Modification of Pea Starch Digestibility through the Complexation with Gallic Acid via High-Pressure Homogenization.

Pea starch and some legume starches are the side streams of plant-based protein production. Structural modification toward moderate digestibility and desirable functionality is a way to increase the economic values of these side-stream starches. We applied an innovative and sustainable technique, high-pressure homogenization, to alter pea starch structure, which resulted in a high level of complexation with the small phenolic acid molecule, gallic acid, to alter starch digestibility. This study showed a great level of disruption of the compact starch structure represented by the decrease in gelatinization temperature, enthalpy change, and relative crystallinity. The addition of a high concentration (10%) of gallic acid contributed to a typical V-type X-ray diffractometry pattern. Data demonstrated a significant decrease (~23%) in the susceptibility to α-amylase and an increase in resistant starch (~13%). In addition, starch functionality was improved with a reduced retrogradation rate. Pea starch responded to the high-pressure homogenization process well. Compared with the rice and maize starch reported in the literature, pea starch required a reduced amount of gallic acid to form a high level of complexation with a significant delay in starch digestion.

Eliminating protein interference when quantifying potato reducing sugars with the miniaturized Somogyi-Nelson assay.

Reducing sugar (RS) quantification is essential in the potato industry because RS content plays a vital role in potato quality, acrylamide formation, post-harvest management, and new variety development. A miniaturized Somogyi-Nelson (SN) analysis can effectively and accurately quantify RS. However, soluble proteins in potatoes interfere with SN analysis. Our research goal was to develop an applicable deprotinization procedure without influencing the precision of the SN analysis. Results showed ethanol effectively removed potato proteins and, unlike other chemicals (salts, acids), ethanol did not affect SN accuracy. Protein removal also can be achieved by heating and pH adjustment, but the ethanol-based procedure provides a simpler alternative. RS content measured by the miniaturized SN assay after deproteinization by ethanol was precise and validated by HPAEC-PAD. Data from 118 potao juicies showed that a commonly used biochemical analyzer obtained a lower reducing sugar content than the deprotinization-SN assay because fructose was not identified by the biochemical analyzer. Results demonstrate the reliability of quantifying potato RS with the SN assay following the ethanol-based deproteinization.

Reduction of falling number in soft white spring wheat caused by an increased proportion of spherical B-type starch granules.

A low falling number (FN) in wheat indicates high α-amylase activity associated with poor end-use quality. We hypothesize starch - the substrate of α-amylase, can directly influence hot flour pasting properties and its susceptibility to α-amylase, which further affects viscosity. We examined the structural characteristics of starch in three soft white spring wheat cultivars grown in Idaho in 2013 (normal FN year) and 2014 (low FN year with pre-harvest rains). Our data surprisingly show that starch in some low FN wheat was not significantly degraded by α-amylase but had developmental changes with an increased proportion of B-type wheat starch. We reconstituted wheat starch and verified that starch with an increase of B-granules has a relatively low viscosity and high susceptibility to wheat α-amylase, which further facilitates the decrease of viscosity. The influence of starch structure and starch-enzyme interaction must be considered while developing a solution to the low FN issue.

Impacts of Starch and the Interactions Between Starch and Other Macromolecules on Wheat Falling Number.

Wheat with a low falling number (FN) has been particularly prevalent in recent years and has resulted in a loss of more than $140 million in a single year in the wheat industry in the Pacific Northwest of the United States. FN measurement is a standard method for the evaluation of grain α-amylase activity, and a low FN indicates a reduction in hot wholemeal paste viscosity due to sprouting damage. Recent studies show that a low FN may result from a developmental change of starch and adverse effects of non-α-amylase macromolecules on wheat. In this review, we describe the principles of FN measurement and the relationship between FN and α-amylase. We also discuss the isozymes, locations, and inhibitors of wheat α-amylase. The effects of various aspects of starch, which is the substrate of α-amylase, on wheat FN are also discussed, including starch structural characteristics (for example, starch granule architecture), starch susceptibility to α-amylase, and the interaction between starch and nonstarch macromolecules (for example, lipids). Studies on the effects of planting environments (for example, temperature) and agronomic practices (for example, irrigation and fertilization) on both starch paste viscosity and FN are also reviewed. This paper highlights the importance of considering the impacts of starch and the interactions of starch and other macromolecules, including wheat α-amylase, on wheat FN, which is important for developing strategies to solve the low FN problem.

Structure and Digestion of Common Complementary Food Starches.

Starch is the major source of dietary glucose for rapid development of children. Starches from various crops naturally differ in molecular structures and properties. Cooking, processing, and storage may change their molecular properties and affect their digestibility and functionality. Starch digestion is affected by its susceptibility to α-amylase and α-glucosidase (maltase), and the susceptibility is determined by starch granule architecture and glucan structures, as well as the interaction between starch and other food components. Starch is given as a complementary feeding to young children in many cultures, and starch or modified starch, is used in special formulae of infant foods or supplements. Although indigestible starch does not provide much energy, it can benefit colonic health.

Improvement in the quantification of reducing sugars by miniaturizing the Somogyi-Nelson assay using a microtiter plate.

Measuring reducing sugar is a common practice in carbohydrate research, and the colorimetric assay developed by Somogyi and Nelson has a high sensitivity in a broad concentration range. However, the method is time-consuming when analyzing a large number of samples. In this study, a modified Somogyi-Nelson assay with excellent accuracy and sensitivity was developed using a 96-well microplate. This microassay greatly improves the analytic capacity and efficacy of the method.

Improved Starch Digestion of Sucrase-deficient Shrews Treated With Oral Glucoamylase Enzyme Supplements.

BACKGROUND AND OBJECTIVE: Although named because of its sucrose hydrolytic activity, this mucosal enzyme plays a leading role in starch digestion because of its maltase and glucoamylase activities. Sucrase-deficient mutant shrews, Suncus murinus, were used as a model to investigate starch digestion in patients with congenital sucrase-isomaltase deficiency.Starch digestion is much more complex than sucrose digestion. Six enzyme activities, 2 α-amylases (Amy), and 4 mucosal α-glucosidases (maltases), including maltase-glucoamylase (Mgam) and sucrase-isomaltase (Si) subunit activities, are needed to digest starch to absorbable free glucose. Amy breaks down insoluble starch to soluble dextrins; mucosal Mgam and Si can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. Starch digestion is reduced because of sucrase deficiency and oral glucoamylase enzyme supplement can correct the starch maldigestion. The aim of the present study was to measure glucogenesis in suc/suc shrews after feeding of starch and improvement of glucogenesis by oral glucoamylase supplements. METHODS: Sucrase mutant (suc/suc) and heterozygous (+/suc) shrews were fed with C-enriched starch diets. Glucogenesis derived from starch was measured as blood C-glucose enrichment and oral recombinant C-terminal Mgam glucoamylase (M20) was supplemented to improve starch digestion. RESULTS: After feedings, suc/suc and +/suc shrews had different starch digestions as shown by blood glucose enrichment and the suc/suc had lower total glucose concentrations. Oral supplements of glucoamylase increased suc/suc total blood glucose and quantitative starch digestion to glucose. CONCLUSIONS: Sucrase deficiency, in this model of congenital sucrase-isomaltase deficiency, reduces blood glucose response to starch feeding. Supplementing the diet with oral recombinant glucoamylase significantly improved starch digestion in the sucrase-deficient shrew.

Contribution of the Individual Small Intestinal α-Glucosidases to Digestion of Unusual α-Linked Glycemic Disaccharides.

The mammalian mucosal α-glucosidase complexes, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), have two catalytic subunits (N- and C-termini). Concurrent with the desire to modulate glycemic response, there has been a focus on di-/oligosaccharides with unusual α-linkages that are digested to glucose slowly by these enzymes. Here, we look at disaccharides with various possible α-linkages and their hydrolysis. Hydrolytic properties of the maltose and sucrose isomers were determined using rat intestinal and individual recombinant α-glucosidases. The individual α-glucosidases had moderate to low hydrolytic activities on all α-linked disaccharides, except trehalose. Maltase (N-terminal MGAM) showed a higher ability to digest α-1,2 and α-1,3 disaccharides, as well as α-1,4, making it the most versatile in α-hydrolytic activity. These findings apply to the development of new glycemic oligosaccharides based on unusual α-linkages for extended glycemic response. It also emphasizes that mammalian mucosal α-glucosidases must be used in in vitro assessment of digestion of such carbohydrates.

Branch pattern of starch internal structure influences the glucogenesis by mucosal Nt-maltase-glucoamylase.

To produce sufficient amounts of glucose from food starch, both α-amylase and mucosal α-glucosidases are required. We found previously that the digestion rate of starch is influenced by its susceptibility to mucosal α-glucosidases. In the present study, six starches and one glycogen were pre-hydrolyzed by α-amylase for various time periods, and then further hydrolyzed with the mucosal α-glucosidase, the N-terminal subunit of maltase-glucoamylase (Nt-MGAM), to generate free glucose. Results showed that α-amylase amplified the Nt-MGAM glucogenesis, and that the amplifications differed in various substrates. The amount of branches within α-amylase hydrolysate substrates was highly related to the rate of Nt-MGAM glucogenesis. After de-branching, the hydrolysates showed three fractions, Fraction 1, 2, and 3, in size exclusion chromatographs. We found that the α-amylase hydrolysates with higher quantity of the Fraction 3 (molecules with relatively short chain-length) and shorter average chain-length of this fraction had lower rates of Nt-MGAM glucogenesis. This study revealed that the branch pattern of α-amylase hydrolysates modulates glucose release by Nt-MGAM. It further supported the hypothesis that the internal structure of starch affects its digestibility at the mucosal α-glucosidase level.

Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation.

SCOPE: The four mucosal α-glucosidases, which differ in their digestive roles, generate glucose from glycemic carbohydrates and accordingly can be viewed as a control point for rate of glucose delivery to the body. In this study, individual recombinant enzymes were used to understand how α-glucan oligomers are digested by each enzyme, and how intermediate α-amylolyzed starches are hydrolyzed, to elucidate a strategy for moderating the glycemic spike of rapidly digestible starchy foods. METHODS AND RESULTS: The C-terminal maltase-glucoamylase (ctMGAM, commonly termed "glucoamylase") was able to rapidly hydrolyze longer maltooligosaccharides, such as maltotetraose and maltopentaose, to glucose. Moreover, it was found to convert larger size maltodextrins, as would be produced early in α-amylase digestion of starch, efficiently to glucose. It is postulated that ctMGAM has the additional capacity to hydrolyze large α-amylase products that are produced immediately on starch digestion in the duodenum and contribute to the rapid generation of glucose from starch-based meals. CONCLUSION: The findings suggest that partial inhibition of ctMGAM, such as by natural inhibitors found in foods, might be used to moderate the early stage of high glycemic response, as well as to extend digestion distally; thereby having relevance in regulating glucose delivery to the body.

Maltase-glucoamylase modulates gluconeogenesis and sucrase-isomaltase dominates starch digestion glucogenesis.

OBJECTIVES: Six enzyme activities are needed to digest starch to absorbable free glucose; 2 luminal α-amylases (AMY) and 4 mucosal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) subunit activities are involved in the digestion. The AMY activities break down starch to soluble oligomeric dextrins; mucosal MGAM and SI can either directly digest starch to glucose or convert the post-α-amylolytic dextrins to glucose. We hypothesized that MGAM, with higher maltase than SI, drives digestion on ad limitum intakes and SI, with lower activity but more abundant amount, constrains ad libitum starch digestion. METHODS: Mgam null and wild-type (WT) mice were fed with starch diets ad libitum and ad limitum. Fractional glucogenesis (fGG) derived from starch was measured and fractional gluconeogenesis and glycogenolysis were calculated. Carbohydrates in small intestine were determined. RESULTS: After ad libitum meals, null and WT had similar increases of blood glucose concentration. At low intakes, null mice had less (f)GG (P = 0.02) than WT mice, demonstrating the role of Mgam activity in ad limitum feeding; null mice did not reduce fGG responses to ad libitum intakes demonstrating the dominant role of SI activity during full feeding. Although fGG was rising after feeding, fractional gluconeogenesis fell, especially for null mice. CONCLUSIONS: The fGNG (endogenous glucogenesis) in null mice complemented the fGG (exogenous glucogenesis) to conserve prandial blood glucose concentrations. The hypotheses that Mgam contributes a high-efficiency activity on ad limitum intakes and SI dominates on ad libitum starch digestion were confirmed.

Mammalian mucosal α-glucosidases coordinate with α-amylase in the initial starch hydrolysis stage to have a role in starch digestion beyond glucogenesis.

Starch digestion in the human body is typically viewed in a sequential manner beginning with α-amylase and followed by α-glucosidase to produce glucose. This report indicates that the two enzyme types can act synergistically to digest granular starch structure. The aim of this study was to investigate how the mucosal α-glucosidases act with α-amylase to digest granular starch. Two types of enzyme extracts, pancreatic and intestinal extracts, were applied. The pancreatic extract containing predominantly α-amylase, and intestinal extract containing a combination of α-amylase and mucosal α-glucosidase activities, were applied to three granular maize starches with different amylose contents in an in vitro system. Relative glucogenesis, released maltooligosaccharide amounts, and structural changes of degraded residues were examined. Pancreatic extract-treated starches showed a hydrolysis limit over the 12 h incubation period with residues having a higher gelatinization temperature than the native starch. α-Amylase combined with the mucosal α-glucosidases in the intestinal extract showed higher glucogenesis as expected, but also higher maltooligosaccharide amounts indicating an overall greater degree of granular starch breakdown. Starch residues after intestinal extract digestion showed more starch fragmentation, higher gelatinization temperature, higher crystallinity (without any change in polymorph), and an increase of intermediate-sized or small-sized fractions of starch molecules, but did not show preferential hydrolysis of either amylose or amylopectin. Direct digestion of granular starch by mammalian recombinant mucosal α-glucosidases was observed which shows that these enzymes may work either independently or together with α-amylase to digest starch. Thus, mucosal α-glucosidases can have a synergistic effect with α-amylase on granular starch digestion, consistent with a role in overall starch digestion beyond their primary glucogenesis function.

Starch source influences dietary glucose generation at the mucosal α-glucosidase level.

The quality of starch digestion, related to the rate and extent of release of dietary glucose, is associated with glycemia-related problems such as diabetes and other metabolic syndrome conditions. Here, we found that the rate of glucose generation from starch is unexpectedly associated with mucosal α-glucosidases and not just α-amylase. This understanding could lead to a new approach to regulate the glycemic response and glucose-related physiologic responses in the human body. There are six digestive enzymes for starch: salivary and pancreatic α-amylases and four mucosal α-glucosidases, including N- and C-terminal subunits of both maltase-glucoamylase and sucrase-isomaltase. Only the mucosal α-glucosidases provide the final hydrolytic activities to produce substantial free glucose. We report here the unique and shared roles of the individual α-glucosidases for α-glucans persisting after starch is extensively hydrolyzed by α-amylase (to produce α-limit dextrins (α-LDx)). All four α-glucosidases share digestion of linear regions of α-LDx, and three can hydrolyze branched fractions. The α-LDx, which were derived from different maize cultivars, were not all equally digested, revealing that the starch source influences glucose generation at the mucosal α-glucosidase level. We further discovered a fraction of α-LDx that was resistant to the extensive digestion by the mucosal α-glucosidases. Our study further challenges the conventional view that α-amylase is the only rate-determining enzyme involved in starch digestion and better defines the roles of individual and collective mucosal α-glucosidases. Strategies to control the rate of glucogenesis at the mucosal level could lead to regulation of the glycemic response and improved glucose management in the human body.

Unexpected high digestion rate of cooked starch by the Ct-maltase-glucoamylase small intestine mucosal α-glucosidase subunit.

For starch digestion to glucose, two luminal α-amylases and four gut mucosal α-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal α-glucosidases on cooked (gelatinized) starch. Gelatinized normal maize starch was digested with N- and C-terminal subunits of recombinant mammalian maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) of varying amounts and digestion periods. Without the aid of α-amylase, Ct-MGAM demonstrated an unexpected rapid and high digestion degree near 80%, while other subunits showed 20 to 30% digestion. These findings suggest that Ct-MGAM assists α-amylase in digesting starch molecules and potentially may compensate for developmental or pathological amylase deficiencies.

Interference prevention in size-exclusion chromatographic analysis of debranched starch glucans by aqueous system.

Branch chain-length distribution of amylopectin plays an important role on the characteristics of starch. One of the adapted protocols for determining the chain-length distribution and mass proportion of starch molecules is that starch is debranched with isoamylase and then analyzed by using high-performance size-exclusion chromatography coupled with multiangle laser-light scattering and refractive index detection (HPSEC-MALS-RI). However, ammonium sulfate in commercial isoamylase and acetate in debranching buffer give significant interferences on the chromatograms because of their undesirable ionic interactions with column sorbent materials. This study deals with development for correcting those interferences. A weak anion-exchange resin or selective precipitation with barium acetate was employed to remove sulfate prior to HPSEC determination. The interference of acetate was overcome by means of high ionic strength eluent, 0.3 M sodium nitrate. The specific refractive index increment (dn/dc) of amylodextrin was determined to be 0.147 using the modified conditions and was applied to calculate the molecular weight distribution of debranched starch molecules.