Daniel J. Gallant

Microscopy of starch: evidence of a new level of granule organization

Abstract Considerable information on starch granule structure may be gathered from a review of published data. Evidence from a range of different (predominantly microscopic) techniques is compared and discussed, allowing the presence of a level of starch granule organization between that of the amylopectin lamellae and the large ‘growth rings’ to be deduced. This structural level of the granule involves the organization of the amylopectin lamellae into effectively spherical ‘blocklets’ which range in diameter from 20 to 500 nm depending on starch botanical type and their location in the granule. The presence of short, radial ‘channels’ of amorphous material within starch granules from some starch varieties is confirmed. The organization and structure of the crystalline and amorphous amylopectin lamellae is also discussed. Consideration of the information regarding starch granule structure and organization to date has significant implications on the internal architecture of the starch granule, and it is evident that the presence of the blockets and amorphous channels play a role in both the resistance of starch to enzymic attack and the structure of the semi-crystalline shells.

Physical and chemical transformations of cereal food during oral digestion in human subjects

Chemical and physical transformations of solid food begin in the mouth, but the oral phase of digestion has rarely been studied. In the present study, twelve healthy volunteers masticated mouthfuls of either bread or spaghetti for a physiologically-determined time, and the levels of particle degradation and starch digestion before swallowing were compared for each food. The amounts of saliva moistening bread and spaghetti before swallowing were, respectively, 220 (SEM 12) v. 39 (SEM 6) g/kg fresh matter. Particle size reduction also differed since bread particles were highly degraded, showing a loss of structure, whereas spaghetti retained its physical structure, with rough and incomplete reduction of particle size. Starch hydrolysis was twice as high for bread as for spaghetti, mainly because of the release of high-molecular-mass alpha-glucans. The production of oligosaccharides was similar after mastication of the two foods, respectively 125 (SEM 8) and 92 (SEM 7) g/kg total starch. Starch hydrolysis, which clearly began in the mouth, depended on the initial structure of the food, as in the breakdown of solid food. These significant physical and chemical degradations of solid foods during oral digestion may influence the entire digestive process.

Spatial and temporal distribution of the major isoforms of puroindolines (puroindoline-a and puroindoline-b) and non specific lipid transfer protein (ns-LTP1e1) of Triticum aestivum seeds. Relationships with their in vitro antifungal properties

In wheat endosperm, the main isoforms of puroindolines (PIN-a and PIN-b) and nonspecific lipid transfer protein (ns-LTP1e1), structurally related lipid binding proteins, were asynchronously synthesized during maturation and are partially degraded during germination. These proteins are not detected in roots and hypocotyls of seedlings, while ns-LTP1e1, but not PINs, was synthesized during germination in the scutellum and/or mesocotyl. In mature wheat seeds, ns-LTP1-e1 was specifically localised within aleurone cells but not in cell walls in marked contrast with most other plant ns-LTP1s. PINs are both located in the starchy endosperm and in the aleurone layer. In the latter cells, PINs and ns-LTP1-e1 were both localised in small inclusions within protein-rich aleurone grains. In the mature starchy endosperm, PINs were localised in the protein matrix and at the interface between starch granules and protein matrix. It was shown that both PIN-a and PIN-b, have antifungal properties in vitro and a synergistic enhancement of the antifungal properties of α-purothionins (α-PTH) was observed in the presence of PINs. This synergism could have biological significance since α-PTH and PINs are both located in the protein matrix of starchy endosperm. ns-LTP1e1 is not capable to inhibit growth of fungi and a synergy rather weak in comparison with PINs was also observed between ns-LTP1e1 and α-PTH.

Extensive degradation of native starch granules by alpha-amylase from aspergillus fumigatus

Abstract Starch granules of various botanical origins were subjected to enzymic degradation by purified alpha -amylases from pig pancreas, Bacillus sp. and Aspergillus fumigatus ( Aspergillus sp. K-27). With the A. fumigatus enzyme, glucose in alpha -anomeric configuration was the sole end degradation product regardless of the starch tested. The efficiency of this enzyme was very high on all native starch granules. Starches from normal and waxy maize, smooth pea and wheat were completely solubilised within 30 h using 1·34 nKat/mg of substrate. High-amylose maize, wrinkled pea and potato starches were degraded to lower extents (50, 70 and 45%, respectively). Such high enzymic efficiency was not observed with alpha -amylases from pig pancreas or Bacillus sp. With alpha -amylase from A. fumigatus , normal and waxy maize starches displayed highly eroded layered structures when observed by scanning or transmission electron microscopy during degradation. In contrast, potato and high-amylose maize starches produced a minor fraction of endo-eroded granules, whereas the rest of the granules exhibited superficial porosity.

Chapter 5 – Structural Features of Starch Granules I

Publisher Summary The amylose and amylopectin molecules, the granule structure, and the natures and amounts of the lipid and protein molecules present in granules vary with the botanical source of the starch. Starch granules are mainly found in seeds, roots, and tubers, but are also found in stems, leaves, fruits, and even pollen. They occur in all shapes and sizes (spheres, ellipsoids, polygons, platelets, irregular tubules) and are long dimensioned. Differences in external granule morphology are generally sufficient to provide unambiguous characterization of the botanical source via optical microscopy. The starch granule is natures chief way of storing energy over long periods in green plants. The granule is well suited to this role, being insoluble in water and densely packed, but still accessible to the plants catabolic enzymes. Native starch granules have a crystallinity varying from 15% to 45%, thus, most native starch granules exhibit a Maltese cross when observed under polarized light. Most starch granules are made up of alternating amorphous and crystalline shells, which are between 100 and 400 nm thick. These structures are termed “growth rings.” Birefringence remains unchanged on both polar and equatorial sections of elongated starch granules. Most starch polymers in the granule are in an amorphous state. Most cereal starches give the so called A-type pattern; some tuber starches (e.g. potato) and cereal starches rich in amylose yield the B-type pattern, while legume starches generally give a C-type pattern. The two major macromolecular components of starch are amylose and amylopectin.

Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.

Factors limiting the biodegradation of Ulva sp cell-wall polysaccharides

The dietary fibres of the seaweed Ulva sp (sea-lettuce) consist of water-soluble ulvan, alkali-soluble β(1,4)-D-glucuronan and β(1,4)-D-glucoxylan, and an insoluble α-cellulose containing xylose residues. They are poorly degraded by human colonic bacteria particularly when associated within the intact plant cell wall. In order to better understand this resistance to microbial attack, their organisation in the cell-wall has been investigated by light and electron microscopy after sequential chemical extractions. Their susceptibility to enzymatic degradation and their accessibility to bacteria and enzyme were also studied. Microscopic localisation in native and sequentially extracted Ulva sp demonstrated that ulvan is in all the cell-walls of the algae and particularly between the two cell layers constituting the thallus. Glucuronan is close to the cytoplasmic membrane facing the outside of the seaweed and between adjacent cells. The xylose and glucose containing polysaccharides form packed layers surrounding the cells. A model of the spatial distribution of the different polysaccharides within the algae is proposed. Ulvan and glucuronan did not limit the xyloglucan and α-cellulose degradation by an endo-xylanase in the whole seaweed and its insoluble dietary fibre but the α-cellulose was not affected by a cellulase. The cell-wall of Ulva sp was accessible to enzymes but poorly to bacteria as assessed from porosity measurements. These results established that the poor fermentation of sea-lettuce by human colonic flora is primarily due to the ubiquitous presence of the degradation-resistant ulvan in the cell wall of Ulva sp. ©1997 SCI

Lipid-protein interactions in wheat gluten: a phosphorus nuclear magnetic resonance spectroscopy and freeze-fracture electron microscopy study

Phosphorus magnetic resonance spectroscopy and freeze-fracture electron microscopy of wheat gluten showed that its lipids are organised in small vesicles (60 to 300 nm in diameter), in which polar lipids exhibit a lamellar liquid crystalline phase. Interactions between phospholipids and proteins are sensitive to heating and to mechanical work. From 50 to 70°C and during cooling from 70 to 25°C these interactions are disrupted due to the expulsion into the aqueous phase of lipid vesicles that are embedded in the protein network. This expulsion can be induced by extensive mechanical work and is prevented in the presence of a reducing agent. Residual starch present in gluten does not play a role. Protein rearrangements during heating and mixing may be responsible for the expulsion of lipid vesicles. These results suggest that the formation of complexes between wheat proteins and lipids such as is found in membranes, does not occur in gluten. It appears that lipid vesicles are embedded in the protein matrix due to the particular properties of wheat proteins, so that gluten may be regarded as a system containing stabilised microemulsions.

Textural Images Analysis of Pasta Protein Networks to Determine Influence of Technological Processes

ABSTRACT The structure of pasta is largely governed by the presence of a structured protein network. This work analyzed the protein network textures of various cooked pasta products through textural image analysis. Six different pasta types were investigated: reference pasta made from durum semolina; pasta enriched with gluten proteins from soft wheat flour at 10 and 20%; autoclaved pasta; soft wheat flour pasta; and pasta made from reconstituted flour fractions. Pasta samples were sectioned, and each crosssection consisted of three distinct zones (central, intermediate, and external) based on the state of swelling of starch granules for each pasta product. Digital images of the protein network in each zone were acquired using confocal laser scanning microscopy. Textural image analysis was then performed. Similarities and differences in protein network texture were assessed by principal component, stepwise discriminant, and variance analyses. With the exception of autoclaved pasta, protein network structu...

Contribution of the digestive tract microflora to amylomaize starch degradation in the rat

To study in vivo the contribution of the bacterial flora to amylomaize starch degradation in the rat, germ-free and conventional rats were fed on a diet containing either a normal maize starch or an amylomaize starch. In germ-free rats maize starch was almost totally digested in the small intestine, whereas 40% of the ingested amylomaize starch reached the caecum and 30% was excreted, despite the very high endogenous amylase activity. Study by transmission electron microscopy of germ-free caecal contents showed an endocorrosion of the starch granule. In conventional rats, as in germ-free rats, digestibility of maize starch reached 98% in the small intestine, whereas that of amylomaize starch was only 60%. In the caecum of these rats amylomaize starch was fermented, and this led to a decrease in caecal pH and to formation of short-chain fatty acids (SCFA), especially propionate. Comparison between conventional rats fed on maize starch or amylomaize starch showed that caecal SCFA concentrations during a circadian cycle varied in the same way whereas total SCFA and lactic acid concentrations were much higher in rats fed on amylomaize starch. Amylase (EC 3.2.1.1) activity was similar in the caecal contents of conventional rats whatever the ingested starch. It was lower in conventional than in germ-free rats, but no starch granule remained in the caecum of conventional rats. These results showed that bacterial amylase was more efficient at degrading resistant amylomaize starch than endogenous amylase.