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.     

Protein Inhibitors ofAlpha-amylase in Mature and Germinating Grain of Rye (Secale cereale)

The activities of endogenous (R-type) and exogenous acting (D-type) protein inhibitors ofalpha-amylase and the activities ofalpha- and total amylase were determined in milling fractions of rye. High D-type amylase inhibitor activities were detected in the embryo (255 IU/g) and in the endosperm fraction (64·9 IU/g), low inhibitor activities were found in the aleurone layer fraction (25·9 IU/g). The highest R-typealpha-amylase inhibitor activity was found in the aleurone layer fraction (32·6 IU/g), and the lowest value in the epidermis containing fraction (5·0 IU/g). The D- and R-typealpha-amylase inhibitor activities varied with growing conditions. D-type amylase inhibitor activities were found to be high in those samples which grew under drought conditions and low in samples cultivated under wet and cool weather. Higher R-typealpha-amylase inhibitor activities were found in rye genotypes cultivated under wet conditions and lower values under dry weather. There were small variations inalpha-amylase inhibitor activities between sprout-stable and sprout-sensitive rye genotypes. The D- and R-typealpha-amylase inhibitor activities of all varieties were stable during 72 h of germination. Similar soil conditions will therefore lead to differentialalpha-amylase inhibitor activities depending on weather conditions during growth.     

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.     

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.     

水稻抗性淀粉突变体抗性淀粉结构的比较研究

利用凝胶渗透色谱法和毛细管电泳法对2个富含抗性淀粉的水稻突变体RS111、AE及其野生型亲本R7954、IR36的抗性淀粉组成结构进行了比较研究，分析了2个突变体与野生型亲本抗性淀粉中直链淀粉和支链淀粉的差异。 RS111的抗性淀粉主要来源于直链淀粉，支链淀粉所占比例很小，直链淀粉与支链淀粉的比率相对于野生型亲本来说大大提高，从抗性淀粉中支链淀粉的链长分布来看，RS111与R7954比较一致，只是RS111中聚合度为10的短链比例明显小于亲本R7954；而另一突变体AE的抗性淀粉来源中，支链淀粉所占比例很大，直链淀粉与支链淀粉的比率相对于野生型亲本来说变化不大，支链淀粉中聚合度为10左右的短链比例大大高于亲本IR36，而聚合度为16、21左右的中长链比例远远低于亲本。     

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.     

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.     

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.     

玉米子粒和果穗离体培养技术及其应用研究进展

本文对玉米籽粒离体培养技术及其在子粒发育生理研究中的应用进行了评述；并提出了果穗离体培养方法．     

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.     

禾生指葡孢霉生物学特性及室内药剂筛选

为了给青稞和燕麦鞘腐病的科学防治提供技术支持,对其病原菌-禾生指葡孢霉的生长温度、适宜培养基种类、适宜pH值、存活时间等生物学特性进行了研究,并选取麦类作物上常用的8种杀菌剂进行了室内药剂筛选。结果表明,禾生指葡孢霉在5~30℃下可生长,适宜生长温度为20~25℃;在PSA、PDA、CA、CMA和OMA培养基平板上均可生长、产孢,适宜病菌生长、产孢的培养基为PDA和PSA;在pH值为4~10的PDA平板上均可生长、产孢,适宜生长的pH值为6~10;在5~10℃冷藏条件下,禾生指葡孢霉存活时间超过5a。在试验浓度(推荐剂量)下,8种杀菌剂对禾生指葡孢霉菌丝生长均具有不同程度的抑制效果,其中50%多菌灵可湿性粉剂(500×)、70%代森锰锌可湿性粉剂(500×)、430g·L-1戊唑醇悬浮剂(5 000×,8 000×)和400g·L-1氟硅唑乳油(5 000×,10 000×)的抑菌效果达100%。     

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).     

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.     

Physico-chemical Characteristics as Indicators of Starch Availability from Milled Rice

The hypothesis was tested that certain physico-chemical characteristics might be used as indicators of total starch availability and rate of starch availability of milled rice. Milled unparboiled (uPB) and parboiled (PB) rice samples (n=93) were characterized using standardized methods of physical tests and chemical analyses and anin vitromethod was used for measuring the rate of starch digestion on a subsample of rice (n=26). The rice varieties were dominated by medium long, bold rice grain with high amylose rice and intermediate gelatinization temperature (GT), but a wide range in all characteristics was measured. Small amounts of resistant starch (RS) were measured in the cooked rice, indicating virtually complete starch availability. The RS of PB rice (0·4 g/100 g rice as eaten) was significantly (P<0·004) higher than the RS of uPB rice (0·1 g/100 g) however. The rate of starch digestion was significantly affected by both variety and parboiling. The starch digestion index (SDI) values of the PB samples (mean value 73·7) were significantly (P<0·001) lower than those of the uPB samples (mean value 79·0). The apparent amylose content (AC) was the strongest determinant for SDI in both uPB and PB rice. The widths and shapes of the raw grains and the elongation after cooking were correlated significantly with SDI values for the uPB rice, while the relatively mild parboiling procedure followed in this study eliminated this correlation. The minimum cooking times were correlated significantly with the SDI values in the uPB samples.     

CerealBeta-Amylases

Cerealbeta-amylases are perhaps best known in terms of the vital role they play in releasing easily fermentable sugars from cereal grain starch to fuel the production of alcohol by yeast in brewing. The extent to which they have been investigated is indeed largely due to their significance in this economically important industry. However, cerealbeta-amylases are also, or could be, employed in many other aspects of the food industry and the analysis of starch, and they constitute valuable markers in cereal assessment and breeding studies. Quite apart from their practical significance, they are rewarding objects of biochemical and physiological research. They are interesting models for the study of enzyme polymorphism, post-translational modification and the differential expression of isoenzymes. In spite of their often high activitiesin situand all that is known about their generation, they are an enigma in that their physiological function, or even necessity, remains unclear. It has been recently recognised that there are two different categories of cerealbeta-amylases which exhibit different tissue and taxonomic specificities and physiological developmental patterns. The «classical»beta-amylases present at high activities in cereal seeds appear to be limited to the endosperm of the species of the Triticeae tribe of the Festucoideae subfamily of the Gramineae (wheat, barley and rye), whereas all cereals exhibit a different, tissue-«ubiquitous» form of the enzyme which is present at much lower activity levels. The physiological phenomenology and the usage of cerealbeta-amylases are discussed in relation to these two categories of enzyme.     

农杆菌介导法将抗草甘膦基因转入玉米自交系

利用农杆菌介导法将抗草甘膦基因(ssu-G₂-aroA)转入优良玉米自交系R18-599和齐319,侵染后的愈伤组织转至潮霉素浓度为10 mg/L的筛选培养基上,继代筛选3轮,每轮3周;得到的抗性愈伤组织在含有潮霉素浓度为3 mg/L的分化培养基上进行分化,R18-599获得30株再生植株,齐319获得18株再生植株。经PCR鉴定后共得到7株转化抗性植株(4株R18-599、3株齐319)。经PCR-Southern及RT-PCR检测结果表明,ssu-G₂-aroA基因已稳定整合到了玉米基因组中,并在RNA水平上得到表达。     

Properties of Thai cooked rice and noodles differing in glycemic index in noninsulin-dependent diabetics

Glycemic index responses of two cooked rices and six types of cooked noodles consumed by eight noninsulin-dependent diabetics correlated positively within vitro starch digestibility of food slurry and negatively with amylose content of the food. Glutinous (waxy) rice had the highest values, and mung bean noodles the lowest.     

The Null-4A Allele at the Waxy Locus in Durum Wheat affects Pasta Cooking Quality

Granule-bound starch synthase, also known as the waxy protein catalyses the synthesis of amylose in wheat endosperm starch. In durum wheats, the genes encoding GBSS are present at the two Wx loci on chromosome 7A and 4A (a segment of 7B that has been translocated). Several null Wx-B1 (missing GBSS protein from chromosome 4A) durum lines were produced from crosses with null-4A bread wheats backcrossed to durum wheats. Semolina milled from 4 normal and 7 null-4A durum wheat lines grown over two seasons (1999 and 2000) in South Australia were analysed for amylose content, starch pasting properties as measured by the Rapid Viscoanalyzer (RVA), swelling power and starch damage, protein content and electrophoretic protein analysis. Spaghetti was prepared with a micro-scale extruder and the cooked pasta evaluated for cooking loss, firmness, stickiness and water absorption. The null-4A lines had significantly lower (ca. 5%) amylose content, higher starch peak viscosities and semolina swelling power. The pasta derived from the null-4A lines had lower cooking loss and in 1999 was more adhesive than the non-waxy lines. Cooking loss was correlated with amylose content, peak starch viscosity, swelling power of semolina and cooked pasta adhesiveness. Semolina swelling power was highly correlated with RVA peak viscosity. Waxy durum wheats appear to have an advantage over the normal types in terms of lower cooking loss, widely used as an indicator of pasta cooking quality.     

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.