Mechanisms Leading to Excess Alpha -Amylase Activity in Wheat (Triticum aestivum, L) Grain in the U.K.

The frequency and mechanisms of four modes of alpha -amylase enzyme accumulation in U.K. wheat, retained pericarp alpha -amylase activity (RPAA), pre-maturity alpha -amylase activity (PMAA), pre-maturity sprouting (PrMS) and post-maturity sprouting (PoMS), were investigated in field and laboratory experiments. Of 56 cultivar site year combinations (four model cultivars grown at up to four sites from 1994–1997), enzyme activity was detected in 32 cases, in 23 cases sufficient to reduce Hagberg falling number (the usual industry measure of alpha -amylase) below the commercial criterion (250 s). The frequency of occurrence of different modes of enzyme accumulation was in the order PoMS>PMAA>PrMS>RPAA. Both PMAA and PrMS were more common than expected and the most usual pattern was for alpha -amylase to accumulate by several modes. Although green grains are rejected as impurities, study of grain colour in relation to pericarp alpha -amylase activity showed that the enzyme could persist in non-green grains in levels sufficient to affect the Hagberg value. Two factors thought to promote PMAA, grain drying rate and transient changes in temperature in early development, were studied in the field and controlled environment cabinets. No significant difference was found in grain drying rate between samples where PMAA was or was not identified. However, out of 19 transfers from a cool (16/10 °C) to a warm (26/20 °C) temperature regime, six led to significant increases in PMAA. No transfers after 45% grain moisture increased PMAA. PrMS occurred as early as 67% grain moisture and susceptibility usually increased with stage of development, being greatest in the grain dough stage. PrMS susceptibility varied with cultivar (in the same order as PoMS sensitivity) and was affected by environmental factors.     

Physicochemical Studies on Resistant StarchIn VitroandIn Vivo

Resistant starch (RS), producedin vitroby hydrolysis of retrograded pea starch gels and amylose gels by porcine pancreaticalpha-amylase, was characterised by X-ray diffraction, size exclusion chromatography and methylation analysis. These techniques showed that RSin vitroconsisted of semi-crystalline, mostly linear material that was present in two main molecular size subfractions (DP_n>100 andDP_n20–30) with a third, minor subfraction (DP_n≤5). The extent of retrogradation of amylose was found to be of primary importance in determining the RS content of starch. Analysis ofin vivoRS, recovered during an ileostomy study, produced results that were similar to those obtained from RSin vitro. Anin vitromodel for the structure of resistant starch is proposed.     

The Role of Dextrins in the Stickiness of Bread Crumb made from Pre-Harvest Sprouted Wheat or Flour Containing ExogenousAlpha-Amylase

Dextrins were extracted in water from bread made from pre-harvest sprouted wheat or standard flour supplemented with exogenousalpha-amylases. The dextrins were separated by gel permeation chromatography and the dextrin content (% of crumb weight) determined for different degree of polymerisation (DP) size classes; DP 1–2, DP 3–10, DP 11–50, DP 51–200 and DP >200. There were significant correlations between the dextrin content in each size class and crumb stickiness (r=0·84–0·91, 22 df ). The most significant correlation (r=0·96) was between total dextrin content and crumb stickiness. Addition of dextrins of various DP ranges from various sources to standard flour produced bread with sticky crumb. Again, the degree of stickiness was generally related to the amount of total dextrin in the crumb and not to size distribution of dextrins. In this instance, extensive enzymic hydrolysis of starch was not necessary to produce sticky crumb; the dextrins caused crumb stickiness directly. Addition of dextrins to reconstituted gluten–starch flour produced bread with unexpectedly low dextrin levels and correspondingly low stickiness scores. It is concluded that, to produce sticky crumb, high levels of dextrin of any size are necessary in the crumb; a sticky mass is produced when dextrins dissolve in the excess «free» water that is normally «bound» to starch, gluten and other insoluble components of bread crumb.     

In VitroDigestion of Wheat Microstructure with Xylanase and Cellulase fromTrichoderma reesei

Resistant starch (RS), producedin vitroby hydrolysis of retrograded pea starch gels and amylose gels by porcine pancreaticalpha-amylase, was characterised by X-ray diffraction, size exclusion chromatography and methylation analysis. These techniques showed that RSin vitroconsisted of semi-crystalline, mostly linear material that was present in two main molecular size subfractions (DP_n>100 andDP_n20–30) with a third, minor subfraction (DP_n≤5). The extent of retrogradation of amylose was found to be of primary importance in determining the RS content of starch. Analysis ofin vivoRS, recovered during an ileostomy study, produced results that were similar to those obtained from RSin vitro. Anin vitromodel for the structure of resistant starch is proposed.     

Alpha-amylase/subtilisin Inhibitor Levels in Australian Barleys

An enzyme-linked immunosorbent assay (ELISA) was used to determine the bifunctional alpha-amylase/subtilisin inhibitor (BASI) content of barley grain from 11 cultivars grown in six diverse locations in Australia. The inhibitor ranged from 119 to 254 μg/g in 57 barley samples. Genotype had a significant (P<0·05) effect on BASI content but there was no effect due to environment. Total protein varied independently of BASI and was influenced by environment and genotype. BASI content was higher (P<0·05) in malting barley than in feed barley and was correlated positively (r=0·29;P<0·05) with alpha-amylase activity in corresponding malts. The ELISA used monoclonal and polyclonal antibodies raised against purified BASI. In immunoblot analysis the monoclonal antibody showed high specificity for the inhibitor in barley and also detected the inhibitor in wheat. Low levels of inhibitor (mean 3·2 μg/g) were found in 12 Australian wheat cultivars using the ELISA developed for barley. The assay had a linear working range of 5–50 ng/mL with a detection limit of 2 ng/mL. Reproducibility between assays was good (CV=4·9%) but mean recoveries were high, ranging from 116–129% when purified inhibitor was added to barley extracts. The ELISA may have useful applications in brewing research and barley breeding programmes.     

Developmental Expression of Amylases During Barley Malting

Amylase activity and qualitative changes in amylase isoenzymes as a function of barley seedling age were investigated in 10 Brazilian barley cultivars. All cultivars showed few isoenzymes in early germination. An increase in general activity ensued in the following days when new isoenzymes were detected and those already observed since early germination had their activity increased. All cultivars disclosed increase in amylase activity until the third or fourth day of germination. Some cultivars maintained this high activity until the last day analysed. Other cultivars presented a decrease in activity in the fifth or sixth day. No electrophoretic pattern or allelomorph responsible for a higher amylase activity were detected. Beta -amylase activity was always superior to alpha -amylase activity. High beta -amylase activity was already observed on the second day of germination while alpha -amylase activity began to increase only from the third day on. The results obtained suggest that, at least for the cultivars analysed, there is a high general amylase activity around the fourth day of germination, indicating that germination could stop at this moment, ensuring that hydrolitic enzyme activity required in the brewing process is met. Beta -amylase was lightly correlated with diastatic power (r=0·565) but no correlation was observed between alpha -amylase and diastatic power (r=-0·128), or neither betweenalpha - and beta -amylase with malting quality (r=0·153 andr =−0·348, respectively). These results indicate that beta -amylase activity in barley grains, more than alpha -amylase, can be a good predictor of diastatic power.     

Comparative Studies of HighMrSubunits of Rye and Wheat. I. Isolation and Biochemical Characterisation and Effects on Gluten Extensibility

The structural features of highM_rglutenin subunits of wheat were compared with those of analogous proteins from rye. Subunits of two rye cultivars (Danko and Halo) and of the wheat cultivar Rektor were isolated from defatted flours by extraction with 50% (v/v) aqueous propan-1-ol under reducing conditions at 60°C followed by precipitation using a 60% concentration of propan-1-ol. The yields of dialysed and freeze-dried subunits were 0·33% and 0·32% (w/w of flour), respectively (rye cultivars), and 0·91% (Rektor). SDS–PAGE revealed that the rye cultivars contained at least five subunits with mobilities corresponding to the x-type subunits of wheat. Separation by RP–HPLC indicated that the rye cultivars did not differ in the qualitative composition of subunits, but in their quantitative proportions. The surface hydrophobicities of the rye subunits were significantly lower than those of wheat subunits. The amino acid compositions of single rye subunits were characterised by high contents of Glx, Gly and Pro, and they were closely related to those of wheat subunits, except that the Glx content was generally lower and the Cys content higher. Notable differences between rye and wheat subunits were found in their contributions to gluten strength. Whereas wheat subunits, reoxidised with potassium bromate and mixed with a standard wheat flour, caused a significant increase in gluten strength, reoxidised rye subunits had the opposite effect.     

Distribution of Enzymes in Wheat Flour Mill Streams

The distribution of alpha -amylase, protease, lipoxygenase, polyphenol oxidase and peroxidase in wheat roller flour mill streams was studied. Break flours had relatively less alpha -amylase and protease activity than reduction flours both on flour weight and a protein basis. Among the different flour streams, the 5^thand 6^threduction passage had the highest alpha -amylase activity, while the 4^threduction passage had the highest protease activity. The lipoxygenase activity was concentrated mostly in the last break and the reduction streams, whereas polyphenol oxidase activity was highest in break flour streams. Peroxidase activity was distributed unevenly among the different mill streams. The lipoxygenase, polyphenol oxidase and peroxidase were highly concentrated in different bran fractions. Except for protease, the other enzymes were concentrated in the «atta», a milling by-product comprising refined flour, bran and shorts; and are least active in semolina (farina).     

Differences in conformational stability of barley alpha-amylase isozymes 1 and 2. Role of charged groups and isozyme 2 specific salt-bridges

Barley alpha-amylase isozymes 1 (AMY1) and 2 (AMY2) have 80% sequence identity but possess different physico-chemical properties. By incubation in the range 37–85 °C T₅₀ is 75.2 °C of AMY1 and 79.2 °C of AMY2. While AMY2 is also most stable in urea at pH 6.7, [urea]₅₀ being 8.2 M compared to 7.9 M for AMY1, AMY1 has highest stability in urea below pH 6 or in the presence of NaCl. Moreover AMY1 is most stable in guanidinium chloride. Charge screening thus destabilises AMY2 but stabilises AMY1. Isozyme sequence comparison suggests that AMY1 lacks four of the 20 salt-bridges identified in the crystal structure of AMY2. The four residues that differ comprise Lys67_AMY2 and Asp267_AMY2, forming salt-bridges on the surface of the catalytic (β/α)₈-barrel (domain A), and Glu96_AMY2 and His344_AMY2 that participate in charged networks between domain A and the small domain B and the C-terminal domain, respectively. Four corresponding AMY2 mimics A68K; D97E; Q269D; N346H were made in AMY1 by site-directed mutagenesis. While D97E and Q269D have slightly improved stability compared to AMY1 wild-type, N346H and, under certain conditions, A68K are destabilised. The four mutants show 22–176% activity (k_cat/K_m) toward 2-chloro-4-nitrophenol β- -maltoheptaoside and amylose DP17 and 43–117% activity for insoluble starch.     

Effect of particle size on kinetics of starch digestion in milled barley and sorghum grains by porcine alpha-amylase

The influence of milled grain particle size on the kinetics of enzymatic starch digestion was examined. Two types of cereals (barley and sorghum) were ground, and the resulting grounds separated by size using sieving, with sizes ranging from 0.1 to 3 mm. In vitro enzymatic digestion was performed, using pancreatic alpha-amylase, amyloglucosidase and protease, to determine fractional-digestion rates over 24 h. The resulting glucose production rate data were well fitted by simple first-order kinetics. For each sieve screen size, the digestion rate of barley was always higher than that of sorghum. The rate coefficients for digestion showed a decrease with increasing size, and could be well fitted by an inverse square relationship. This is consistent with the supposition that starch digestion in these systems is controlled by diffusion of enzyme through the grain fragment. Apparent diffusion coefficients of alpha-amylase obtained by fitting the size dependence were 0.76 (sorghum) and 1.7 (barley) × 10⁻⁷ cm² s⁻¹, 9 (sorghum) and 4 (barley) times slower than predicted for a molecule of the size of alpha-amylase in water.     

Comparative Enzyme Kinetics of Two Allelic Forms of Barley (Hordeum vulgare L.) Beta -amylase

The barley (Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta -amylase (EC 3.2.1.2) encoded on chromosome 4H by the Bmy 1-Sd1 and Bmy 1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2Lbeta -amylases after germination. The K_mvalue for green malt beta -amylase was less than that of mature grain beta -amylase for both varieties when potato starch was used as a substrate, although V_maxwas similar. This indicated that proteolysis after germination increased the affinity of beta -amylase for potato starch. No significant kinetic differences were observed between beta -amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta -amylase. Sd1 beta -amylase from green malt exhibited a lower K_mvalue for potato starch than Sd2L beta -amylase, demonstrating that at non-saturating starch concentrations Sd1 beta -amylase is better able to hydrolyse starch than Sd2L beta -amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K_mvalues for beta -amylase increased, whereas V_maxvalues decreased. Maltose, the hydrolytic product of beta -amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta -amylases with respect to potato starch and amylose. Taken together the kinetic observations for bet a-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process.     

Hydrothermal Processing of Barley (cv. Blenheim): Optimisation of Phytate Degradation and Increase of FreeMyo-inositol

Optimal conditions were developed for hydrothermal processing of whole barley kernels (cv. Blenheim) to degrade phytate (myo-inositol hexaphosphate) and to increase the content of freemyo-inositol. The hydrothermal treatment comprised of two wet steeps, where lactic acid solution of different concentrations was used, and two dry steeps followed by successive drying. Experiments were performed as a central composite design and evaluated by multiple linear regression. The variables in the experiments were temperature in the first wet and dry steep (T₁), temperature in the second wet and dry steep (T₂) and lactic acid solution concentration in both wet steeps (C) and mathematical models were developed in these variables. Optimal conditions for maximal phytate degradation and for maximal increase of freemyo-inositol wereT₁=48 °C,T₂=48–50 °C andC=0·8%, at these conditions the amount of phytate was reduced by 95–96% and the freemyo-inositol concentration was increased from 0·56 to 2·45 μmol/g d.m. We conclude that this hydrothermal process can be used to produce a barley product (cv. Blenheim) with a low phytate content and a high level of freemyo-inositol.     

Beta-amylase degradation by serine endoproteinases from green barley malt

Proteolytic degradation of barley proteins is examined in green (unkilned) malt and germinating seeds from Hordeum vulgare L. cv. Harrington. Zymographic analysis of the Harrington green malt extracts using commercial preparations of barley beta-amylase incorporated as a proteolytic substrate in 2-D SDS gels shows multiple proteolytic activities. A developmental study shows that the several green malt beta-amylase-degrading activities appear at around day 2 of germination. The several activities appear to increase and decrease through 7 days of germination in a coordinated fashion. Gels treated with class-specific proteinase inhibitors show that serine-class proteinase activities are responsible for barley beta-amylase degradation seen on the zymograms. Western blot analysis also shows that proteolytic enzymes recovered from 1-D electrophoretic gels degrade barley beta-amylase, and that the degradation is inhibited by PMSF. This is the first demonstration that malt proteinases are capable of degrading important metabolic enzymes in germinating barley, and the first postulated physiological role for the serine class proteinases in barley malt.     

Production of Proteases by Fusarium Species Grown on Barley Grains and in Media Containing Cereal Proteins

The production of proteases by the cereal plant pathogens Fusarium culmorum, F. graminearum and F. poae was followed through seven days of cultivation. The fungi were grown in mineral and in gluten culture media, and on autoclaved barley grains. The proteolytic activities of each sample were analysed at pH 2·2, 5·0 and 8·0 and the pH optima of the most active proteases were determined. All of the fungi grown in the gluten medium produced proteases that were active at pH levels between 6 and 10 and were most active at about pH 9·0. Fusarium poae also produced acid protease(s) with pH optima between 3.0 and 3.5 when grown in the gluten medium. No protease activity was detected in the cultures that were grown in the mineral medium, except that a small amount was formed after the glucose substrate was depleted. When grown on the barley grain medium the Fusarium species produced protease activities that were similar to the neutral and alkaline ones present in the gluten cultures, but no pH 2·2 protease activity was detected. The alkaline proteases had some characteristics that were similar to those of chymotrypsin.     

Variation of Beta -Amylase Activity in Barley as Affected by Cultivar and Environment and its Relation to Protein Content and Grain Weight

The cultivar and environmental variation of beta -amylase activity was studied using two barley cultivars with contrasting growth properties. There was a significant difference in beta -amylase activity between the two cultivars used, 92-11 being significantly higher than Xiumai 3. A significant variation in beta -amylase activity was detected between grains at different positions within a spike. The two cultivars showed the same pattern, with top grains showing the highest and bottom ones the lowest activities. The relative difference within a spike varied between the cultivars, with 92-11 being larger than Xiumai 3. Both seeding rate and timing of N application dramatically affected the beta -amylase activity. With N application at the booting stage, beta -amylase activity increased, mainly due to the significantly increased beta-amylase activity in the topmost grains. The bottom grains showed a lower response to timing of N application. The variation in protein content and grain weight between cultivars and among the various treatments was also examined. The possible influence of these factors on beta -amylase activity are discussed.     

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.     

Variation in Amylase Activities in Radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) Cultivars

The radish (Raphanus sativus) is a root vegetable of the Brassicaceae family which shows amylolytic activity in the taproot. However, there is little information about differences in these amylolytic activities among radish cultivars. We analyzed the amylase activities and starch contents of 7 kinds of radish cultivars. The Koshin cultivar showed the highest amylase activity, with a level approximately 6 times higher than that of the Sobutori cultivar, which had the lowest. Cultivars with higher amylase activities showed higher starch contents. These results suggest that there are intraspecies variations in amylolytic activities in radishes, and positive correlations between amylase activity and starch content.     

Functional Properties of Low Mr Wheat Proteins. I. Isolation, Characterization and Comparison with Other Reported Low Mr wheat Proteins

A group of low M_r wheat proteins with characteristic extractability behavior was isolated using two different isolation procedures. The proteins were extractable with water, salt solution and 70% (v/v) ethanol. After water extraction of flour and separation of gluten, a substantial proportion of these proteins was still extractable from gluten using 70% (v/v) ethanol. Based in their amino acid compositions, M_rs and IEF patterns, the isolated proteins resemble closely most of the alpha -amylase/protease inhibitors described in the literature. This was confirmed by enzyme inhibition studies in which it was shown that they inhibited mammalian, but not wheat, bacterial and fungal alpha-amylases. All proteases tested were inhibited by the low M_r proteins. Their M_rs and their high cysteine contents (6·5-8·1 mol%) indicated that the proteins contain four to five disulphide bonds. Free thiol groups were not detected in the proteins. Upon reduction, the M_r increased from 7-8000 to 14-19000. Furthermore, the disulphide bonds were highly reactive as determined by their reaction with the thiol-specific label monobromobimane. This suggests that the low M_r wheat proteins may play a role in thiol group/disulphide bond exchange in wheat proteins.