Contribution of wheat endogenous and wheat kernel associated microbial endoxylanases to changes in the arabinoxylan population during breadmaking

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.     

Impact of wheat flour-associated endoxylanases on arabinoxylan in dough after mixing and resting

The impact of varying levels of endoxylanase activity in wheat flour on arabinoxylan (AX) in mixed and rested dough was studied using eight industrially milled wheat flour fractions with varying endoxylanase activity levels. Analysis of the levels of reducing end xylose (RX) and solubilized AX (S-AX) formed during mixing and resting and their correlation with the endoxylanase activity in the flour milling fractions showed that solubilization of AX during the mixing phase is mainly due to mechanical forces, while solubilization of AX during resting is caused by endoxylanase activity. Moreover, solubilization of AX during the dough resting phase is more outspoken than that during the mixing phase. Besides endoxylanase activity, there were significant xylosidase and arabinofuranosidase activities during the dough resting phase. The results indicate that wheat flour-associated endoxylanases can alter part of the AX in dough, thereby changing their functionality in bread making and potentially affecting dough and end product properties.     

Insight into the distribution of arabinoxylans, endoxylanases, and endoxylanase inhibitors in industrial wheat roller mill streams

To gain insight into the distribution of arabinoxylans (AX), endoxylanases, and endoxylanase inhibitors in industrial wheat roller milling, all streams, that is, 54 flour fractions, 4 bran fractions, and the germ, were analyzed for ash, starch, and protein contents, alpha-amylase activity levels, total (TOT-AX) and water-extractable arabinoxylan (WE-AX) contents, endoxylanase activity levels, and endoxylanase inhibitor (TAXI and XIP) contents. In general, bran fractions were significantly richer in TOT-AX and WE-AX contents, endoxylanase activity levels, and endoxylanase inhibitor contents than germ and, even more so, than flour fractions. In the 54 different flour fractions, minimal and maximal values for TOT-AX and WE-AX contents differed by ca. 2-fold, whereas they differed by ca. 15-fold for endoxylanase activity levels. The latter were positively correlated with ash and negatively correlated with starch content, suggesting that the endoxylanase activity in flour is strongly influenced by the level of bran contamination. TAXI contents in the flour fractions varied ca. 4-fold and were strongly correlated with bran-related parameters such as ash content and enzyme activity levels, whereas XIP contents varied ca. 3-fold and were not correlated with any of the parameters measured in this study. The results can be valuable in blending and optimizing wheat flour fractions to obtain flours with specific technological and nutritional benefits.     

TAXI type endoxylanase inhibitors in different cereals

An affinity-based purification procedure with the immobilized family 11 Bacillus subtilis endoxylanase XynA allowed us to obtain high yields of highly pure endoxylanase inhibitor fractions from rye, barley, and durum wheat. In contrast, no inhibitors interacting with the B. subtilis endoxylanase affinity column are present in corn, buckwheat, rice, and oats. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and inhibitor specificity showed that the isolated inhibitors belonged to the TAXI endoxylanase inhibitor family, thus providing a view on the diversity of this cereal inhibitor family. The isolated inhibitors are basic proteins of ca. 40 kDa, occurring in two molecular forms, with pI values of ca. 8.5 (durum wheat) and ca. 9.0 (rye, barley). They are, in general, strong inhibitors of family 11 endoxylanases but not of family 10 endoxylanases. Because cereal endogenous endoxylanases belong to the latter family, this probably indicates that they do not influence cereal metabolic processes. For the first time, endoxylanase inhibitors, similar to TAXI I and TAXI II from wheat, were isolated from durum wheat and characterized. For each cereal, high-resolution cation exchange chromatography revealed the presence of multiple isoinhibitors, each of which occurs in two molecular forms. However, in durum wheat and barley, a single isoform is predominantly present.     

Endoxylanase Inhibition Activity in Different European Wheat Cultivars and Milling Fractions

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 different endoxylanase inhibitors with different endoxylanase specificities in the dormant wheat grains are also linearly related or that one (or more) of these inhibitors are predominantly present or has much higher specific activity, consequently causing almost all of the inhibition activity measured. Wheat flour accounted for ≈57% of the total inhibition activity in wheat grains, while the shorts and bran fractions each contained ≈21% of the total activity. On dry weight basis, the inhibition activities were about three times higher in shorts and about two times higher in bran than in flour. The results obtained may be useful in explaining differences in functionality of different endoxylanases in biotechnological processes in which wheats of different cultivars, or fractions thereof, are used as well as in screening endoxylanases for applications in wheat‐based processes.     

Enzymic degradability of hull-less barley flour alkali-solubilized arabinoxylan fractions by endoxylanases

The impacts of the arabinose to xylose (A/X) ratio of arabinoxylans (AX) and the endoxylanase substrate specificity on the enzymic degradability of hull-less barley flour AX by endoxylanases were studied by using alkali-solubilized AX (AS-AX) fractions with different A/X ratio, on the one hand, and glycoside hydrolase family 10 and 11 endoxylanases of Aspergillus aculeatus (XAA) and Bacillus subtilis (XBS), respectively, on the other hand. AS-AX were obtained by saturated barium hydroxide treatment of hull-less barley flour water-unextractable AX. Fractionation of AS-AX by stepwise ethanol precipitation resulted in structurally different hull-less barley flour AS-AX fractions. Their A/X ratios increased with increasing ethanol concentration, and this increase in A/X ratio was reflected in their xylose substitution levels. For both XAA and XBS, the enzymic degradability of AX and apparent specific endoxylanase activity decreased with increasing A/X ratio of the AS-AX substrates, implying that both endoxylanases were sterically hindered by arabinose substituents. However, for all AS-AX fractions, hydrolysis end products of lower average degree of polymerization were obtained after incubation with XAA than with XBS, indicating that the former enzyme has a lower substrate specificity toward hull-less barley flour AS-AX than the latter. In addition, apparent specific endoxylanase activities indicated that XBS was approximately 2 times more sensitive to variations in the A/X ratio of AS-AX fractions than XAA. Furthermore, AS-AX with higher A/X ratio were relatively resistant to degradation by XBS.     

Fractionation-reconstitution experiments provide insight into the role of endoxylanases in bread-making.

The impact mechanism of endoxylanases in straight dough bread-making was investigated in fractionation-reconstitution experiments. To this end, two European flours with different bread-making characteristics were separated in gluten, prime starch, a squeegee fraction (SQF), and a water-extractable fraction. Whereas the former fractions contained negligible levels of arabinoxylan (AX), the latter contained, respectively, most of the water-unextractable AX (WU-AX) and all of the water-extractable AX (WE-AX). In vitro modification with a Bacillus subtilis endoxylanase allowed controlled solubilization of WU-AX from SQF and controlled degradation of solubilized AX and WE-AX from the water-extractables. It followed from bread-making tests with the reconstituted flours that endoxylanases exert positive loaf volume effects in bread-making by lowering the concentration of WU-AX and increasing that of total soluble AX. Limited degradation of WE-AX and significant breakdown of solubilized AX by endoxylanases, on the other hand, resulted in volume losses when compared to their nondegraded counterparts. The volume increasing effects of endoxylanases are therefore related to their ratio of solubilizing to degrading activity and thus to their substrate specificity.     

Endoxylanases in durum wheat semolina processing: solubilization of arabinoxylans, action of endogenous inhibitors, and effects on rheological properties

Endoxylanases seriously affect the rheological properties of durum wheat (Triticum durum Desf.) semolina spaghetti doughs prepared with, and as evaluated, by the farinograph. Under the experimental conditions, control doughs (34.9% moisture content) made from two semolinas (semA and semB) yielded a maximal consistency of 525 and 517 farinograph units (FU), with, respectively, 19.4 and 16.4% of the total level of arabinoxylans (TOT-AX) being water-extractable (WE-AX). When 75.4 Somogyi units/50 g of semolina of the endoxylanases from Trichoderma viride (XTV), rumen microorganisms (XRM), Bacillus subtilis (XBS), and Aspergillus niger (XAN) were used, the maximal consistencies at 34.9% moisture decreased for semA to 467, 436, 448, and 417 FU, respectively. This was accompanied by increased WE-AX contents of 60.8, 71.2, 70.7, and 73.0%, respectively. Similar results were observed for semB. By reducing the total water content of doughs, it was possible to recover the maximal consistency of the original doughs. Both the decrease in maximal consistency and the amount of water to be omitted were significantly related to the decrease in molecular weight (MW) of the WE-AX and the percentage of WE-AX solubilized as a result of the enzymic action. At the same time, it was clear that endogenous endoxylanase inhibitors were present in the durum wheat semolinas and that they inhibited the endoxylanases used to different degrees. Part of the differences in effects between the different endoxylanases (decrease in maximal consistency, amount of AX solubilized, MWs of the WE-AX, and amount of water that could be omitted) could be ascribed to the differences in inhibition of the endoxylanases by endogenous inhibitors.     

Effect of Wheat Grain Steaming and Washing on Lipase Activity in Whole Grain Flour

During whole grain flour (WGF) storage, lipase activity causes partial loss of its functionality and the sensory acceptability of products produced from it. The objective of this research was to evaluate the effect of steaming and washing on lipase activity in (fractions of) wheat. Steam treatment conditions were optimized for wheat grains and their bran, shorts, and flour fractions. Lipase activities were determined colorimetrically, as were peroxidase, endoxylanase, and α‐amylase activities. Steaming grains for 180 s effectively inactivated lipase, peroxidase, endoxylanase, and part of the α‐amylase without gelatinizing starch. The work further demonstrated that lipase is mainly, if not only, located in the bran fraction. Separate bran treatment holds promise for obtaining WGF with reduced lipase activity but without altered functional properties. Washing grains did not reduce WGF lipase activity.     

Fermented Wheat Bran as a Functional Ingredient in Baking

The aim of the current study was to identify factors influencing the technological functionality of fermented bran. The influences of fermentation type and type of wheat bran on the microbial community, bioactivity, arabinoxylans (AX), and activity of xylanases were studied in the bran ferments. Furthermore, technological quality of ferments was established by using them to replace wheat in baking with a 20% substitution level. Solubilization of AX and endogenous xylanase activity of bran were influenced by the type of bran, fermentation type, and conditions. Peeled bran had a clearly reduced microbial load and different microbial community in comparison to native bran. Bran from peeled kernels contained 10‐fold lower activities of endogenous xylanases in comparison to native bran. Yeast fermentation of bran from peeled kernels increased the level of folates (+40%), free phenolic acids (+500%), and soluble AX (+60%). Bread containing yeast‐fermented peeled bran had improved volume (+10–15%) and crumb softness (25–35% softer) in comparison to unfermented counterparts. Solubilization of AX during the 20 hr fermentation and decreased endogenous xylanase activity are proposed as the main reasons for the improved technological functionality of fermented bran.     

Isolation and characterization of two endoxylanases from Fusarium graminearum

This paper reports the first isolation from cultures of two endoxylanases secreted by Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch]. When F. graminearum is grown on wheat bran hydrated with a modified synthetic medium, high xylanase activity can be extracted. The two endoxylanases were identified by LC-MS/MS as the products of genes FGSG_6445 (Genbank gene id 2788192 ) (xylanase 1) and FGSG_3624 (GenBank accession no. AJ863566 ) (xylanase 2) with 61 and 51% sequence coverage, respectively. Both enzymes showed a pH optimum at pH 6, with xylanase 1 exhibiting a wider active pH range (5.5-9) than xlylanase 2 (5.5-7.5). Their temperature dependences were similar, >60% between 35 and 60 °C, with optimal temperatures of 45 °C for xylanase 1 and 50 °C for xylanase 2. Kinetic studies found that both enzymes had a lower K(m) for linear beachwood xylan than arabinoxylan. For xylanase 2, the V(max) increased with arabinoxylan, but decreased for xylanase 1.     

Impact of inhibition sensitivity on endoxylanase functionality in wheat flour breadmaking

A Bacillus subtilis endoxylanase (XBS(i)) sensitive to inhibition by Triticum aestivum L. endoxylanase inhibitor (TAXI) and a mutant thereof (XBS(ni)), uninhibited by TAXI, were used in straight-dough breadmaking to assess the importance of endoxylanase inhibition sensitivity on endoxylanase functionality in the process. With two European wheat flours, the loaf volume improving effect of XBS(ni) at much lower enzyme dosages was substantially larger than that brought about by XBS(i). This coincided with differences in arabinoxylan (AX) hydrolysis. Although XBS(ni) had a lower substrate selectivity for water-unextractable arabinoxylan (WU-AX) than XBS(i), the former solubilized significantly more WU-AX than XBS(i). Because of inhibition, XBS(i) solubilized most of the WU-AX during mixing, whereas, with XBS(ni), the rate of solubilization decreased less with increasing processing time than that with XBS(i). During fermentation and baking and at the highest dosage (600 U/kg of flour of XBS(i) and 60 U/kg of flour of XBS(ni)), XBS(ni) induced a stronger degradation of enzymically solubilized and water-extractable AX than XBS(i). Taken together, the data clearly demonstrate that endoxylanases, which in vitro are inhibited by endoxylanase inhibitors and still are active in the breadmaking process, as demonstrated by their functional (bread volume) enhancing effect, gradually lose their activity in the process.     

Purification and Partial Characterization of an Endoxylanase Inhibitor from Barley

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.     

Extraction and Activation of Wheat Polyphenol Oxidase by Detergents: Biochemistry and Applications

Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in undesirable brown discolorations of food products. It has been demonstrated that these enzymes are often present in a latent form or are membrane‐associated, necessitating detergent or other treatments to obtain fully active preparations. Here, the influence of different detergents on wheat meal and flour PPOs was investigated. Extraction in presence of 50 mM SDS led to a 5‐ to 15‐fold increase in PPO activity, making quantitative assays in flour from low‐PPO lines more robust. Among a series of additional nonionic, anionic, and cationic detergents tested, only n ‐lauroylsarcosine increased extractable PPO activity to a degree comparable to that of SDS. Additional experiments suggested that a large fraction of wheat meal PPOs may be membrane‐associated and that SDS is able to activate PPOs extracted from high‐activity but not from low‐activity wheat lines. PPO activities assayed after SDS extraction of meal and flour were highly correlated with each other and with activity determined in whole (intact) kernels in absence of SDS. Correlation coefficients between PPO activities measured with all these methods and noodle brightness were about equal, indicating that activities assayed after SDS extraction are useful for germplasm screening and quality prediction.     

Combined Effects of Endoxylanases and Reduced Water Levels in Pasta Production

The combined effects on pasta properties of 1) varying dosages of endoxylanases (EC 3.2.1.8) from Aspergillus aculeatus and Bacillus subtilis and 2) lower levels of water during pasta dough processing were studied. The A. aculeatus endoxylanase has high selectivity toward water‐extractable arabinoxylan (WE‐AX), whereas B. subtilis endoxylanase preferentially hydrolyzes water‐unextractable arabinoxylan (WU‐AX). Pasta was produced on a microscale (50.0 g) from the semolinas of both a strong (AC Navigator) and a moderately strong (AC Avonlea) durum wheat cultivar. The levels of added water in endoxylanase‐treated pastas were adjusted to obtain the same maximal farinograph consistencies as for the control pastas. The extruded pastas were dried with drying cycles at 40, 70, or 90°C. Apart from increasing levels of solubilized arabinoxylans, these treatments had little effect on the color, optimal cooking time, and firmness of the resulting pasta. High enzyme concentrations and low (40°C) drying temperature resulted in clearly or much less checked final products for the B. subtilis and A. aculeatus enzyme, respectively. Upon cooking, the enzymically formed low molecular weight arabinoxylans were retained better in the pasta strands than their equally low molecular weight arabinogalactan counterparts.     

Solubilization of Arabinoxylans from Isolated Water-Unextractable Pentosans and Wheat Flour Doughs by Cell-Wall-Degrading Enzymes

Water-unextractable pentosans (WUP) isolated from the flours of three wheat cultivars (Apollo, Soissons, Thésée) were treated with enzymes to solubilize the arabinoxylans. The water-unextractable arabinoxylans from the three cultivars had similar susceptibility to solubilization by enzymes: Grindamyl S 100 (GS100), a commercial preparation for baking, rich in pentosanase activities that originated from an Aspergillus niger culture; and three endoxylanases (E1, E2, E3), an arabinofuranosidase (Af), a β- glucanase (βG), and a ferulate esterase (FAE) purified from GS100. A cellulase (C) and a pure endoglucanase (eG) from Trichoderma reesei were also used. GS100 was able to solubilize high molecular weight arabinoxylans (HMWAX) from WUP that markedly enhance the viscosity of the reaction mixture supernatants. The endoxylanase E1 was responsible for this solubilizing activity of GS100, whereas E2 and E3 made only a very low contribution. Combining E1 with FAE led to a limited increase in the arabinoxylan-solubilizing effect. Also, enzymes hydrolyzing cellulose and β-glucans slightly improved the arabinoxylan solubilization from WUP when combined with GS100 or E1, but produced arabinoxylans of lower intrinsic viscosity. Similar effects of the enzymes were observed on arabinoxylan solubilization when applied to dough instead of isolated WUP.     

Impacts of long-term application of buctril super (bromoxynil) herbicide on microbial population,enzymes activity,nitrate nitrogen,Olsen-P and total organic carbon in soil

Long-term impact of buctril super (bromoxynil) herbicide in the wheat fields on soil microbial population, nitrate-N, Olsen-P, total organic carbon (TOC) and enzyme activities was evaluated in 18 sites in Pakistan. Nine sites were randomly selected from those places where bromoxynil herbicide had been used for the last 10 years designated as soil ‘X’ and other nine where no herbicide was used in that period designated as soil ‘Y’. Very importantly, it was found that long-term application of this herbicide in wheat fields reduced the actinomycetes and fungi population up to 19.7 and 14.3%, respectively, urease and dehydrogenase activity by 17.5 and 28.2%, respectively, and reduced nitrate-N, Olsen-P and TOC up to 55, 17 and 28.57%, respectively. Presence of high clay and organic matter contents enhanced the detrimental effect of herbicide by prolonging its persistence as compared to light-textured soils with low organic matter. As in Pakistan this herbicide is being used most frequently in wheat fields, data are scarce on the long-term effect of this herbicide on soil microbial activities and soil health. These findings could give new insights about the use of alternate herbicide in wheat fields, particularly in clay-textured and high organic matter contained soils for maintaining soil health.     

Influence of Arabinoxylans and Endoxylanases on Pasta Processing and Quality. Production of High‐Quality Pasta with Increased Levels of Soluble Fiber

As part of a general study aiming to clarify the role of arabinoxylans (AX) in pasta processing and quality, AX were modified by the addition of endoxylanases during pasta processing. The influence on processing parameters and quality were determined. Pasta (800 g) was produced from two commercial semolinas (semA and semB) using dosages of Bacillus subtilis (XBS) and Aspergillus niger (XAN) endoxylanases of 0–0.225 Somogyi units/g of semolina. Increased dosages resulted in a drop of extrusion pressure. The endoxylanase treatments had no great effect on the resulting pasta quality (color of dry products and surface condition, viscoelastic index, and resistance to longitudinal deformations of cooked products). High dosages of XAN and XBS resulted in high levels of solubilized AX (as an extra source of soluble dietary fiber) of low molecular weight which were expected to easily leach out during the cooking process of pasta. Surprisingly, only low levels of AX were found in the cooking water, even with extremely high dosages of endoxylanases used and cooking beyond optimum time. A method is provided to obtain high‐quality pasta with increased levels of soluble fiber.     

Detection,Localization, and Variability of Endogenous β‐Glucanase in Wheat Kernels

Clinical studies with isolates of β‐glucan have shown that the health benefits are regulated not only by the polysaccharide concentration but also by the molecular weight and concentration in solution, because these health benefits are controlled, inter alia, by viscosity in the gut. The degradation of β‐glucan in baked products is likely caused by baking ingredients or processes, or by endogenous enzymes in wheat flour. The objectives of the present study were to quantify β‐glucanase in wheat kernels and to determine factors that influence the levels of this enzyme. A modified protocol to quantify β‐glucanase was developed and then confirmed through high‐performance size‐exclusion chromatography (HPSEC) with Calcofluor detection. Under this protocol, it was shown that the concentration of β‐glucanase activity was the highest in the bran fraction of the kernel in ungerminated wheats, whereas it was distributed throughout the entire kernel following germination. Furthermore, investigation on different wheat cultivars planted in the same and different locations showed that genotype, environment, and agronomic practice all can have an effect on β‐glucanase activity level in wheat kernels.     

秸秆还田对土壤氮素养分及微生物量氮动态变化的影响

通过定位试验研究玉米秸秆全量粉碎还田及小麦秸秆旋耕施肥播种同步完成的前提下,秸秆还田循环利用对小麦玉米两熟制土壤氮素养分及土壤微生物量氮的动态变化。结果表明小麦、玉米秸秆还田能满足小麦旺盛生长阶段拔节期对氮素养分的需求,秸秆还田处理或施肥处理的土壤全氮量总体上在小麦拔节期处于最低值,而既无秸秆还田又没有施肥的对照处理土壤全氮含量最低值出现的时期延后,在小麦开花期出现,持续至小麦的灌浆期。对于麦玉秸秆还田但不施氮磷钾肥而言,小麦生长后期(小麦开花期以后)土壤脱氮比较严重。秸秆还田后土壤碱解氮含量在小麦整个生长发育时期呈现上升的趋势。单独施肥或秸秆还田对提高土壤微生物量氮均有一定的作用,但是仅仅施肥其后效不足。秸秆还田并且施肥显著地促进了土壤微生物的活动,能持续地增加土壤微生物量氮含量。