Germination of Wheat Grains at Various Temperatures in Relation to the Activities of α-Amylase and Endoprotease

Abstract

Germination percentages of wheat grains sampled at 3 grain-filling stages : yellow-ripe stage (water content 45-50%), dough-ripe stage (35-40%), and full-ripe stage (25-30%), and imbibed in water at 12°C and 20°C were examined in relation to the activities of α-amylase and endoprotease. Wheat varieties studied were Chihoku-komugi, which is susceptible to pre-harvest sprouting, and Satanta, which is resistant. Germination percentage was higher at 12°C than at 20°C in all grains sampled at all stages in both varieties, and was higher in Chihoku-komugi than in Satanta at 20°C. The activity of α-amylase in the grains at the yellow-ripe stage was higher at 12°C than at 20°C in both varieties, but that at the other 2 stages was higher only in Satanta. Endoprotease increased rapidly from 7 to 10 days after the start of imbibition, and exceeded 12 units only at 12°C in Chihoku-komugi grains at the dough and full-ripe stages. The results showed that α-amylase activity was lower than the value equivalent to 300 brabender unit (BU) in amylography when the germination percentage was 0%. Endoprotease activity exceeded 6 units when the germination percentage exceeded 90%.     

Some Characteristics of β-D-Xylopyranosidases, α-L-Arabinofuranosidases and an Arabinoxylan α-L-arabinofuranohydrolase from Wheat Bran and Germinated Wheat

Enzymes from wheat bran and germinated wheat involved in the degradation of arabinoxylan and arabinoxylooligosaccharides were investigated. Fourp-nitrophenyl-α-l-arabinofuranoside hydrolysing activities (ArafI-IV) and threep-nitrophenyl-β-d-xylopyranoside hydrolysing activities (XylpI-III) were identified in wheat kernels and germinating wheat. Two of these activities, ArafI and XylpII, were purified about 10 000-fold from wheat bran. Both enzymes were inactive towards polymeric arabinoxylan. ArafI produced no arabinose but some xylose from arabinoxylooligosaccharides, while XylpII gave only xylose upon incubation with this substrate. An arabinoxylan arabinofuranohydrolase (AXH) was found in wheat bran and germinated wheat. This enzyme was active towards the polymeric substrate, but was unable to hydrolysep-nitrophenyl-α-l-arabinofuranoside. TheM_rs of these enzymes were determined by size exclusion chromatography and were in the range of 40–50 000, except for ArafIII, for which aM_rof 104 000 was determined. During germination, the levels of these enzymes increased markedly between the third and fifth day, after which some of them decreased again by the seventh day. ArafI-IV were inhibited strongly by arabinonic acid-γ-lactone, while xylonic acid-γ-lactone was a good inhibitor of XylpI-III. The latter lactone also inhibited ArafI. Neither of these lactones inhibited AXH. Endoxylanase activity was demonstrated but not quantified.     

Inhibition of Key Enzymes Linked to Obesity by Preparations From Mediterranean Dietary Plants: Effects on α-Amylase and Pancreatic Lipase Activities

One of the most important strategy in the treatment of obesity includes the development of nutrient digestion and absorption inhibitors. Inhibition of digestive enzymes is one of the most widely studied mechanisms used to determine the potential efficacy of natural products as hypolipidemic and hypoglycaemic agents. In vitro studies here reported were performed to evaluate the inhibitory activity of five species (as hydroalcoholic extracts) of edible plants from Calabria region (Italy) on amylase and lipase by monitoring the hydrolysis of p-NPC and the hydrolysis of glycoside bonds in digestible carbohydrate foods. The formulation obtained from Clematis vitalba L. exhibited the strongest inhibitory effect on pancreatic lipase (IC₅₀?=?0.99 mg/ml) and on α-amylase (IC₅₀?=?31.52 μg/ml). In order to explore metabolome production HPTLC analysis of the extracts was performed, revealing the predominance of (±)-catechin, caffeic acid and chlorogenic acid in C. vitalba formulation at concentration of 23.18?±?3.14, 13.63?±?0.65 and 18.88?±?0.76 mg/g, respectively. GC/MS analysis was used to identify fatty acids and terpene composition.     

Elongation and insolubilisation of α-glucans by the action of Neisseria polysaccharea amylosucrase

Amylosucrase (E.C. 2.4.1.4) from Neisseria polysaccharea catalyses a transglycosylation reaction using sucrose as a glucose donor to synthesise an insoluble (1→4)-α-glucan, releasing fructose in the solution. If glycogen is used as a glucosyl unit acceptor, amylosucrase elongates polymer branches from their non-reducing ends. Testing of a large series of acceptors with various molar masses, glycosidic linkages, branching degrees and solubilities showed that chain elongation occurred only on polymers with (1→4)-α- or (1→4)-α- and (1→6)-α- linkages. Synthesis yields were better for completely soluble polymers (1.45 glucosyl units grafted per glucosyl unit for polymers with (1→4)-α- and (1→6)-α- linkages), than for partially soluble polymers (0.27–0.97 glucosyl units grafted per glucosyl unit for polymers with either (1→4)-α- linkages or (1→4)-α- and (1→6)-α- linkages). Elongation occurred randomly at the non-reducing ends of some external chains. Elongation of soluble branched molecules led to rapid gel formation favourable to gelation control inside suspensions and solutions. These polymers showed resistant starch contents (22% (w/w) to 57%) after elongation with amylosucrase.     

Impact of α-Amylase During Breadmaking on In Vitro Kinetics of Starch Hydrolysis and Glycaemic Index of Enriched Bread with Bran

Nowadays, the use of enzymes has become a common practice in the bakery industry, as they can improve dough quality and texture of final product. However, the use of α-amylases could have a negative effect in the glycaemic load of product, due to the released sugars from the starch hydrolysis that are not used by yeasts during the fermentation process. This study evaluated the effect of the addition of α-amylase in bakery products with bran on in vitro kinetics of starch hydrolysis. The use of flour with a high degree of extraction or high bran amount could decrease the GI even with the inclusion of α-amylase in the formulation. It should be taken into account the amount of bran and α-amylase when formulating breads in order to obtain products with lower GI than white bread. However, the fact that kinetics of starch hydrolysis remained unaltered indicates that the use of α-amylase in bread-making processes could provide technological advantages improving quality of breads without markedly changes in their glycaemic index.     

Content of starch and sugars and in vitro digestion of starch by α-amylase in five minor millets

Five varieties of minor millets were studied for their amylose, soluble amylose, amylopectin, soluble amylopectin, reducing sugar, total sugar and starch contents. Pure starch was isolated from each variety and the enzymic degradation of starch by porcine pancreatic -amylase were examined with and without gelatinisation. Gelatinised sample ofEchinochloa frumentacea (var. K2) showed minimal hydrolysis and gelatinised sample ofPanicum miliaceum (var. CO3) showed maximum hydrolysis of starch by porcine pancreatic -amylase. Gelatinised starch was highly susceptible to enzymic digestion when compared to ungelatinised starch. The extent of starch degradation varied from 71 to 85 percent in gelatinised samples and starch degradation in ungelatinised sample varied from 10 to 18 percent.     

Effect of Betulin-containing Extract from Birch Tree Bark on α-Amylase Activity In vitro and on Weight Gain of Broiler Chickens In vivo

In vitro effect of betulin-containing extract from Betula pendula Roth. bark on alpha-amylase activity was studied, the kinetic mechanism of interaction was proposed and in vivo effect of betulin-containing extract on weight gain and meat quality of broiler chickens was evaluated. The highest level of inhibitory activity (20 %) was detected in extract concentration of 1,000 mg/L. Increased extract concentration did not lead to increased enzyme inhibition. Using Dixon and Cornish-Bowden coordinates, the competitive mechanism of inhibition was demonstrated. Calculated kinetic parameters were: K_m equal to 0.6 mg/mL, V_max equal to 2.6 and 2.1 mM/min from Lineweaver-Burk and Dixon coordinates, respectively and K_i equal to 3,670?±?230 mg/mL. The partial inhibition of enzyme indicates the existence of low concentration of active inhibitory form, which reaches saturation level with increased extract concentration in applied suspension. Therefore, K_i has an apparent constant character. This partial inhibition of amylase activity observed in in vitro assay did not affect weight gain and meat quality of broiler chickens during in vivo assay. Rather, the tendency to increase the weight of edible parts and muscles compared to diet without additive suggests that the extract may be a potential food additive in poultry farming. Additionally, it could be a source for further pharmaceutical and pharmacological research.     

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH₂), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 3₁₀-helix from residues Pro⁷ to His¹⁰ and a type I β-turn from residues Pro¹⁴ to Cys¹⁷. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC₅₀ of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu² by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser¹² with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.     

Prediction of the Physicochemical and Nutraceutical Characteristics of ‘Hass’ Avocado Seeds by Correlating the Physicochemical Avocado Fruit Properties According to Their Ripening State

Plant Foods for Human Nutrition - Vegetal wastes are currently a source of pollution due to the excess of organic compounds in the environment. Seeds are the main by-product of the avocado industry...     

Biological and chemical evaluation of chick pea seed proteins as affected by germination,extraction and α-amylase treatment

The effects of germination, extraction (double extraction with 70% ethanol and water at isoelectric point) and -amylase treatments of chick pea seed flours on crude protein, total carbohydrate, protein efficiency ratio (PER), biological value (BV), true digestibility (TD), net protein utilization (NPU), essential amino acid composition, in-vitro protein digestibility (IVPD) and actual amino acid indices (essential amino acid index or amino acid score) were evaluated. Crude protein content was increased (8–149%), while total carbohydrate was decreased (11–62%) by germination, extraction and -amylase treatments. Alpha-amylase treatment was more efficient in reducing total carbohydrate and increasing the protein content than that of extraction treatment. The protein quality of chick pea flours as measured by PER, BV, TD, NPU, IVPD and corrected amino acid indices (actual amino acid indices×IVPD) was significantly improved by these treatments. The protein quality of germinated--amylase treatment was comparble with casein, while germinated--amylase treaded seeds appeared nutritionally superior to casein. The results indicate that the germinated--amylase and germinated--amylase-extracted treatments could be used successfully as a source of concentrated high quality protein for baby food production. The corrected amino acid indices gave better prediction of PER, BV, TD and NPU (r=93 to 97) than actual amino acid indices (r=45 to 71). PER was highly correlated with corrected amino acid score (r=0.93). The PER could be predicted from the following simple regression equation: PER=–1.827+0.0561×corrected amino acid score.     

Inhibition study of red rice polyphenols on pancreatic α-amylase activity by kinetic analysis and molecular docking

This study sought to investigate the possible inhibition mechanism of red rice polyphenols (RRP) on pancreatic α-amylase (PA) activity. RRP showed strong inhibition against PA activity and the half-inhibitory concentration (IC₅₀) value was 3.61 μg/mL. The fluorescence quenching of PA by RRP was a combination of static quenching and dynamic quenching. RRP could aggregate with PA and the physiochemical properties of the aggregates were closely related to the concentration of RRP. Kinetic analysis suggested that the inhibition mode of RRP on PA was reversible inhibition, which was a mixing of competitive inhibition and noncompetitive inhibition. Molecular docking speculated that RRP could form hydrogen bonds with PA by binding to the catalytic active sites (ASP197, GLU233 and ASP300) and the microenvironments of TRP58 and TRP59 were altered, thus inhibiting PA activity.     

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23°C and 3°C. II. Evaluation of protein patterns

Summary The changes in polypeptide profiles (2D-PAGE) occurring in the soluble and microsomal fractions of parenchymatic tissue of potato (Solanum tuberosum L.) tubers were studied during the last 30 d of maturation and during storage at 23°C and 3°C. The major changes were observed in the last period of tuber maturation, when several polypeptides disappeared and new ones appeared. At both 23°C and 3°C specific polypeptides disappeared in dormant tubers and new polypeptides appeared during storage. At 3°C specific changes in protein composition occurred, particularly in the microsomal fraction. The changes in polypeptide profiles are discussed in relation to the transition from “sink” to “source” of the tuber, the onset of dormancy and of sprouting ability and the activation of cold acclimation responses. The results are also discussed on the basis of the physiological and biochemical changes that occur in the parenchymatic tissue.     

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23°C and 3°C. I. Biochemical and physiological parameters

Summary In the last 30 d of potato (Solanum tuberosum L.) tuber growth metabolic activity decreased. Levels of glucose-6-P and sucrose in whole tuber tissues declined and in tuber slices there was a decrease in the uptake from the medium and in the incorporation into macromolecules of [U-¹⁴C]sucrose. During storage at 23°C only the uptake of [U-¹⁴C]sucrose increased concomitant with tuber sprouting, indicating a possible involvement of the transport mechanisms in dormancy breaking. At 3°C, levels of reducing sugars and sucrose increased in response to the low temperature and increased release of K⁺ and malondialdehyde levels indicated cell membrane damage. The cell membrane functionality was restored at sprouting. The sprouting potential of the tubers was evaluated using the sprouting ability of single-bud explants (“seedcores”) in response to water, GA₃ or ABA dips. This sprouting potential of tubers changed with stage of tuber growth and storage duration and temperature, indicating that the tissue hormonal state changed strongly throughout tuber life, probably in relation with the “sink” to “source” transition.     

Synthesis of poly(norbornene ester)s by using (<Emphasis Type="Italic">η</Emphasis><Superscript>3</Superscript>-substituted allyl) palladium (NHC) complex as catalyst and investigation of their chemical structure - Glass transition temperature - Refractive index relationships

The synthesis of poly(norbornene ester)s by using a (η ³-substituted allyl) palladium (N-heterocyclic carbene (NHC)) complex as catalyst was performed and the relationship between chemical structure and glass transition temperature or refractive index of poly(norbornene ester)s was investigated. Norbornene ester monomers were synthesized via esterification of 5-norbornene-2-methyl alcohol and aromatic carboxylic acids. The polymerization catalyst, (η ³-substituted allyl) palladium (NHC) complex, was synthesized according to a published procedure. ¹H-NMR spectroscopy was performed to determine chemical structure of monomers and polymers. The molecular weight of the polymers was measured via gel permeation chromatography and the thermal properties were analyzed via thermogravimetric analysis and dynamic mechanical analysis. Refractive indices of polymer films were measured using a prism coupler. Polymers with the highest M _n (between 100 kg/mol and 300 kg/mol) were synthesized when the ratio of monomer to catalyst was 2000:1. The glass transition temperature of synthesized polymers was about 100 °C lower than that of conventional norbornene polymers. Among the six polymers of different chemical structures, four polymers exhibited a refractive index of 1.6 or more at a wavelength in the visible light region.