Affinity Chromatography with Immobilised Endoxylanases Separates TAXI- and XIP-type Endoxylanase Inhibitors from Wheat (Triticum aestivum L.)

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.     

Starch gelatinization and amylose–lipid interactions during rice parboiling investigated by temperature resolved wide angle X-ray scattering and differential scanning calorimetry

Starch gelatinization and formation of crystalline amylose–lipid complexes during the heat/moisture treatment step in rice parboiling were studied with temperature resolved wide angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC) using flour from Puntal (24% apparent amylose) and Jacinto (12% apparent amylose) rice samples [66, 40 or 25% moisture content (mc)]. Temperature resolved WAXS showed that the crystallinity index (CI, i.e. the relative amount of A-type crystals) of non-parboiled rice flours at 66% mc, monotonically decreased between 65 °C (Puntal) and 70 °C (Jacinto) and 90 °C (both Puntal and Jacinto). These temperatures were in agreement with the respective onset and conclusion temperatures of the M₁ endotherm measured by DSC. At 40% mc, the CI decreased monotonically from 65 °C (Puntal) and 70 °C (Jacinto) until 105 °C. In DSC both M₁ and M₂ endotherms were present. The conclusion temperature of the M₂ endotherm was higher than 105 °C. At 25% mc, the CI decreased very gradually and A-type crystals were no longer present at 145 °C. Under these conditions, no DSC endotherms were detected. No type II amylose–lipid complexes were formed during heating at 66% mc. In contrast, at 40 and 25% mc, V_h-type crystals were formed from 100 and 130 °C, respectively. Non-parboiled white rice flour had a strong A-type pattern. Mildly parboiled rice had a clear A-type, with a weak V_h-type and B-type pattern. Severe parboiling resulted in partially crystalline systems with superimposed A-type, V_h-type and B-type crystals. It was concluded that the rice variety, the combination of mc and the moisture distribution in the rice kernel and the temperature during parboiling all impact the level and the types of crystals in the parboiled rice.     

Impact of Starch Gelatinization and Kernel Fissuring on the Milling Breakage Susceptibility of Parboiled Brown Rice

Parboiling, a hydrothermal treatment of paddy or brown rice, improves the texture and nutritional characteristics of cooked rice. We investigated milling breakage susceptibility of brown rice parboiled under different soaking and steaming conditions, resulting in samples with different degrees of starch gelatinization and levels of fissured grains and white bellies, that is, parboiled grains with translucent outer layers and an undesirable opaque center. The milling breakage susceptibility was 2.1% for raw rice and ranged from less than 1% up to 11.3% for parboiled rice. Parboiled samples with increased milling breakage susceptibility contained higher levels of white bellies and fissured grains. In white bellies, starch gelatinization is incomplete. Scanning electron microscopy revealed inhomogeneities in individual white bellies and fissured rice grains, indicating moisture gradients inside the grains during parboiling. Starch needs to be completely gelatinized to ensure the absence of white bellies and minimal fissured grain levels in the parboiled end product and, as a consequence, a decreased milling breakage.     

A simple and accurate method for determining wheat grain fructan content and average degree of polymerization

An improved method for the measurement of fructans in wheat grains is presented. A mild acid treatment is used for fructan hydrolysis, followed by analysis of the released glucose and fructose with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Not only the amount of fructose set free from fructans but also the released glucose can be quantified accurately, allowing determination of the average degree of polymerization of fructans (DP(av)). Application of the mild acid treatment to different grain samples demonstrated that a correction should be made for the presence of sucrose and raffinose, but not for stachyose or higher raffinose oligosaccharides. The fructan content and DP(av) of spelt flour, wheat flour, and whole wheat flour were 0.6%, 1.2%, and 1.8% of the total weight and 4, 5, and 6, respectively. Validation experiments demonstrate that the proposed quantification method is accurate and repeatable and that also the DP(av) determination is precise.     

Quantification of Wheat TAXI and XIP Type Xylanase Inhibitors: A Comparison of Analytical Techniques

To date, three different techniques are available for the quantification of TAXI and XIP type proteinaceous xylanase inhibitors in cereals. A first approach is based on the determination of the residual activities of xylanases (also referred to as endo‐1,4‐β‐d ‐xylanases, EC 3.2.1.8), which are specifically inhibited by these inhibitors, after incubation with sample containing the inhibitors. The other two techniques are immunoblotting and ELISA which are based on recognition of TAXI and XIP proteins by specific antibodies. TAXI, as well as XIP, are easily extracted by aqueous buffers. Hence, the large difference in their concentrations (2–10 fold higher for XIP than for TAXI in whole meal) is not caused by differences in extractability. The repeatabilities of the three techniques are comparable. The intra‐assay and inter‐assay coefficients of variation were 6–7 and 10–14%, respectively, which is in the range of values described for methods to quantify other compounds in plant and animal tissues. The three methods give comparable results, suggesting they have similar accuracies. The choice of the technique to be used will depend not only on the sensitivity and dynamic range needed, but also on its technical simplicity and the need for high‐throughput analysis.     

In Situ Production of Prebiotic AXOS by Hyperthermophilic Xylanase B from Thermotoga maritima in High‐Quality Bread

In situ enrichment of bread with arabinoxylan‐oligosaccharides (AXOS) through enzymic degradation of wheat flour arabinoxylan (AX) by the hyperthermophilic xylanase B from Thermotoga maritima (rXTMB) was studied. The xylanolytic activity of rXTMB during breadmaking was essentially restricted to the baking phase. This prevented problems with dough processability and bread quality that generally are associated with thorough hydrolysis of the flour AX during dough mixing and fermentation. rXTMB action did not affect loaf volume. Bread with a dry matter AXOS content of 1.5% was obtained. Further increase in bread AXOS levels was achieved by combining rXTMB with xylanases from Pseudoalteromonas haloplanktis or Bacillus subtilis. Remarkably, such a combination synergistically increased the specific bread loaf volume. Assuming an average daily consumption of 180 g of fresh bread, the bread AXOS levels suffice to provide a substantial part of the AXOS intake leading to desired physiological effects in humans.     

Amylases and bread firming – an integrated view

Despite much research, bread crumb firming during storage and amylase anti-staling properties are still ill understood. We present a coherent view on the topic based on literature, experimental data, and food polymer science-related concepts. During bread storage, the gelatinised starch (amylopectin) network, present in soft, fresh bread, is gradually transformed into an extensive, partially crystalline, permanent amylopectin network, with amylopectin crystallites acting as junction zones. This network increasingly accounts for the bulk rheological behaviour of aging bread crumb. Furthermore, as amylopectin retrogradation proceeds, moisture migration within the crumb structure occurs, and more and more water is immobilised within amylopectin crystallites. The crystalline hydrate water can no longer plasticise the different networks, which goes hand in hand with increased crumb firmness and decreased crumb resilience, due to a less flexible gluten network. The efficiency of anti-staling amylases can be related to the extent they limit the formation and the strength of the permanent amylopectin network, and the water immobilisation. Conventional alpha-amylases weaken the amylopectin network by cutting the long polymer chains connecting the crystalline regions, but have little effect on amylopectin recrystallisation. In contrast, maltogenic alpha-amylase primarily shortens the amylopectin side chains, thus hindering amylopectin recrystallisation, and the concomitant network formation and water immobilisation.