Dietary fibre (DF) is a non-digestible nutrient which has important roles in the digestive system including mantaining regularity, and reducing the risk of certain cancers and non-communicable diseases, such as metabolic syndrome. Even though the positive health effects of DF have long been established, it has been shown that DF intake for children and adults in Australia is below the recommended range – less than 20% of adults met the suggested intake for reducing risk of chronic diseases(1). Plantago ovata, also known as psyllium, is widely used as DF supplement with evidence showing positive effects on weight control, hyperglycaemic response, cholesterol levels, and irritable bowel syndrome(2). P. ovata seed husk produces a highly viscous gel called mucilage when seeds are exposed to moisture. This mucilage is nearly pure DF and has an intricately layered structure which can be further fractionated and studied as a proxy for different gelling systems. Interestingly, Australia is home to many mucilage-producing Plantago species, most of which are underexplored and underutilised, but show remarkable gelling properties and hypoglycaemic potential(3). In this work, we compare structural and functional properties of fractionated DF from P. ovata, and two promising Australian native relatives, P. turrifera and P. drummondii, and their effect on enzymatic hydrolysis in potato starch gels. Using a 3-step fractionation method, we have separated distinct fractions and explored their individual properties(4). P. turrifera and P. drummondii have higher water absorbing capacity, DF yield, and viscosity compared to P. ovata. Monosaccharide composition of all three species is similar – they are highly substituted heteroxylans with minor pectic component. Notably, arabinose to xylose ratio in all species increases with further extraction steps, which is different from cereal arabinoxylans. In an attempt to explore impact of DF in starch-rich systems, we have fabricated DF-potato starch gels and measured enzymatic hydrolysis (with porcine pancreatic α-amylase), freeze-thaw stability, and colour change. Addition of DF reduced syneresis (water separation) during 15 day freeze-thaw cycle measurement, which can lead to prolonged storage stability and has positive implications for shelf life. Colour change was most noticeable when P. drumondii DF were added, while colour of P. ovata and P. turiferra DF gels was similar to control potato starch gel. Effects on α-amylase starch hydrolysis were significant as well, and depended on species and fractions. Certain DFs had impacts on constant k (speed of hydrolysis), while effects on the extent of hydrolysis are still being explored. In conclusion, utility of Australia native P. turrifera and P. drumondii DFs are evident when applied to starch gels, and should be further explored in food products such as bread to increase DF intake and possibly lower glycaemic index.

The aim of this study is to determine to what extent the addition of chitinase to black soldier fly (BSF) larval meal enriched or not with long-chain PUFA (LC-PUFA) could improve growth, protein digestion processes and gut microbial composition in Nile tilapia. Two different types of BSF meal were produced, in which larvae were reared on substrates formulated with vegetable culture substrate (VGS) or marine fish offal substrate (FOS). The BSF raised on VGS was enriched in α-linolenic acid (ALA), while that raised on FOS was enriched in ALA + EPA + DHA. Six BSF-based diets, enriched or not with chitinase, were formulated and compared with a control diet based on fishmeal and fish oil (FMFO). Two doses (D) of chitinase from Aspergillus niger (2 g and 5 g/kg feed) were added to the BSF larval diets (VGD0 and FOD0) to obtain four additional diets: VGD2, VGD5, FOD2 and FOD5. After 53 d of feeding, results showed that the BSF/FOS-based diets induced feed utilisation, protein efficiency and digestibility, as well as growth comparable to the FMFO control diet, but better than the BSF/VGS-based diets. The supplementation of chitinase to BSF/FOS increased in fish intestine the relative abundance of beneficial microbiota such as those of the Bacillaceae family. The results showed that LC-PUFA-enriched BSF meal associated with chitinase could be used as an effective alternative to fishmeal in order to improve protein digestion processes, beneficial microbiota and ultimately fish growth rate.

To alleviate the growth inhibition, and intestinal damage of Chinese mitten crab (Eriocheir sinensis) induced by low fishmeal diets (LF), an 8-week feeding trial was conducted to evaluate the addition of dietary soybean-derived bioactive peptides (SBP) in LF diets on the regulation of growth, digestion and intestinal health. The crabs were fed isonitrogenous and isoenergetic conventional diet and LF diets (10 % fishmeal replaced by soybean meal, LF) supplemented with 0, 1 %, 2 %, 4 % and 6 % SBP, respectively. The results showed that LF diet inhibited growth while inclusion of SBP quadratically remitted the growth inhibition induced by LF. For digestive function, increasing addition level of SBP quadratically improved the α-amylase and trypsin activities. For antioxidant function, LF group significantly increased the malondialdehyde content, while SBP linearly decreased the malondialdehyde level and cubically increased the anti-superoxide anion activity and total antioxidant capacity level. For intestinal health, the peritrophic membrane (PM) almost completely separated from the inner wall of the intestinal lumen, the epithelial cells reduced, the muscularis became thinner and the apoptotic signals increased in LF group; with SBP addition, the intestinal morphology was improved, with the PM adhering to the inner wall of the intestinal lumen, an increase in the number of epithelial cells and an increase in the thickness of the muscularis. Additionally, there was a decrease in apoptotic signals. Dietary SBP also increased the expression of PT and Crustin1 quadratically and decreased the expression of ALF1 linearly, ALF3 and ILF2 quadratically.

White kidney bean extract (WKBE) is a nutraceutical often advocated as an anti-obesity agent. The main proposed mechanism for these effects is alpha-amylase inhibition, thereby slowing carbohydrate digestion and absorption. Thus, it is possible that WKBE could impact the gut microbiota and modulate gut health. We investigated the effects of supplementing 20 healthy adults with WKBE for 1 week in a randomised, placebo-controlled crossover trial on the composition of the gut microbiota, gastrointestinal (GI) inflammation (faecal calprotectin), GI symptoms, and stool habits. We conducted in vitro experiments and used a gut model system to explore potential inhibition of alpha-amylase. We gained qualitative insight into participant experiences of using WKBE via focus groups. WKBE supplementation decreased the relative abundance of Bacteroidetes and increased that of Firmicutes, however, there were no significant differences in post-intervention gut microbiota measurements between the WKBE and control. There were no significant effects on GI inflammation or symptoms related to constipation, or stool consistency or frequency. Our in vitro and gut model system analyses showed no effects of WKBE on alpha-amylase activity. Our findings suggest that WKBE may modulate the gut microbiota in healthy adults, however, the underlying mechanism is unlikely due to active site inhibition of alpha-amylase.

Genetic susceptibility to late maturity alpha-amylase (LMA) in wheat (Triticum aestivum L.) results in increased alpha-amylase activity in mature grain when cool conditions occur during late grain maturation. Farmers are forced to sell wheat grain with elevated alpha-amylase at a discount because it has an increased risk of poor end-product quality. This problem can result from either LMA or preharvest sprouting, grain germination on the mother plant when rain occurs before harvest. Whereas preharvest sprouting is a well-understood problem, little is known about the risk LMA poses to North American wheat crops. To examine this, LMA susceptibility was characterized in a panel of 251 North American hard spring wheat lines, representing ten geographical areas. It appears that there is substantial LMA susceptibility in North American wheat since only 27% of the lines showed reproducible LMA resistance following cold-induction experiments. A preliminary genome-wide association study detected six significant marker-trait associations. LMA in North American wheat may result from genetic mechanisms similar to those previously observed in Australian and International Maize and Wheat Improvement Center (CIMMYT) germplasm since two of the detected QTLs, QLMA.wsu.7B and QLMA.wsu.6B, co-localized with previously reported loci. The Reduced height (Rht) loci also influenced LMA. Elevated alpha-amylase levels were significantly associated with the presence of both wild-type and tall height, rht-B1a and rht-D1a, loci in both cold-treated and untreated samples.

α-amylase was immobilized on acid-activated montmorillonite K-10 via adsorption and covalent linkage. The immobilized enzymes were characterized by X-ray diffraction (XRD), surface area measurements, 27Al nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM). Surface area measurements indicate pore blockage due to linking of the enzyme in the vicinity of the pore mouth. The XRD demonstrates intercalation of enzyme upon immobilization. The NMR studies indicate that, during adsorption, tetrahedral Al sites are involved, while covalent binding occurs exclusively on the octahedral Al sites. The SEM images depict the changed morphology of the clay surface due to immobilization. The efficiency of immobilized enzymes for starch hydrolysis was tested in a batch and a fixed-bed reactor and the performances were compared. The immobilized α-amylase showed a broad pH profile and improved stability characteristics in both reactor types when compared to the free enzyme. The effectiveness factor increased in the fixed-bed reactor, implying that diffusional restrictions to mass transfer operate in the heterogeneous reaction and the use of a fixed-bed reactor leads to a reduction in these diffusional resistances. In the continuous run, 100% initial activity was maintained for 72 h, and after 96 h, >80% activity was retained.

The diamondback moth (Plutella xylostella) and small white cabbage butterfly (Pieris rapae) are the two main serious pests of cruciferous crops (Brassicaceae) that have developed resistance to chemical control methods. In order to avoid such resistance and also the adverse effects of chemical pesticides on the environment, alternative methods have usually been suggested, including the use of plant enzyme inhibitors. Here, the inhibitory effects of proteinaceous inhibitors extracted from wheat, canola, sesame, bean and triticale were evaluated against the digestive α-amylases, larval growth, development and nutritional indecs of the diamondback moth and small white cabbage butterfly. Our results indicated that triticale and wheat extracts inhibited α-amylolytic activity in an alkaline pH, which is in accordance with the moth and butterfly gut α-amylase optimum pH. Dose-dependent inhibition of two crucifer pests by triticale and wheat was observed using spectrophotometry and gel electrophoresis. Implementation of specificity studies showed that wheat and triticale-proteinaceous extract were inactive against Chinese and purple cabbage amylase. Triticale and wheat were resistant against insects’ gut proteases. Results of the feeding bioassay indicated that triticale-proteinaceous extract could cause a significant reduction in survival and larval body mass. The results of the nutritional indecs also showed larvae of both species that fed on a Triticale proteinaceous inhibitor-treated diet had the lowest values for the efficiency of conversion of ingested food and relative growth rate. Our observations suggested that triticale shows promise for use in the management of crucifer pests.

Helicoverpa armigera is a significant agricultural pest and particularly notorious for its resistance to many types of common insecticides. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing and trigged by double-strand RNA (dsRNA), has become a widely used reverse genetics and potent tool for insect pest control. In this study, the effect of ingestion and injection delivery methods of dsRNA related two important enzyme genes, α-amylase (HaAMY48, Ha-AMY49) and juvenile hormone esterase (Ha-JHE), were examined on growth and development of H. armigera. After 24, 48, 72 and 96 h of feeding bioassay, significant down regulation was observed about; 56, 68, 78, 80.75% for HaAMY48, 60, 70, 86.5 and 96.75%, for Ha-AMY49 and 14, 27.5, 23 and 31.7% for Ha-JHE, respectively. The results for injection assay was 61.5, 71.5, 74 and 95.8% for Ha-AMY48; 70, 88, 91.5 and 97.7% for Ha-AMY49 and 22, 61, 75 and 74% for Ha-JHE after 24, 48 and 72 h of last injecting, respectively. Larvae that treated with dsRNA, fed or injected, lost more than half of their weight. 50% mortality in treated larvae was observed in the case injection bioassay with dsHa-JHE and 59% of larvae that fed of dsRNA-treated cubes survived. DsHa-AMY48 and 49 have significant mortality, but mixing of them is more effective in both bioassays. Injection bioassay has a potent inhibitory effect on α-amylase-specific activity about more than 87% in treated larvae with mix of dsHa-AMY48 and 49. Adult malformation percent was evaluated for feeding (28, 35.5 and 43% for Ha-AMY48, 49 and Ha-JHE, respectively) and injection bioassay (23, 42 and 29% for Ha-AMY48, 49 and Ha-JHE, respectively). All these finding suggest that Ha-AMY48, Ha-AMY49 and Ha-JHE can be new candidates to scheming effective dsRNAs pesticide for H. armigera control.

Polyphenol- and fibre-rich foods (PFRF) have the potential to affect postprandial glycaemic responses by reducing glucose absorption, and thus decreasing the glycaemic response of foods when consumed together. A randomised, single-blind, cross-over study was conducted on sixteen healthy volunteers to test whether PFRF could attenuate postprandial blood glucose in healthy volunteers when added to a source of carbohydrate (starch in bread). This is the first study to examine the effects of a meal comprised of components to inhibit each stage of the biochemical pathway, leading up to the appearance of glucose in the blood. The volunteers were fasted and attended four visits: two control visits (bread, water, balancing sugars) and two test visits (single and double dose of PFRF) where they consumed bread, water and PFRF. Blood samples were collected at 0 (fasted), 15, 30, 45, 60, 90, 120, 150 and 180 min after consumption. The PFRF components were tested for α-amylase and α-glucosidase inhibitory potential in vitro. Plasma glucose was lower after consumption of both doses compared with controls: lower dose, change in mean incremental areas under the glucose curves (IAUC)=−27·4 (sd 7·5) %, P<0·001; higher dose, IAUC=−49·0 (sd 15·3) %, P<0·001; insulin IAUC was also attenuated by−46·9 (sd 13·4) %, P<0·01. Consistent with this, the polyphenol components of the PFRF inhibited α-amylase (green tea, strawberry, blackberry and blackcurrant) and α-glucosidase (green tea) activities in vitro. The PFRF have a pronounced and significant lowering effect on postprandial blood glucose and insulin response in humans, due in part to inhibition of α-amylase and α-glucosidase, as well as glucose transport.

Replacing dairy components from milk replacer (MR) with vegetable products has been previously associated with decreased protein and fat digestibility in milk-fed calves resulting in lower live weight gain. In this experiment, the major carbohydrate source in MR, lactose, was partly replaced with gelatinized corn starch (GCS) to determine the effect on protein and fat digestibility in milk-fed calves. In total, 16 male Holstein-Friesian calves received either MR with lactose as the carbohydrate source (control) or 18% GCS at the expense of lactose. In the adaptation period, calves were exposed to an increasing dose of GCS for 14 weeks. The indigestible marker cobalt ethylenediaminetetraacetic acid was incorporated into the MR for calculating apparent nutrient digestibility, whereas a pulse dose of chromium (Cr) chloride was fed with the last MR meal 4 h before slaughter as an indicator of passage rates. The calves were anesthetized and exsanguinated at 30 weeks of age. The small intestine was divided in three; small intestine 1 and 2 (SI1 and SI2, respectively) and the terminal ileum (last ~100 cm of small intestine) and samples of digesta were collected. Small intestinal digesta was analysed for α-amylase, lipase and trypsin activity. Digestibility of protein was determined for SI1, SI2, ileum and total tract, whereas digestibility of fat was determined for SI1, SI2 and total tract. Apparent protein digestibility in the small intestine did not differ between treatments but was higher in control calves at total tract level. Apparent crude fat digestibility tended to be increased in SI1 and SI2 for GCS calves, but no difference was found at total tract level. Activity of α-amylase in SI2 and lipase in both SI1 and SI2 was higher in GCS calves. Activity of trypsin tended to be higher in control calves and was higher in SI1 compared with SI2. A lower recovery of Cr in SI2 and a higher recovery of Cr in the large intestine suggest an increased rate of passage for GCS calves. Including 18% of GCS in a milk replacer at the expense of lactose increased passage rate and decreased apparent total tract protein digestibility. In the small intestine, protein digestion did not decrease when feeding GCS and fat digestion even tended to increase. Overall, effects on digestion might be levelled when partially replacing lactose with GCS, because starch digestion is lower than that of lactose but fat digestion may be slightly increased when feeding GCS.

Bacillus cereus can produce α-amylase, which has important industrial production value and can endure high temperatures. The enzymatic characteristics of two α-amylases from B. cereus (B905 and B904) were studied. The results showed that both preserved their activity at 90–100°C. Their thermal stability and enzymatic activity did not depend on Ca2+. However, their protein molecular weights were obviously different. The structural genes of amy905 and amy904 were cloned successfully using the polymerase chain reaction (PCR) and expressed in Escherichia coli. The amy904 and amy905 genes were 1362 bp and 1761 bp long and their protein molecular weights were about 55 kDa and 68 kDa, respectively.

Wheat (Triticum aestivum L) seeds contain a proteinaceous α-amylase inhibitor. We estimated the amount of the inhibitor that can bind to α-amylase rather than the total amount of the inhibitor, using an ELISA method. During 48 h of imbibition, the amount of the inhibitor did not change, while α-amylase activity increased rapidly. These results suggest that α-amylase synthesized de novo does not bind to the inhibitor. To examine further the interaction between α-amylase and its inhibitor, we localized immunohistochemically both α-amylase and the inhibitor in germinating seeds. In the embryo, we observed α-amylase only in the scutellar epithelial cells at 24 h after the start of imbibition, and later in the scutellar parenchyma cells. The inhibitor, on the other hand, was present between the scutellar parenchyma cells of the embryo at 0 h and 24 h after the start of imbibition, and disappeared later. α-amylase is separated from the inhibitor in the embryo. In the endosperm, we detected α-amylase in the aleurone cells at 24 h after the start of imbibition and later in the starchy endosperm. The inhibitor was present in the aleurone cells and starchy endosperm of the dry seed. During imbibition, the inhibitor in the starchy endosperm disappeared from the area to which α-amylase was secreted. It appears that α-amylase in the starchy endosperm also does not interact with the inhibitor. The aleurone cells are the only place that α-amylase can come into contact with the inhibitor. But, early secretion of α-amylase from the aleurone cells after imbibition started, and rapid disappearance of the inhibitor in the aleurone cells suggest that the α-amylase inhibitor does not work as an effective inhibitor of the endogenous α-amylase synthesized following germination.

The biochemical properties of the digestive alpha-amylase from Tecia solanivora larvae, an important and invasive insect pest of potato (Solanum tuberosum), were studied. This insect has three major digestive α-amylases with isoelectric points 5.30, 5.70 and 5.98, respectively, which were separated using native and isoelectric focusing gels. The alpha-amylase activity has an optimum pH between 7.0 and 10.0 with a peak at pH 9.0. The enzymes are stable when heated to 50°C and were inhibited by proteinaceous inhibitors from Phaseolus coccineus (70% inhibition) and P. vulgaris cv. Radical (87% inhibition) at pH 6.0. The inhibitors present in an amaranth hybrid inhibited 80% of the activity at pH 9.0. The results show that the alpha-amylase inhibitor from amaranth seeds may be a better candidate to make genetically-modified potatoes resistant to this insect than inhibitors from common bean seeds.

The effects of methanol extract of Peganum harmala L. seeds were evaluated against Tribolium castaneum (Herbst). Larval growth was inhibited and food intake reduced when the extract was incorporated into the diet at concentrations of 2.5, 5 and 10%. All doses caused an increase in the duration of larval period and completely inhibited F1 progeny production. The main body metabolites were determined in last-instar larvae after feeding for 8 days on the diet treated with different doses. Protein and lipid contents were decreased with concentrations >2.5%. Glycogen content was reduced at a concentration of 10%. Tribolium castaneum larvae possessed two major and one minor α-amylase isoforms as determined by gel assays. Larvae fed the treated diet presented less activity of the major isoforms, while activity of the minor isoform was totally inhibited. The activity of this enzyme is not inhibited in vitro by seed extracts. Histological studies showed that P. harmala extracts caused a severe cytotoxicity on the epithelial cells of the midgut resulting in marked vacuolization of the cytoplasm and a large intercellular space. These findings suggest that seed extracts of P. harmala exert their toxic effect by severely damaging the midgut epithelial cells. This naturally occurring plant extract could be useful for control of T. castaneum.

The salivary glands of the cowpea pod-sucking bugs Anoplocnemis curvipes (Fabricius), Clavigralla tomentosicollis Stål, Clavigralla shadabi Dolling, Riptortus dentipes (Fabricius) and Mirperus jaculus (Thunberg) are described and illustrated. Extracts of the glands were assayed for the presence of proteinases, α-amylase, β-amylase and amyloglucosidase. The salivary glands consist of four-lobed principal glands and a tubular accessory gland, and they differ in shape and size among the different species except for the median lobe, which is more or less spherical. The sickle-shaped nature of the posterior lobe reported before for Riptortus linearis (Linnaeus) was also observed in R. dentipes, suggestive of a probable generic characteristic. Anoplocnemis curvipes has the biggest salivary gland, and it is followed by R. dentipes, M. jaculus, C. tomentosicollis and C. shadabi in descending order. Assays of the gland extracts revealed that the four digestive enzymes tested are present in the five coreoid species. Proteinases were preponderant (1.4–94.9 μg/ml), while amylases were detected in traces [(0.5–11.0) × 10− 3 μg/ml]. Proteinases may be largely responsible for the feeding damage caused by the coreoid species and their concentration in the glands of the different species correlates well with their known capacity to inflict pod damage and yield reduction in cowpea. Fourth instar nymphs of the different species also had higher concentrations of the digestive enzymes compared with their respective adults; and this correlates with the known ability of the nymphs to cause greater pod damage and yield reduction than adults.

Abstract In isolated embryos from dormant barley grains, synergistic effects of fusicoccin (FC) and gibberellic acid (GA3) were observed on the induction of α-amylase mRNA expression. However, no α-amylase mRNA expression could be induced by both agents in embryos from non-dormant grains. Both light- and electron-microscopy studies demonstrated that there were large numbers of starch granules present in mature embryos (mainly in scutellum) from dormant barley grains but none or almost none in embryos from non-dormant grains. Furthermore, the content of reducing sugars in embryos from dormant grains was about half of that from non-dormant grains. In contrast to GA3, FC was able to induce a strong acidification of extracellular pH (pHe). Clamping the pHe to prevent FC-induced acidification, by using 50 mM MES buffer (pH 5.6), caused an inhibition of GA3- or FC-induced α-amylase mRNA expression but did not affect the germination of embryos from dormant grains. In addition, in MES buffer, addition of FC or a combination of FC and GA3increased the germination rate of embryos isolated from dormant grains, though large numbers of starch granules were still present in these embryos. Based on these observations, the presence of starch granules and a low reducing sugar level in embryos from dormant grains is not a factor for control of grain dormancy and germination.

Cherry tomato (Lycopersicon esculentum Mill.) seeds harvested from fruits at four stages of development [2, 4, 6 and 8 weeks after flowering (WAF)] were washed (1% HCl) and germinated after 72 h of desiccation at 20°C, and 76%, 49% or 12% RH. Seed α-amylase activity was determined at each stage of development and correlated with seed germination. Desiccation at 76% and 49% RH had no significant effect on the germination of seeds at 4, 6 and 8 WAF, whereas it improved germination of seeds at 2 WAF. Low RH (12%) significantly reduced seed germination at all stages of development. There was a positive correlation (r = 0.68, P ≤ 0.05) between germination and α-amylase activity during seed development. Scanning electron microscopy also revealed a correlation between starch grain occurrence in the endosperm and α-amylase activity. Placenta water potential decreased with fruit and seed maturation, and correlated negatively with improved seed germination and α-amylase activity. Promotion of seed germination by desiccation in developing cherry tomato seeds was lost 4 weeks prior to mass maturity (80 ± 3% seed water content and –2.3 ± 0.1 MPa placenta water potential). It is proposed that the water potential differential between the placenta and the seeds influences α-amylase activity and germination behaviour during development.