Wheat-kernel-associated endoxylanases consist of a majority of microbial and a minority of wheat endogenous endoxylanases

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.     

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.     

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.     

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.     

Effect of Kernel Size and Mill Type on Protein,Milling Yield,and Baking Quality of Hard Red Spring Wheat

Optimization of flour yield and quality is important in the milling industry. The objective of this study was to determine the effect of kernel size and mill type on flour yield and end‐use quality. A hard red spring wheat composite sample was segregated, based on kernel size, into large, medium, and small kernels, as well as unsorted kernels. The four fractions were milled in three roller mills: Brabender Quadrumat Jr., Quadrumat Sr., and Bühler MLU‐202 laboratory mills. Large kernels had consistently higher flour yield than small kernels across mills, with the Quadrumat Jr. mill showing the lowest flour yield. Mill type and kernel size significantly affected variation in flour protein molecular weight distribution. When compared with larger kernels, flour milled from the small‐kernel fraction contained a higher gliadin fraction and SDS‐unextractable high‐molecular‐weight polymeric proteins, which had positive correlations with bread loaf volume (r = 0.61, P < 0.05) and mixograph peak time (r = 0.84, P < 0.001). Overall, small kernels could contribute to enhancing flour breadmaking quality while having a detrimental effect on milling yield.     

Use of Two Endoxylanases with Different Substrate Selectivity for Understanding Arabinoxylan Functionality in Wheat Flour Breadmaking

A Bacillus subtilis endoxylanase (XBS) with a strong selectivity for hydrolysis of water‐unextractable arabinoxylan (WU‐AX) and an Aspergillus aculeatus endoxylanase (XAA) with a strong selectivity for hydrolysis of water‐extractable arabinoxylan (WE‐AX) were used in straight‐dough breadmaking with two European wheat flours. Dough, fermented dough, and bread characteristics with different levels of enzyme addition were evaluated with a strong emphasis on the arabinoxylan (AX) population. The WU‐AX solubilized by XBS during breadmaking were mainly released during mixing and had higher molecular weight, in contrast to their counterparts solubilized by XAA, which were mainly released during fermentation and had lower molecular weight. This coincided with increased loaf volume with XBS and a negative to positive loaf volume response with XAA. Bread firmness and dough extract viscosity also were affected by endoxylanase addition. Results confirmed that WU‐AX are detrimental for breadmaking, while WE‐AX and solubilized AX with medium to high molecular weight have a positive impact on loaf volume.     

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.     

Effect of Dark,Hard, and Vitreous Kernel Content on Protein Molecular Weight Distribution and on Milling and Breadmaking Quality Characteristics for Hard Spring Wheat Samples from Diverse Growing Regions

Kernel vitreousness is an important grading characteristic for segregation of subclasses of hard red spring (HRS) wheat in the United States. This research investigated the protein molecular weight distribution (MWD) and the flour and baking quality characteristics of different HRS wheat market subclasses. The U.S. regional crop quality survey samples obtained from six regions for three consecutive growing years were used for subclass segregation based on the dark, hard, and vitreous (DHV) kernel percentage. Flour milled from HRS wheat with greater percentages of DHV kernel showed higher water absorption capacity for breadmaking. Protein MWD parameters could be related to the association between DHV kernel level and water absorption. Specifically, flour protein fractions rich in gliadins and high‐molecular‐weight polymeric proteins in the SDS‐unextractable fraction were identified to have significant and positive correlations with both DHV kernels and flour water absorption levels. An example further showed the importance of flour water absorption on potential economic incentives that can be gained with having a greater percentage of vitreous kernels. This information could help the flour milling and baking industry to segregate the different subclasses of HRS wheat with varying DHV content for their intended end‐use applications.     

Physical and Milling Characteristics of Wheat Kernels After Enzyme and Acid Treatments

The current wheat milling process separates bran from endosperm by passing tempered wheat kernels through successive break rolls and sifters. Using hydrolytic enzymes during tempering degrades bran and aleurone layers and can improve milling efficiency and yield. This study was conducted to evaluate the effects of chemical and enzymatic treatments of wheat kernels before milling on physical and milling characteristics of the resulting wheat and flour quality. Hard wheat kernels were soaked in dilute acid or water and dried back to original moisture before being tempered with enzymes in water. Kernel physical and milling characteristics (600 g) were evaluated. Dilute acid soaking did not affect the 1,000‐kernel weight and diameter but softened treated kernels. When treated kernels were pearled, bran removal was mostly from ends; and the reducing sugar content in enzyme‐treated bran was significantly higher than the control. Compared with the control, acid‐soaked enzyme‐tempered kernels showed small but significant improvement in straight flour yield, with virtually no difference in protein content, and flour color. Chemical and enzyme treatment resulted in higher ash in flour. These differences were not seen in milling of larger batches (1,500 g) of kernels.     

Influence of Kernel Size and Shriveling on Soft Wheat Milling and Baking Quality

Small kernels of soft wheat are sometimes considered to be harder than larger kernels and to have inferior milling and baking characteristics. This study distinguished between kernel size and kernel shriveling. Nine cultivars were separated into large, medium, and small kernels that had no shriveling. Eleven cultivars were separated into sound, moderate, and severely shriveled kernels. Shriveling greatly decreased the amount of flour produced during milling. It adversely affected all other milling quality characteristics (ash content, endosperm separation index, and friability). Shriveled kernels produced flour that had inferior soft wheat baking qualities (smaller cookie diameter and higher alkaline water retention capacity). In contrast, test weight and milling qualities were independent of kernel size. Small, nonshriveled kernels had slightly better baking quality (larger cookie diameter) than larger nonshriveled kernels. Small kernels were softer than large kernels (measured by break flour yield, particle size index, and flour particle size). Small nonshriveled kernels did not have diminished total flour yield potential or other reduced flour milling characteristics. Those observations suggest a possibility of separating small sound kernels from small shriveled kernels to improve flour yield and the need to improve dockage testing estimation techniques to distinguish between small shriveled and small nonshriveled kernels.     

Classification of wheat varieties based on structural features of arabinoxylans as revealed by endoxylanase treatment of flour and grain

Arabinoxylans (AX) are cell wall polysaccharides of complex structure involved in many aspects of wheat flour end uses. The study of the variations of AX structure can lead to the identification of genes involved in their biosynthesis, and thus in the control of the various aspects of grain quality related to their presence. A method is proposed to identify AX variations directly in whole grain by enzymatic degradation. An endoxylanase from Trichoderma viride was used to extract AX from a collection of 20 wheat cultivars (Triticum aestivum L.). Enzymatic degradation products were analyzed by HPAEC and multivariate analysis techniques (principal component analysis, canonical correlation analysis, and cluster analysis) were applied to analyze chromatographic data. The method evidenced variations in the proportion of mono- and disubstitution of the xylan backbone by arabinose side chains, allowing classification of the different varieties according to the structural features of AX. A similar classification was obtained starting from flour or whole grain, indicating that the method was specific of AX from endosperm tissues. In conclusion, the method combining endoxylanase treatment of wheat grain and the analysis of degradation products, e.g., enzymatic fingerprinting, can be applied to collections of wheat cultivars, and possibly other cereals in order to establish quantitative trait loci related to the biosynthesis of AX.     

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.     

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.     

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.     

Description of a Micromill with Instrumentation for Measuring Grinding Characteristics of Wheat Grain

A new method for characterizing the grinding characteristics of wheat grain is described. A micromill was designed for this purpose and equipped with on‐line torque transducers to obtain accurate measurements of mechanical energy consumption during milling. This micromill can be used for testing the milling performance of small quantities of grain (100 g). It can distinguish between different types of wheat grain (soft wheat, hard wheat, durum wheat) on the basis of total specific energy during milling. Wheat characterization can be enhanced by taking particle sizes of the milled products into account. A milling index based on energy consumption and particle size reduction was developed to characterize wheat behavior during milling. This index had a high discriminatory potential, ranging from 100 kJ/kg for soft wheat flour to 600 kJ/kg for durum wheat flour. This micromill directly measures the grinding resistance of wheat kernels as a function of both the kernel hardness and vitreousness, contrary to standard kernel hardness measurements obtained by particle size index and near‐infrared reflectance analysis techniques that only reflect the fracture mode (fine particle reduction potential).     

Use of Aspiration and the Single Kernel Characterization System to Evaluate the Puffed and Shriveled Condition of Soft Wheat Grain

Shriveled kernels lower wheat test weight and reduce milling flour yields. Test weight is also lowered by rain-dry cycles that cause kernels to puff (exhibit, in part, loosened layers of pericarp). A numeric score was developed for degree of puffing and for degree of shriveling based on simple measurement devices. Wheat samples were evaluated for test weight and Single Kernel Characterization System (SKCS) hardness index, SKCS kernel weight, milling flour yield, and kernel density (hexane displacement). Those evaluations were performed before and after samples were air-aspirated to remove all shriveled kernels. Test weight, SKCS hardness index, and density of aspirated samples were used to develop a puffing score. Changes (resulting from aspiration) in test weight, SKCS kernel weight, and flour yield were used to develop a shriveling score. Higher puffing scores were related to elevated α-amylase activity. Puffed kernels were softer and were not associated with decreased flour yield. Puffing and shriveling scores were independent (poorly correlated), but together predicted 95% of the variation in original, nonaspirated test weight.     

Comparison of Quality Characteristics and Breadmaking Functionality of Hard Red Winter and Hard Red Spring Wheat

Various whole‐kernel, milling, flour, dough, and breadmaking quality parameters were compared between hard red winter (HRW) and hard red spring (HRS) wheat. From the 50 quality parameters evaluated, values of only nine quality characteristics were found to be similar for both classes. These were test weight, grain moisture content, kernel size, polyphenol oxidase content, average gluten index, insoluble polymeric protein (%), free nonpolar lipids, loaf volume potential, and mixograph tolerance. Some of the quality characteristics that had significantly higher levels in HRS than in HRW wheat samples included grain protein content, grain hardness, most milling and flour quality measurements, most dough physicochemical properties, and most baking characteristics. When HRW and HRS wheat samples were grouped to be within the same wheat protein content range (11.4–15.8%), the average value of many grain and breadmaking quality characteristics were similar for both wheat classes but significant differences still existed. Values that were higher for HRW wheat flour were color b*, free polar lipids content, falling number, and farinograph tolerance. Values that were higher for HRS wheat flour were geometric mean diameter, quantity of insoluble polymeric proteins and gliadins, mixograph mix time, alveograph configuration ratio, dough weight, crumb grain score, and SDS sedimentation volume. This research showed that the grain and flour quality of HRS wheat generally exceeds that of HRW wheat whether or not samples are grouped to include a similar protein content range.     

Einkorn Characterization for Bread and Cookie Production in Relation to Protein Subunit Composition

Twenty-four einkorns were evaluated for agronomic traits in Italy and in Germany in replicated plot trials. After dehulling and milling, the harvested kernels, flour protein content, sedimentation volume, falling number, carotenoid, and dry gluten content were determined. Farinograph profiles were obtained with a farinograph and baking and cookie quality were evaluated with standard microtests. Significant differences in yield potential were observed between the two locations, with a three-fold increase in Germany as compared with Italy. One of the einkorn lines (ID529) had farinograph stability and degree of softening indices better than those of the control bread wheat. All the samples analyzed for breadmaking aptitude showed some degree of stickiness, but it was possible to handle the dough during the different steps of breadmaking. On average, cookies produced with einkorn flour were larger in diameter and thinner than those produced with soft wheat flour. The composition in α-, β- and γ-gliadins and in high molecular weight glutenin subunits was similar in all the lines. In contrast, the pattern exhibited in low molecular weight glutenin subunits correlated strictly with baking quality. In particular, the lines with bands arbitrarily designated a and b showed a high breadmaking potential, while the lines lacking these bands had an ample range of variability but, on average, a much lower baking potential. Our data point to a simple genetic control of the breadmaking aptitude and indicate einkorn not only as a promising source of specialty foods but also as an ideal species for genetic investigations on wheat quality.     

Distribution of Redox Enzymes in Millstreams and Relationships to Chemical and Baking Properties of Flour

Millstream flours, bran, pollard, and germ fractions were prepared from two Australian and two New Zealand wheat cultivars using a pilot‐scale roller mill. The distribution of six redox enzymes in milling fractions and the relationship of the enzymes to baking parameters were investigated. Lipoxygenase (LOX), dehydroascorbate reductase (DAR), and protein disulfide isomerase (PDI) tended to be higher in the tail‐end fractions of break and reduction flour streams, but the highest levels were in the bran, pollard, and germ fractions. These enzymes had moderate to strong correlations with ash content of flour. These results indicated that a considerable amount of these enzymes in the tail‐end flour streams were likely to be derived from contamination with bran, aleurone, or germ components of grain. Peroxidase (POX) tended to be higher in the break flours, but polyphenol oxidase (PPO) and ascorbate oxidase (AOX) tended to be evenly distributed in the millstream flours. These three enzymes generally had poor correlations with ash and baking parameters. LOX and DAR had a negative correlation with the baking quality of bread made in the absence of ascorbic acid (AA) but a poor correlation with improvement of bread quality made with AA. The negative correlation probably reflects the high content of ash (hence trichomes), glutathione, and protein thiols in those fractions that have high LOX and DAR, and these high‐reducing‐power components and trichomes in flour may be the actual cause of poor quality bread. PDI generally had a poor correlation with bread quality in the absence of AA but a significant positive correlation with improvement in the quality of bread made with AA. It thus seems that the endogenous levels of these six enzymes were not a limiting factor in the breadmaking process, except for PDI, the levels of which may have positively influenced breadmaking in the presence of AA.     

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.