Effect of Phytate and Other Myo-Inositol Phosphate Esters on Lipase Activity

The effects of phytate and other myoinositol phosphate esters (containing one or more phosphate groups) on lipase activity were evaluated by an in vitro procedure. Porcine pancreatic lipase was treated with four concentrations of phytate (0–4 mM) and myo-inositol-2-monophosphate (0–12 mM). The lipase was also treated with six myoinositol phosphate ester fractions (4 mM) isolated from hydrolyzed phytate. The enzyme was incubated (0.5 hr) with the esters prior to addition of substrate and measurement of fatty acid production. Phytate at 4 mM and 1-2-MP at 12 mM caused significant inhibition of lipase, 14.5% and 8.2%, respectively. The isolated inositol phosphate ester fractions caused significant decreases in lipase activity. The decrease in enzymatic activity was highly correlated (r = 0.980) with degree of inositol phosphorylation.     

Raw Starch Digestion by α-Amylase and Glucoamylase from Chalara paradoxa

Glucoamylase and α-amylase of Chalara paradoxa were separated by hydrophobic column chromatography using butyl-Toyopearl 650M. The α-amylase showed the highest activity at pH 5.5 and 45°C, and was stable in the pH range of 5.5–6.5 and at temperatures lower than 40°C. The glucoamylase showed the highest activity at pH 5.0 and 45°C, and was stable in the pH range of 4.0–7.5 and at temperatures lower than 45°C. The molecular mass of the α-amylase and glucoamylase estimated by SDS polyacrylamide gel electrophoresis was 80,000 and 68,000, respectively. Both glucoamylase and α-amylase could digest more effectively raw rice starch and raw corn starch than raw sago starch and raw potato starch. 2% raw rice starch in 10 ml solution was digested by more than 90% by 100 units of each amylase. When these amylases were used combined, raw corn starch was more effectively digested than they were used singly. This cooperative action in raw corn starch digestion was also observed when. C. paradoxa α-amylase and R. niveus glucoamylase were combined.     

Sweetpotato α- and β-Amylases: Characterization and Kinetic Studies with Endogenous Inhibitors

Sweetpotato α- and β-amylases were characterized to assist optimization of direct hydrolysis of starch by endogenous amylases. In kinetic studies purified starch was substrate, and ascorbate, oxalate, phenolics, phytate and sweetpotato extracts were assayed for inhibitory activity. α-Amylase had optimum pH between 5.8 and 6.4 and was stable from pH 5.0 to 9.0. Optimum activity occurred at 71.5°, but it was inactivated by heat in the absence of Ca²⁺ at > 63°. The Km for soluble starch was 2.08 mg/mL. The molecular weight was 45000 daltons. α-Amylase activity was reduced up to 70% by 0.2 mM K-ascorbate and moderately by Na-oxalate and Na-phytate. β-Amylase had optimum pH between 5.3 and 5.8, and was stable from pH 4.0 to 8.0. Its maximum activity was at 53° and it was inactivated at 60°. Km for soluble starch was 3.71 mg/mL. At 0.08 mM, K-ascorbate strongly inhibited β-amylase activity.     

Physicochemical properties, α-amylase and α-glucosidase inhibitory effects of the polysaccharide from leaves of Morus alba L. under simulated gastro-intestinal digestion and its fermentation capability in vitro by human gut microbiota

The investigation aimed at determining the impact of sequential simulated digestion on the physicochemical properties and digestive enzymes inhibitory effects of the polysaccharides fraction (MLP-2) of Morus alba L. leaves as well as its in vitro fermentation behaviours. After artificial salivary, gastric and intestinal digestions, the chemical components and microstructure of MLP-2 were altered with significantly (P < 0.05) decreased molecular weight. The α-amylase and α-glucosidase inhibitory activities of MLP-2 were significantly (P < 0.05) improved throughout simulated digestion. MLP-2I, the intestinal digested fraction of MLP-2, could significantly (P < 0.05) decrease the pH value of fermented culture and increase the short-chain fatty acids (SCFA) concentrations, especially acetic, propionic and butyric acids. In conclusion, MLP-2 could be gradually degraded under simulated digestion with altered physicochemical properties and enhanced α-amylase and α-glucosidase inhibitory effects, and further utilised by human gut microbiota to decrease pH value and promote SCFA production.     

Screening of α-Amylase Suitable for Evaluating the Degree of Starch Retrogradation

To estimate the degrees of starch retrogradation in the complex foods, an enzymatic method using α-amylase from Bacillus subtilis was proposed in the previous report (Tsuge, H. et al.: Starch/Stärke 42 (1990), 213–216). However, actual digestibility of the enzyme for the native starch granules was not checked at that time. A comparative study to see the digestibility of native starch granules was carried out using four different α-amylase preparations and digestion of retrograded wheat starch was tested by two α-amylase preparations. Pancreas α-amylase preparation digested some native starch granules to a great extent, while Aspergillus oryzae enzyme did not digest native starch granules virtually. In conclusion, α-amylase preparation from A. oryzae was an ideal enzyme as the tool to distinguish between raw and gelatinized starches. It was justified for the use of A. oryzae enzyme as well as B. subtilis α-amylase to evaluate the retrograded starch contents in the complex foods.     

Biochemical changes in phosphorus compounds and in the activity of phytase and α-amylase in the rice (Oryza sativa) grain during germination

Changes in the phytic acid, inorganic phosphorus and ATP contents, and in the activity of phytase and α-amylase in rice (Oryza sativa L) grains were determined during 18 days of germination in a dark room. The effect of phytic acid on α-amylase activity was studied in vitro. Rice grains immersed in sterilised deionised water at 14^°C germinated on the fifth day. Phytase activity, detected in the ripening rice grains, increased linearly until the eighth day and reached a maximum on the tenth day. There was a marked decrease in phytate and an increase in inorganic phosphorus accompanying germination. There was a good inverse correlation between the levels of both phytase activity and inorganic phosphorus, and phytate breakdown. α-Amylase activity was detected on the fourth day and increased markedly from the 12th to the 16th day of germination. ATP level increased from the second to the fourth day and slightly decreased from the fourth to the eighth day; it increased rapidly again from the eighth to the 18th day of germination. α-Amylase activity was influenced by both pH and phytic acid concentration in the assay system. At 75 mM phytic acid, α-amylase activity was lowered by 23%, 93% and 52% at pH 4–0, 5–0 and 6–0 respectively. When the enzyme, phytate and Ca²⁺ were incubated together at pH 5–0, the inhibition of α-amylase by phytic acid was markedly decreased by addition of Ca²⁺. The chemical affinity of Ca²⁺ for phytic acid was higher in the reaction at pH 5–0 than in those at pH 4–0 and pH 6–0, and over 98% of Ca²⁺ in the reaction system was precipitated as Ca-phytate.     

A Study of Native and Chemically Modified Potato Starch. Part I: Analysis and Enzymic Availability in vitro

The availability of native and chemically modified potato starch to α-amylase was studied in vitro (distarch phosphate, acetylated distarch phosphate and hydroxypropyl distarch phosphate). Enzyme availability was also related to results obtained during analysis of starch and dietary fibre, The degree of substitution (DS) was determined by ¹HNMR spectroscopy and the substitution sites were evaluated by gasliquid chromatography and mass spectrometry. Substitution with acetyl- and hydroxypropyl groups reduced the availability of drum-dried starch derivatives to α-amylase. In contrast, the susceptibility of raw starch increased after introduction of hydroxypropyl groups. Cross-linking with phosphate only had but minor effects on enzyme availability. The recovery of starch during analysis depended on (a) degree of hydrolysis to glucose (b) whether or not the substitution groups were removed. The lowest yields were observed when analyzing substituted derivatives after boiling and enzymic hydrolysis (60-70%). Enzymically unavailable starch was not recovered as dietary fibre when using an enzymic gravimetric assay.     

Peniophora lycii phytase is stabile and degrades phytate and solubilises minerals in vitro during simulation of gastrointestinal digestion in the pig

BACKGROUND: Microbial phytases (EC 3.1.3) are widely used in diets for monogastric animals to hydrolyse phytate present in the feed and thereby increase phosphorus and mineral availability. Previous work has shown that phytate solubility is strongly affected by calcium in the feed and by pH in the gastrointestinal (GI) tract, which may have an effect on phytase efficacy. An in vitro model simulating the GI tract of pigs was used to study the survival of Peniophora lycii phytase and the effect of the phytase on phytate degradation, inositol phosphate formation and mineral solubilisation during in vitro digestion of a 30:70 soybean meal/maize meal blend with different calcium levels. RESULTS: The phytase retained 76 and 80% of its initial activity throughout the gastric in vitro digestion. Total phytate hydrolysis by P. lycii phytase was in the same range at total calcium levels of 1.2 and 6.2 mg g^?1 dry matter (DM), despite very large differences in phytate solubility at these calcium levels. However, at 11.2 and 21.2 mg Ca g^?1 DM, phytate hydrolysis was significantly lower. The amount of soluble mineral was generally increased by P. lycii phytase. CONCLUSION: Stability of P. lycii phytase during gastric digestion was not found to be critical for phytate hydrolysis. Furthermore, original phytate solubility was not an absolute requirement for phytate degradation; phytate solubility seemed to be in a steady state, allowing insoluble phytate to solubilise as soluble phytate was degraded. This is new and interesting knowledge that adds to the current understanding of phytate–phytase interaction. Copyright © 2007 Society of Chemical Industry     

In vitro and in vivo Digestion of High-Amylose Type Starch Granules

In vitro and in vivo digestion of high-amylose type starch granules from sugary-2 (su₂), amylose-extender (ae), and dull (du) maize endosperms by salivary and pancreatic α-amylase of rats were investigated. From the results of in vitro starch granule digestion by both α-amylases, we found a strong correlation between the starch-granule digestibility and the wavelength at the maximum absorbance λ_max, nm of the absorption spectra of starch-iodine complexes. While the digestibility of the starch granules, which were isolated from the stomach, small intestine, and cecum of rats fed on diets containing either normal, su₂, or ae starch-granules, during passage through the mouth to the stomach of rats was calculated by using the regression equation obtained from the in vitro trials. We calculated 32–40,47–68, and 7–15% degradation of starch-granules from normal, su₂, and ae endosperms, respectively. The relative order of digestibility of starch-granules in vivo was su₂normalae, respectively, which agrees with the previous in vitro studies.     

Inhibition of phenolics on the in vitro digestion of noodles from the view of phenolics release

The inhibition mechanism of buckwheat phenolics on in vitro starch digestion was investigated using extruded noodles with buckwheat starch and phenolic extract (0.50%–2.00%). The cooking quality and reducing sugar released during in vitro digestion were studied, and the extractable phenolic content along digestion was also monitored to reveal a dose–effect relationship between reducing sugar released and the release of phenolics. Noodles containing increased phenolics released less reducing sugar (230–188 mg g⁻¹) during digestion. Cooking and digestion made phenolics more extractable, and most of the phenolics were released at the end of the gastric phase (85.6%–94.8%) compared with during the small intestinal digestion. The IC₅₀ of buckwheat phenolic extract (0.102 mg mL⁻¹) was four times that of acarbose (0.032 mg mL⁻¹). The inhibitory mechanism was further analysed using molecular docking, in which the activity of α-amylase was inhibited by phenolics that bind with active sites of α-amylase through hydrogen bonds and hydrophobic interaction. Phenolics interacting with starch and the released phenolics can both explain the reduction in starch digestion.     

Comparative Studies on the in vitro alpha-Amylolysis of Different Wheat Starch Products

The time dependent α-amylolytic degradation of different wheat starch products was investigated in vitro. Starch digests were analysed by gel filtration chromatography and measurement of reducing end groups. The hydrolytic activity of crystalline porcine pancreatic α-amylase was compared to that of human salivary α-amylase in cell-free pooled saliva. With conditions similar to those of the human oral cavity wheat starch solutions, whole wheat bread and wheat starch biscuit were hydrolyzed rapidly to mainly maltose, maltotriose and limit dextrins. The distribution of the main degradation products was different depending on the source of the α-amylase. Independent of the enzyme source the rate of hydrolysis decreased from wheat starch solution to bread and biscuit.     

In vitro Digestibility of Native Starch Granules of Samai and Sanwa

The in vitro digestibility of native samai and sanwa starch granules with glucoamylase and salivary α-amylase has revealed a range of enzyme degradation patterns on scanning electron microscopy.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM RYE (SECALE CEREALE) FLOUR

An α-amylase inhibitor from rye (Secale cereale) flour has been purified to homogeneity by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE- and CM-cellulose. The isoelectric point was pH 5.8, and the molecular weight 28,000 by polyacrylamide gel electrophoresis with different gel concentrations and 27,000 by sedimentation equilibrium centrifugation. Under denaturating conditions the molecular weight was about 14,000, indicating two subunits identical in size. The inhibitor was active towards human salivary and hog pancreatic α-amylases but inactive towards Bacillus subtilis and Aspergillus oryzae α-amylases. The pH optimum for the reaction between the rye inhibitor and human salivary α-amylase was 6.0. The inhibitor did not change activity when exposed to pH 2 (0.01M HCl), but prolonged digestion by trypsin destroyed the inhibitor. The rye α-amylase inhibitor lost about 80% of its activity after 10 min at 100°C.     

Natural plant enzyme inhibitors. Purification and properties of an amylase inhibitor from yam (Dioscorea alata)

A high-molecular-weight α-amylase inhibitor has been isolated from mature tubers of Dioscorea alata by extraction with 0.02M acetate buffer (pH 5.0), negative absorption on CM-cellulose and ultracentrifuging. The inhibitor was fairly heat stable and was active against human salivary, human pancreatic and pig pancreatic amylases. The inhibitor had no action on Bacillus subtilis and Aspergillus oryzae amylases. Trypsin and α-chymotrypsin inactivated the inhibitor. Pre-incubation of the inhibitor with starch or concanavalin A resulted in complete abolition of its activity. Chemical modification of the amino groups with trinitrobenzene sulphonic acid led to loss of activity. The inhibitor was found to be a glycoprotein with 64% carbohydrate. The monosaccharide units present were glucose, mannose and galactose in a molar ratio of 5.5:3.8:1.     

Bioactive Phaseolus lunatus peptides release from maltodextrin/gum arabic microcapsules obtained by spray drying after simulated gastrointestinal digestion

Bioactive peptides can be protected from the gastrointestinal environment by microencapsulation. The aim was to assess the effect of simulated gastrointestinal digestion on bioactive properties of P. lunatus peptides (ACE-I, α-glucosidase, α-amylase, and dipeptidyl peptidase-IV inhibition) microencapsulated by spray drying using maltodextrin/gun arabic in optimal formula. Microcapsules were formulated using a 2² factorial design. The simultaneous effect of P. lunatus hydrolysate (4 and 10 g 100 g⁻¹) and proportion of maltodextrin/gum arabic (25:75 and 75:25 g 100 g⁻¹) on yield, protein efficiency, size, protein release at pH 2.0 and pH 7.0 of the different microcapsules was studied. Microcapsules with maximum yield, protein efficiency, and protein release at pH 7.0, and minimum protein release at pH 2.0 were subjected to simulated gastrointestinal digestion. Microcapsules obtained in optimal formula preserved the α-glucosidase, α-amylase, and dipeptidyl peptidase-IV inhibitory activity of P. lunatus peptides after in vitro gastrointestinal digestion.     

Characterization of Two α-Amylase Inhibitors from Black Bean (Phaseolus vulgaris)

Two inhibitors of α-amylase, I-1 and I-2, were purified from the black bean (Phaseolus vulgaris). The rate of complexation with porcine pancreatic α-amylase was very slow. I-l required 12 hr and I-2 6 hr for maximum inhibition at 30°C. The stoichiometry of complexation of α-amylase with I-1 was 2:1 and 1:1 with I-2. Optimum pH for complexation with I-1 ranged from 4.5 to 5.0 and 5.0 to 5.5 for I-2 and both were most stable at pH 3.0 to 4.0. An increase in ionic strength of the preincubation buffer from 0.106 to 0.906 enhanced the rate of inhibition 3 fold for both inhibitors. Maltose, a competitive inhibitor of α-amylase, added to the preincubation solution prevented complex formation for both inhibitors; however, it did not reverse the inhibition of the preformed complex. The dissociation constant for I-1 was 2.2 × 10^?9M and 3.8 × 10^?9M for I-2 at 30°C and pH 6.9. Both inhibitors functioned via a noncompetitive mechanism. I-2 was also stable at pH 2.0. I-2 was more stable to proteolytic digestion by trypsin than I-1 and was also stable to pepsin digestion.     

Interactions of starch with a cyanidin–catechin pigment (vignacyanidin) isolated from Vigna angularis bean

A cyanidin–catechin pigment isolated from adzuki bean (vignacyanidin) interacted with starch. The pigment had absorption maxima at 530 and 540 nm at pH 2.0 and 6.8, respectively, and starch (10 and 100 mg ml⁻¹) increased the absorbance, shifting the absorption maxima to longer wavelengths. Nitrite oxidised vignacyanidin at pH 2.0, and the oxidation resulted in the production of nitric oxide (NO). Rates of the oxidation and the NO production were enhanced by starch. Vignacyanidin inhibited α-amylase-catalysed digestion of starch at pH 6.8, and amylose digestion was more effectively inhibited than amylopectin digestion. The above results suggest (i) that binding of the pigment to starch increased the accessibility of nitrous acid to the pigment, and (ii) that the binding reduced the digestibility of starch by α-amylase. Possible functions of the pigment in the stomach and the intestine are postulated, taking the above results into account.     

Monitoring the stepwise phytate degradation in the upper gastrointestinal tract of pigs

The degradation and formation of inositol phosphates as affected by microbial phytase and gastrointestinal enzyme activities during the passage of phytate through the stomach and small intestine were studied in two experiments with four barrows and three collection periods. The degradation and formation of inositol phosphates were measured at the duodenal and ileal sites using Cr‐NDR, TiO₂ and Co‐EDTA as indigestible markers. In experiment 1, the effect of graded doses of Aspergillus niger phytase (0, 150 and 900 FTU Natuphos^® kg^?1), added to a maize–soybean meal‐based diet with very low intrinsic phytase activity on the degradation of phytate and the formation of inositol phosphates during digestion in the stomach and small intestine was investigated. In experiment 2, three different mixtures of inositol phosphates, produced by Aspergillus niger phytase, containing mainly high, intermediate and low phosphorylated inositol phosphates, were added to the same maize–soybean meal‐based diet as used in experiment I. The fate of the inositol phosphates during digestion in the stomach and small intestine was studied. Experiment 1 showed that the extent of phytate degradation was dependent of the graded dietary phytase activities. At high phytase activity (900 FTU kg^?1 of diet), strong phytate degradation occurred and the once hydrolysed phytate was rapidly dephosphorylated to lower inositol phosphates (mainly inositol di‐ and triphosphates). Intermediate inositol phosphates, such as inositol tetraphosphates, were quantitatively unimportant in duodenal and ileal digesta. At a phytase activity of 150 FTU kg^?1 of diet, a broader spectrum of intermediate inositol phosphates was determined, which was probably due to a slower breakdown of phytate. Experiment 2 showed as a predominant result that lower inositol phosphates such InsP₄ and InsP₃ were degraded, whereas InsP₂ accumulated in the duodenal and ileal digesta. No substantial disappearance of phytate from the stomach and small intestine was found when high concentrations of soluble phytate were added to the diet, which indicates that no substantial phytate absorption occurs in the upper part of the pig gut. Copyright © 2005 Society of Chemical Industry     

PURIFICATION AND PHYSICOCHEMICAL PROPERTIES OF AN EXTRA CELLULAR AMYLASE FROM A STRAIN OF BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM DRY ONION POWDER

An extracellular α-amylase from Bacillus amyloliquefaciens, isolated from dry onion powder, has been purified to homogeneity by ammonium sulfate fractionation, adsorption on starch, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100 column. The enzyme consisted of one polypeptide chain with a molecular weight of 60,000. The isoelectric point was pH 5.2, the pH optimum 5.5 and the temperature optimum ranging from 50°-70°C. Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 6.9 mg/ml. The enzyme was quite stable at 50°C, but lost about 85% of its activity at 60° after 30 min (pH 6.0).     

Anti-Diabetic Potential of Phenolic Compounds: A Review

Starch is the main carbohydrate in human nutrition. Starch digestibility can vary from a rapid digestion to indigestibility. Therefore, postprandial glycaemic control in type 2 diabetics is of great interest in the context of worldwide health concerns. Although powerful synthetic inhibitors of starch digestive enzymes, such as acarbose, are available to control postprandial hyperglycemia, plant-based enzyme inhibitors are potentially safer. Natural enzyme inhibitors, such as wheat albumin, the Phaseolus vulgaris α-amylase inhibitor, and several phenolic compounds, have the potential to serve as a remedy against hyperglycemia-induced chronic diseases. The inhibition of α-amylase and α-glucosidase is mediated by different phenolics found in varieties of raspberry. Maltase inhibitory activities of chebulagic acid and chebulinic acid from fruit of Terminalia chebula are comparable to that of acarbose. The Nepalese herb Pakhanbhed (Bergenia ciliata) phenolics, (-)-3-O-galloylepicatechin and (-)-3-O-galloylcatechin, showed effective inhibition against starch digesting enzymes. In separate studies, oral administration of starch and maltose with persimmon (Diospyros kaki) leaf tea proanthocyanidins [containing (-)-epigallocatechin-3-O-gallate] and black/bitter cumin (Centratherum anthelminticum) seed phenolics, respectively, resulted in a significant and dose-dependent decrease in the blood glucose level in Wistar rats. Co-application of phenolics with synthetic enzyme inhibitors may reduce the effective dose of synthetic inhibitors required in the regulation of starch digestion. Several phenolic compounds might be useful functional food components and could contribute to manage both hyperglycemia and proper cellular redox status. Human dose-selecting studies and well-controlled long-term human studies would help to optimize the beneficial effects of phenolic compounds.