einkorn, emmer, and durum wheats contained about half the level of mixed-linkage beta-glucan (0.25-0.45% of dm) present in winter, spring, and spelt wheats (0.50-0.95% of dm). All wheat types had Klason lignin, the levels of which varied from 1.40 to 3.25% of dm. The arabinoxylan contents in bran and the dietary fiber contents in wholemeal were inversely and positively related with bran yield, respectively. Aqueous wholemeal extract viscosity, a measure for the level of soluble dietary fiber, was determined to large extent by the level of water-extractable arabinoxylan. In conclusion, the present study revealed substantial variation in the contents of dietary fiber and constituents thereof between different wheat types and varieties.

One hundred and fifty bread wheat lines and 50 other lines of small-grain cereals (spelt, durum wheat, Triticum monococcum, Triticum dicoccum, oats, rye, and barley) were selected for diversity in their geographical origin, age, and characteristics. They were grown on a single site in Hungary in 2004−2005, harvested, milled, and analyzed for a range of phytochemicals (tocols, sterols, phenolic acids, folates, alkylresorcinols) and fiber components that are considered to have health benefits. Detailed analyses of these components in the different species are reported in a series of accompanying papers. The present paper discusses the comparative levels of the bioactive components in the different species, showing differences in both ranges and mean amounts. Furthermore, detailed comparisons of the bread wheat lines show that it is possible to identify lines in which high levels of phytochemicals and dietary fiber components are combined with good yield and processing quality. This means that commercially competitive lines with high levels of bioactive components are a realistic goal for plant breeders.

A xylanase inhibitor of the xylanase-inhibiting protein (XIP) type was detected in sorghum (Sorghum bicolor L. Moench) whole meal using Western blot and immunoprobing with polyclonal anti-XIP antibodies. No detectable levels of Triticum aestivum xylanase inhibitor (TAXI) and thaumatin-like xylanase inhibitor (TLXI) type xylanase inhibitors were present. Trichoderma longibrachiatum xylanase affinity chromatography (AC) was used for the purification of sorghum XIP. Biochemical characteristics and protein sequence data show that sorghum XIP strongly corresponds to wheat (T. aestivum L.) XIP-I. Like wheat XIP-I, it inhibits both glycoside hydrolase family (GH) 10 and 11 xylanases, indicating that the XIP-I active site residues are well conserved in sorghum XIP. However, in contrast to wheat XIP-I, the inhibitor is unable to affect Aspergillus niger GH 11 xylanase activity. Its specific inhibition activities towards the other xylanases tested are comparable to those of wheat XIP-I. Based on the available sorghum expressed sequence tag (EST) database, XIP is expressed in sorghum in different tissues and developmental stages. Also expression in the presence of several plant hormones and under biotic as well as abiotic stress conditions is suggested.

Fungicide treatment had a significant impact on endoxylanase activity and XIP levels, but did not affect arabinoxylan (AX) and TAXI levels. The different response of TAXI and XIP type inhibitors to fungicide treatment is interesting. N-fertilisation did not affect AX levels, but significantly increased TAXI and XIP type inhibitor levels. Wheat-associated microbial endoxylanase activity levels were also affected by nitrogen supply, but levels of the endogenous enzyme did not change, except when sprouting occurred. The weather conditions before harvest had no influence on total AX (TOT-AX) and inhibitor levels, but had a large impact on both microbial and endogenous endoxylanase activity and water extractable AX (WE-AX) levels. Under most conditions, endoxylanase activity levels were related to those of α-amylases, liquefaction numbers (LN) and specific weights. WE-AX levels were often weakly but significantly correlated with endoxylanase activity levels, indicating that it is possible that part of the WE-AX in wheat originates from AX degradation by endoxylanases in the field. These results clearly indicate that agronomic circumstances significantly affect the levels of AX, endoxylanases and their inhibitors in wheat, and consequently could affect wheat quality.

The effects of genotype, harvest year and their interaction on the levels of arabinoxylans (AX), endoxylanases and endoxylanase inhibitors in wheat were studied using 14 varieties grown in three successive growing periods with diverse climatological conditions. Relations with more commonly evaluated wheat characteristics such as yield, thousand kernel weight, specific weight, protein level, Hagberg falling number (HFN) and α -amylase activity level were examined. Water extractable arabinoxylan (WE-AX) levels in wheat varied much more than total arabinoxylan (TOT-AX) levels. This variability was mainly genetically determined, but harvest year also had an important effect. Total endoxylanase activity levels varied more than a factor of 20 between the different wheat samples. Endogenous endoxylanases typically accounted for only 10–15% of this activity, while wheat-associated microbial endoxylanases accounted for the remaining 85–90%. However, when preharvest sprouting occurred, the contribution of endogenous endoxylanases could sometimes amount to over 40% of this total activity. Endogenous endoxylanase activity levels were mainly determined by the interaction of genotype and harvest year, while wheat-associated microbial endoxylanase activity levels were predominantly determined by genotype alone. Endogenous and microbial endoxylanase activity levels were strongly correlated, suggesting that wheat varieties which are susceptible to preharvest sprouting are often also susceptible to microbial contamination. The TAXI and XIP-type endoxylanase inhibitor levels varied by a factor of 8 and 1.8, respectively. They were mainly determined by genotype and were rather similar in the different growing periods.

Wheat kernel associated endoxylanases consist of a majority of microbial endoxylanases and a minority of endogenous endoxylanases. At least part of these enzymes can be expected to end up in wheat flour upon milling. In this study, the contribution of both types of these endoxylanases to changes in the arabinoxylan (AX) population during wheat flour breadmaking was assessed. To this end, wheat flour produced from two wheat varieties with different endoxylanase activity levels, both before and after sodium hypochlorite surface treatment of the wheat kernels, was used in a straight dough breadmaking procedure. Monitoring of the AX population during the breadmaking process showed that changes in AX are to a large extent caused by endogenous endoxylanases, whereas the contribution of microbial endoxylanases to these changes was generally very low. The latter points to a limited contamination of wheat flour with microbial enzymes during milling or to an extensive inactivation of these wheat flour associated microbial endoxylanases by endoxylanase inhibitors, present in wheat flour. When all wheat kernel associated microbial endoxylanases were first washed from the kernels and then added to the bread recipe, they drastically affected the AX population, suggesting that they can have a large impact on whole meal breadmaking.

Endo-beta-1,4-xylanase X-I is a major hydrolase produced by the aleurone tissue of germinating barley grain. It was previously reported that this cytosolic enzyme is synthesized as an inactive precursor which is proteolytically processed to active forms upon its programmed cell death dependent release. We here demonstrate, however, that the precursor form of X-I is an active enzyme. Purified recombinant precursor X-I was characterised with respect to its molecular weight, iso-electric point and temperature and pH activity and stability. Analysis of the hydrolysis products showed that it is an endo-acting enzyme which has the striking ability to release xylose from both polymeric xylan as well as from small xylo-oligosaccharides. The implications of these findings in relation to the putative role of the N-terminal propeptide as a carbohydrate binding module and the possible consequences for the way X-I fulfils its role in the germination process, are discussed.

Polyclonal antibodies (PAbs) were raised against wheat (Triticum aestivum L.) TAXI- and XIP-type xylanase inhibitors by rabbit immunization. A small contaminant in both antigens, not detected by SDS-PAGE and later identified through Western blot as a recently discovered third type of xylanase inhibitor from wheat, i.e. thaumatin-like xylanase inhibitor (TL-XI), led to the coproduction of PAbs against this protein in the rabbits. To obtain inhibitor-specific PAbs, the PAbs against TAXI, XIP and TL-XI were separated by affinity chromatography using immobilised recombinant and native xylanase inhibitors. The purified PAbs allowed the immunoquantification of each type of wheat xylanase inhibitor using Western blot and densitometric analysis against purified inhibitor standards. The method allowed the detection of the purified inhibitors at the 20 ng level. As the PAbs against the wheat xylanase inhibitors cross-reacted with their homologous targets from other cereals, immunoprobing allowed identification of XIP homologues in oats (Avena sativa L.) and TL-XI homologues in durum wheat (Triticum durum Desf.) and rye (Secale cereale L.).

The endoxylanases associated with wheat kernels consist of wheat endogenous endoxylanases on one hand and kernel-associated microbial endoxylanases on the other hand. Assessment of their presence, based on analysis of their enzymic activity, can be expected to be hampered by the presence in wheat of high levels of endogenous endoxylanase inhibitors, which are able to inhibit the wheat-kernel-associated microbial endoxylanases. On the basis of preliminary experiments aimed at clarifying the distribution of the wheat-associated endoxylanases, a method to estimate total endoxylanase activities in wheat kernels was developed. Extensive washing of wheat kernels with universal buffer of pH 8.0 provided near-quantitative separation of the microbial endoxylanases located on the surface of wheat kernels from the endogenous endoxylanases and endoxylanase inhibitors located in such kernels. The microbial or endogenous nature of the endoxylanases was confirmed by making use of the inhibition specificity of endoxylanase inhibitors. Determination of the endoxylanase activity in the washing liquid, corresponding to the microbial endoxylanase population, and the washed kernels, corresponding to the endogenous endoxylanase population, allowed estimation of the total endoxylanase activities associated with the wheat kernel. Results showed that microbial endoxylanases can account for over 90% of the total wheat-associated endoxylanase activity and that the latter can be at least 5 times higher than the apparent endoxylanase activity.

The influence of wheat variety, harvest year and harvest date on the levels of wheat kernel-associated endogenous and microbial endoxylanases and wheat endogenous endoxylanase inhibitors was investigated using ten winter wheat varieties grown in 2002 and 2003. Three of the varieties were harvested on five different dates in a 1 month period spanning the optimal harvest period. Introductory experiments indicated that endoxylanase activity was very heterogeneously distributed among the different wheat kernels, necessitating the use of relatively large amounts of kernels, i.e. 100 g, to obtain representative wholemeal samples. Apparent endoxylanase activities in these samples largely depended on variety and weather conditions prior to harvesting. In contrast, apparent endoxylanase inhibitor levels were less dependent on climatological circumstances and were mainly determined by genetic factors. The data derived from this study may contribute to explaining the differences observed in functionality between wheats.

Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.

A recent approach based on affinity chromatography with immobilised endoxylanase inhibitors was used to isolate two endoxylanases (EC 3.2.1.8) with different bread-making functionalities from an Aspergillus niger fermentation broth. TAXI ( Triticum aestivum endoxylanase inhibitor) affinity chromatography yielded a TAXI- and XIP (endoxylanase inhibiting protein)-sensitive family 11 endoxylanase (24 kDa, p I 3.5), XIP affinity chromatography subsequently yielded a family 10 endoxylanase (36 kDa), only inhibited by XIP. While the first enzyme improves bread volume, the latter enzyme has no effect on bread quality whatsoever. The bread-making positive endoxylanase rather selectively hydrolyses water-unextractable arabinoxylan in an in vitro screening method, still performs/is active during bread-making and produces soluble arabinoxylan of high (>11.2 × 10 4 Da) and low molecular mass (≤11.2 × 10 4 Da). In contrast, the bread-making neutral endoxylanase in the in vitro assay displays a bias for water-extractable arabinoxylan and is immediately and almost completely inhibited during the early stages of bread-making. The results show that the functionalities of the purified A. niger endoxylanases in wheat bread-making are strongly dictated by their sensitivities towards wheat endoxylanase inhibitors.

The vast majority of bread is traditionally produced from wheat flour. Apart from its major constituent starch, wheat flour also contains many other types of substances of which the gluten, the non-starch polysaccharides as well as the lipids are the most important in terms of their impact on the processability of the raw material and in terms of the quality of the final products. We here provide the basics on the processability and quality determining wheat flour constituents and present common concepts on their fate during the breadmaking process as well as on approaches targeted to influence their functionality.

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.

Plants developed a diverse battery of defense mechanisms in response to continual challenges by a broad spectrum of pathogenic microorganisms. Their defense arsenal includes inhibitors of cell wall-degrading enzymes, which hinder a possible invasion and colonization by antagonists. The structure of Triticum aestivum xylanase inhibitor-I (TAXI-I), a first member of potent TAXI-type inhibitors of fungal and bacterial family 11 xylanases, has been determined to 1.7-A resolution. Surprisingly, TAXI-I displays structural homology with the pepsin-like family of aspartic proteases but is proteolytically nonfunctional, because one or more residues of the essential catalytical triad are absent. The structure of the TAXI-I. Aspergillus niger xylanase I complex, at a resolution of 1.8 A, illustrates the ability of tight binding and inhibition with subnanomolar affinity and indicates the importance of the C-terminal end for the differences in xylanase specificity among different TAXI-type inhibitors.

Debranning of wheat to remove the outer 7% of the kernel, prior to grinding or milling reduced xylanase activity in wheat wholemeal and wheat endosperm flour by up to 80 and 60%, respectively, whereas there was no significant reduction of xylanase inhibiting activity. Flours obtained after debranning and milling showed no major differences in moisture content, whereas ash content decreased and protein and arabinoxylan content decreased slightly with increasing debranning degree. Part of the xylanase activity in the flour was lost on addition of Triticum aestivum xylanase inhibitor (TAXI). Since TAXI specifically inhibits glycosyl hydrolase family 11 xylanases and since endogenous cereal xylanases belong exclusively to family 10, part of the xylanase activity in the flour is most likely of microbial origin. Debranning also significantly reduced alpha-amylase activities in wheat wholemeal and wheat flour. Debranning prior to milling can, therefore, impact on flour functionality.

Cereals contain proteinaceous inhibitors of endoxylanases, which affect the efficiency and functionality of these enzymes in cereal processing. This review relates their first discovery in wheat and the subsequent purification of two distinct classes of endoxylanase inhibitors, namely Triticum aestivum xylanase inhibitor (TAXI)-type and xylanase inhibitor protein (XIP)-type inhibitors in cereals. Both inhibitor classes occur in monocots as multi-isoform families. The reported data provide an overview of the relative quantitative and qualitative variation of these inhibitors in cereals. Wheat and rye are particularly rich in TAXI-type and XIP-type inhibitors with the latter inhibitors being more abundant. Lower inhibitor levels are present in durum wheat and barley, while maize contains solely XIP-type inhibitors. No inhibitors have been isolated from rice, oats and buckwheat.

Highly pure XIP-type (for Xylanase Inhibiting Protein) endoxylanase inhibitor fractions were selectively obtained with a high yield from rye, durum wheat, barley, and maize extracts by affinity chromatography with immobilised Aspergillus niger endoxylanase Xyn1 following removal of the TAXI-type (for Triticum aestivum xylanase inhibitor) endoxylanase inhibitors by affinity chromatography with immobilised Bacillus subtilis endoxylanase XynA. No inhibitors belonging to the XIP family occur in rice, oats, and buckwheat. N-terminal amino acid sequences of the non-wheat XIP-type inhibitors were very similar or identical to those of wheat XIP-1, a chitinase homologue. The isolated inhibitors are basic, monomeric proteins of ca. 30 kDa with pI values of at least 8.5 (rye, durum wheat, and barley XIP) and ca. 7.0 (maize XIP). They are, in general, active against fungal endoxylanases and do not hydrolyse chitin. SDS–PAGE analysis and high-resolution cation exchange chromatography suggest the presence of multiple XIP-type isoinhibitors in the different cereals.

An affinity-based purification procedure allowed the resolution of two distinct groups of endoxylanase inhibitors with different molecular structures and endoxylanase specificities from wheat wholemeal. The first group comprises the so-called Triticum aestivum L. Endoxylanase inhibitor (TAXI)-type proteins which are of approx. M r 40 000 and occur in two different molecular forms. These inhibitors were removed from a concentrated cation exchange chromatography fraction from wheat wholemeal on a Bacillus subtilis endoxylanase affinity column. The second group of structurally different endoxylanase inhibitors, the so-called xylanase inhibiting protein (XIP)-type, of approx. M r 29 000–32 000, with pI values varying between 8·8 and 9·2, was purified from the unbound fraction from the B. subtilis endoxylanase affinity column by chromatography on an Aspergillus niger endoxylanase affinity column followed by gel permeation chromatography. The XIP-type inhibitors are not active against the B. subtilis endoxylanase and were consequently not retained on the B. subtilis endoxylanase column. Further analysis of the XIP-type proteins by high-resolution cation exchange chromatography, SDS-PAGE and iso-electrofocusing, revealed several forms. They had similar endoxylanase specificities and N-terminal amino acid sequences.

Four Secale cereale L. xylanase inhibitors (SCXI) from rye were purified to homogeneity. The inhibitor proteins all occur in two molecular forms, i.e. a c. 40 kDa monomeric protein and, presumably following proteolytic modification, a heterodimer consisting of two disulphide linked subunits of c. 30 and c. 10 kDa. These basic proteins all have isoelectric points of at least 9·0 and a highly homologous N -terminal amino acid sequence. The isolated proteins strongly inhibited the activity of Aspergillus niger, Bacillus subtilis and Trichoderma viride family 11 endoxylanases, but showed no activity towards an Aspergillus aculeatus family 10 endoxylanase. These characteristics demonstrate that rye contains a family of isoinhibitors with similar structures and specificities, that are homologous with Triticum aestivum L. xylanase inhibitor I (TAXI I) from wheat andHordeum vulgare L. xylanase inhibitor (HVXI) from barley.

Twenty-three wheat samples from 19 different European wheat cultivars (Triticum aestivum L.) were tested for their quantitative and qualitative variation in inhibition activity against family 11 endoxylanases of Aspergillus niger, Bacillus subtilis, and Trichoderma viride and a family 10 endoxylanase of A. aculeatus. Under the experimental conditions, the A. aculeatus enzyme was not inhibited by the wheat extracts, the A. niger and B. subtilis endoxylanases were affected to a similar extent, while the T. viride enzyme was much more inhibited. The inhibition activities in the different wheat samples against the A. niger, B. subtilis, and T. viride endoxylanases varied between 36.0 and 11.7, 34.0 and 12.9, and 86.2 and 46.6 IU/100 mg of dry whole meal, respectively. One IU (inhibition unit) corresponds to the amount of inhibitor resulting in 50% inhibition of endoxylanase activity under the conditions of the assay. The inhibitor activities were linearly related, indicating that the levels of differ...

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation. of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase. ispartof: Journal of enzyme inhibition and medicinal chemistry vol:17 issue:1 pages:61-68 ispartof: location:England status: published

Hordeum vulgare L. xylanase inhibitor (HVXI), an endoxylanase inhibitor with a protein structure, was purified to homogeneity from barley (Hordeum vulgare L.). HVXI is a nonglycosylated monomeric protein, with a molecular weight of ≈40,000 and a pI ≥ 9.3. Although it inhibits different endoxylanases to a varying degree, the activities of an α-L-arabinofuranosidase and a β-d-xylosidase were not inhibited. Apparently, HVXI occurs in two molecular forms. These characteristics and the N-terminal sequences of the composing polypeptides show that HVXI is homologous with Triticum aestivum L. xylanase inhibitor I, an endoxylanase inhibitor from wheat flour.

J. Am. Soc. Brew. Chem. 72(1):12-21, 2014 Gushing is the vigorous overfoaming of carbonated beverages when the bottle is opened. Primary gushing in beer is mostly caused by a group of proteins called hydrophobins secreted by filamentous fungi, which contaminate CO2 gaseous molecules during carbonation and form nanobubbles. The influence of hop oil antifoam on primary gushing showed a complete suppressing effect in sparkling water, a decreasing effect in wort, and no influence on gushing-positive beers. This shows the importance of the critical point of addition of this product in the brewing process. GC and GC-MS analysis show that commercial available lipophilic hop extract comprises fatty acids, either saturated fatty acids (SFA) or unsaturated fatty acids (UFA) (both free and incorporated in mono-, di-, and triglycerides), waxes (long-chain alkanes), and steroid compounds. Gushing analysis of each compound showed that SFA and UFA behave in a different manner regarding gushing. In contrast to SFA and trans-form UFA, cis-form UFAs do not induce gushing. Long-chain alkanes provide sufficient hydrophobic structures to interact with gaseous CO2 molecules and induce gushing. Because hop antifoam molecules are hydrophobic, they interact with hydrophobins and prevent interaction with CO2 and, consequently, inhibit the possibilities of development of the explosive nanobubble structure.

The cell wall polysaccharides of wheat affect the quality for food processing, livestock feed and distilling, and also form a major source of dietary fibre (DF) for human health. Multisite field trials of wheat genotypes show extensive variation in the content of the major types of cell wall polysaccharide in wheat, arabinoxylan and β-glucan, much of which is heritable and hence available for exploitation by plant breeders. Furthermore, contents of DF components have not declined as a result of intensive wheat breeding. The identification of candidate genes for DF synthesis using bioinformatics and RNAi suppression in transgenic wheat will allow the content and composition of DF components to be fine-tuned for specific end uses.

The wheat grain comprises three groups of major components, starch, protein, and cell wall polysaccharides (dietary fiber), and a range of minor components that may confer benefits to human health. Detailed analyses of dietary fiber and other bioactive components were carried out under the EU FP6 HEALTHGRAIN program on 150 bread wheat lines grown on a single site, 50 lines of other wheat species and other cereals grown on the same site, and 23−26 bread wheat lines grown in six environments. Principal component analysis allowed the 150 bread wheat lines to be classified on the basis of differences in their contents of bioactive components and wheat species (bread, durum, spelt, emmer, and einkorn wheats) to be clearly separated from related cereals (barley, rye, and oats). Such multivariate analyses could be used to define substantial equivalence when novel (including transgenic) cereals are considered.