Whole Proteome Analyses on Ruminiclostridium cellulolyticum Show a Modulation of the Cellulolysis Machinery in Response to Cellulosic Materials with Subtle Differences in Chemical and Structural Properties.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the "xyl-doc" cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.

Physicochemical and crystalline properties of standard maize starch hydrothermally treated by direct steaming.

The changes in physicochemical properties of standard maize starch (SMS) by three hydrothermal treatments; DV-HMT (Direct Vapor-Heat Moisture Treatment), RP-HMT (Reduced Pressurized-Heat Moisture Treatment) and DIC (instantaneous controlled pressure drop) were investigated at different processing conditions; steam pressure (SP) varied from 1 to 3bar during 20min. Starch was steamed by direct contact, whose interest was to intensify the heat transfer phenomenon but also the water transfer. The physicochemical changes of SMS depended on process conditions and their extent followed this order: DIC>RP-HMT>DV-HMT. All treatments significantly increased gelatinization temperatures and decreased the enthalpies, confirmed by loss of granules birefringence. From 2bar, the crystalline structure changed from A-type to Vh-type, revealing formation of amylose-lipid complexes during steaming. The results clearly showed that the particle size distribution depends on the melting extent of crystalline structure during treatment. At severe processing conditions the melted fraction increased and more complex aggregates of different sizes have been formed.

Shape-memory effect in amorphous potato starch: The influence of local orders and paracrystallinity.

In this paper, a detailed characterization of the mechanisms at the origin of the shape-memory effect in amorphous potato starch is presented. Using different treatments (annealing) and preparation methods (hot casting and extrusion), the local structures responsible for the shape-memory were disrupted, as evidenced in the first part of the article detailing the macroscopic properties: mechanical, calorimetric and shape-memory. In the second part the macromolecular scale is investigated using X-rays diffraction and CP-MAS NMR, and thus allows making the link between the structural differences and the macroscopic properties. Finally we discuss the origin of shape-memory in amorphous starch.

Multivariate model to characterise relations between maize mutant starches and hydrolysis kinetics.

The many studies about amylolysis have collected considerable information regarding the contribution of the starch physico-chemical properties. But the inherent elaborate and variable structure of granular starch and, consequently, the multifactorial condition of the system hinders the interpretation of the experimental results. The immediate benefit of multivariate statistical analysis approaches with that regard is twofold: considering the factors, possibly interrelated, all together and not independently, providing a first estimation of the magnitude and confidence level of the relations between factors and amylolysis kinetic parameters. Based on data of amylolysis of 13 starch samples from wild type, single and double mutants of maize by porcine pancreatic α-amylase (PPA), a multivariate analysis is proposed. Amylolysis progress-curves were fitted by a Weibull function, as proposed in a previous work, to extract three kinetic parameters: the reaction rate coefficient during the first time-unit, k, the reaction rate retardation over time, h, and the final hydrolysis extent, X∞. Multivariate models relate the macromolecular composition and the fractions of crystalline polymorphic types to the kinetic parameters. h and X∞ are found to be highly related to the measured properties. Thus the amylose content appears to be significantly correlated to the hydrolysis rate retardation, which sheds some light on the probable contribution of the amylose molecules contained in the granules. The multivariate models give correct prediction performances except for k whose a part of variability remains unexplained. A further analysis points out the extent of the characterisation effort of the granule structure needed to extend the fraction of explained variability.

Amylolysis of maize mutant starches described with a fractal-like kinetics model.

Two empirical models, a conventional first-order kinetics and a fractal-like first-order kinetic model were tested for analysing the hydrolysis of 13 wild type, single and double mutants of maize starch by porcine pancreatic α-amylase (PPA). The major difference between the two models is an additional parameter, the fractal kinetics exponent h, which enables to characterise a decrease of the reaction rate coefficient over time. The fractal-like kinetic model should be preferred to characterise the amylolysis for 10 mutants out of 13 as sugary-2 and amylose-extender curves exhibit clear reaction rate retardation, unlike normal maize and waxy maize. Analysis of the model parameter values reveals two groups of kinetics for the maize mutants: amylose-extender, normal and waxy on one hand, sugary-2 on the other hand. Possible relations between the parameters of the model and granule composition and structure are discussed.

Crystallization and chain reorganization of debranched rice starches in relation to resistant starch formation.

The effects of chain distribution, concentration, temperature and hydrothermal treatments on the recrystallization behavior and formation of resistant starch (RS) were investigated. Waxy and normal rice starches were debranched at 10 and 21% w/w solid concentrations, incubated at 25 or 50 °C, and further subjected to annealing or heat moisture treatment (HMT) to enhance RS formation. The crystallization at 25 °C favored the formation of the B-type structure, whereas crystallization at 50 °C led to the A-type structure with a higher melting temperature (100-120 °C) and a higher RS content (52%). All incubated samples showed an increase in RS content after subsequent hydrothermal treatments. The sample incubated at a high temperature contained the highest RS content (74.5%) after HMT with larger/perfect crystallites. These results suggested that the RS formation could be manipulated by crystallization conditions and improved by hydrothermal treatments which are dependent on the initial crystalline perfection.

Shape memory starch-clay bionanocomposites.

1-10% starch/clay bionanocomposites with shape memory properties were obtained by melt processing. X-ray diffraction (XRD) and TEM evidenced the presence of a major fraction of clay tactoids, consisting of 4-5 stacked crystalline layers, with a thickness of 6.8 nm. A significant orientation of the nanoparticles induced by extrusion was also observed. Tensile tests performed above the glass transition of the materials showed that the presence of clay nanoparticles leads to higher elastic modulus and maximum stress, without significant loss in elongation at break which typically reached 100%. Samples submitted to a 50% elongation and cooled below the glass transition showed shape memory behavior. Like unreinforced starch, the bionanocomposites showed complete shape recovery in unconstrained conditions. In mechanically constrained conditions, the maximum recovered stress was significantly improved for the bionanocomposites compared to unreinforced starch, opening promising perspectives for the design of sensors and actuators.

The deposition and characterization of starch in Brachypodium distachyon.

Brachypodium distachyon is a non-domesticated cereal. Nonetheless, Brachypodium was recently introduced as a model plant for temperate cereals. This study compares grain starch metabolism in Brachypodium and barley (Hordeum vulgare). In Brachypodium, we identified and annotated 28 genes involved in starch metabolism and identified important motifs including transit peptides and putative carbohydrate-binding modules (CBMs) of the families CBM20, CBM45, CBM48, and CBM53. Starch content was markedly lower in Brachypodium grains (12%) compared to barley grains (47%). Brachypodium starch granules were doughnut shaped and bimodally distributed into distinct small B-type (2.5-10 µm) and very small C-type (0.5-2.5 µm) granules. Large A-type granules, typical of cereals, were absent. Starch-bound phosphate, important for starch degradation, was 2-fold lower in Brachypodium compared with barley indicating different requirements for starch mobilization. The amylopectin branch profiles were similar and the amylose content was only slightly higher compared with barley cv. Golden Promise. The crystallinity of Brachypodium starch granules was low (10%) compared to barley (20%) as determined by wide-angle X-ray scattering (WAXS) and molecular disorder was confirmed by differential scanning calorimetry (DSC). The expression profiles in grain for most genes were distinctly different for Brachypodium compared to barley, typically showing earlier decline during the course of development, which can explain the low starch content and differences in starch molecular structure and granule characteristics. High transitory starch levels were observed in leaves of Brachypodium (2.8% after 14h of light) compared to barley (1.9% after 14h of light). The data suggest important pre-domesticated features of cereals.

Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping.

BACKGROUND: Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2-0.5%) and proteins (0.1-0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis. METHODS: Synchrotron micro-X-ray fluorescence (µXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme. RESULTS: Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3-5µm in diameter. CONCLUSIONS: Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before. GENERAL SIGNIFICANCE: µXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.

Characterization of hyperbranched glycopolymers produced in vitro using enzymes.

Asymmetrical flow field flow fractionation (AF4) has proven to be a very powerful and quantitative method for the determination of the macromolecular structure of high molar mass branched biopolymers, when coupled with multi-angle laser light scattering (MALLS). This work describes a detailed investigation of the macromolecular structure of native glycogens and hyperbranched α-glucans (HBPs), with average molar mass ranging from 2 × 10(6) to 4.3 × 10(7) g mol(-1), which are not well fractionated by means of classical size-exclusion chromatography. HBPs were enzymatically produced from sucrose by the tandem action of an amylosucrase and a branching enzyme mimicking in vitro the elongation and branching steps involved in glycogen biosynthesis. Size and molar mass distributions were studied by AF4, coupled with online quasi-elastic light scattering (QELS) and transmission electron microscopy. AF4-MALLS-QELS has shown a remarkable agreement between hydrodynamic radii obtained by online QELS and by AF4 theory in normal mode with constant cross flow. Molar mass, size, and dispersity were shown to significantly increase with initial sucrose concentration, and to decrease when the branching enzyme activity increases. Several populations with different size range were observed: the amount of small size molecules decreasing with increasing sucrose concentration. The spherical and dense global conformation thus highlighted was partly similar to native glycogens. A more detailed study of HBPs synthesized from low and high initial sucrose concentrations was performed using complementary enzymatic hydrolysis of external chains and chromatography. It emphasized a more homogeneous branching pattern than native glycogens with a denser core and shorter external chains.

Influence of amylopectin structure and degree of phosphorylation on the molecular composition of potato starch lintners.

Morphology, molecular structure, and thermal properties of potato starch granules with low to high phosphate content were studied as an effect of mild acid hydrolysis (lintnerization) to 80% solubilization at two temperatures (25 and 45°C). Light microscopy showed that the lintners contained apparently intact granules, which disintegrated into fragments upon dehydration. Transmission electron microscopy of rehydrated lintners revealed lacy networks of smaller subunits. The molecular composition of the lintners suggested that they largely consisted of remnants of crystalline lamellae. When lintnerization was performed at 45°C, the lintners contained more of branched dextrins compared to 25°C in both low and intermediate phosphate-containing samples. High-phosphate-containing starch was, however, unaffected by temperature and this was probably due to an altered amylopectin structure rather than the phosphate content. After lintnerization, the melting endotherms were broad with decreased onset and increased peak melting temperatures. The relative crystallinity was lower in lintners prepared at 45°C. A hypothesis that combines the kinetics of lintnerization with the molecular and thermal characteristics of the lintners is presented.

Molecular and supra-molecular structure of waxy starches developed from cassava (Manihot esculenta Crantz).

The aim of this work was to characterize the amylopectin of low amylose content cassava starches obtained from transgenesis comparatively with a natural waxy cassava starch (WXN) discovered recently in CIAT (International Center for Tropical Agriculture). Macromolecular features, starch granule morphology, crystallinity and thermal properties of these starches were determined. M¯(w) of amylopectin from the transgenic varieties are lower than WXN. Branched and debranched chain distributions analyses revealed slight differences in the branching degree and structure of these amylopectins, principally on DP 6-9 and DP>37. For the first time, a deep structural characterization of a series of transgenic lines of waxy cassava was carried out and the link between structural features and the mutated gene expression approached. The transgenesis allows to silenced partially or totally the GBSSI, without changing deeply the starch granule ultrastructure and allows to produce clones with similar amylopectin as parental cassava clone.

In vitro synthesis of hyperbranched α-glucans using a biomimetic enzymatic toolbox.

Glycogen biosynthesis requires the coordinated action of elongating and branching enzymes, of which the synergetic action is still not clearly understood. We have designed an experimental plan to develop and fully exploit a biomimetic system reproducing in vitro the activities involved in the formation of α(1,4) and α(1,6) glycosidic linkages during glycogen biosynthesis. This method is based on the use of two bacterial transglucosidases, the amylosucrase from Neisseria polysaccharea and the branching enzyme from Rhodothermus obamensis . The α-glucans synthesized from sucrose, a low cost agroresource, by the tandem action of the two enzymes, have been characterized by using complementary enzymatic, chromatographic, and imaging techniques. In a single step, linear and branched α-glucans were obtained, whose proportions, morphology, molar mass, and branching degree depended on both the initial sucrose concentration and the ratio between elongating and branching enzymes. In particular, spherical hyperbranched α-glucans with a controlled mean diameter (ranging from 10 to 150 nm), branching degree (from 10 to 13%), and weight-average molar mass (3.7 × 10(6) to 4.4 × 10(7) g.mol(-1)) were synthesized. Despite their structure, which is similar to that of natural glycogens, the mechanisms involved in their in vitro synthesis appeared to be different from those involved in the biosynthesis of native hyperbranched α-glucans.

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis.

BACKGROUND: Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. METHODS: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure. RESULTS: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family. CONCLUSIONS: RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. GENERAL SIGNIFICANCE: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.

Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules.

BACKGROUND: Starch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed "resistant starch" (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss. RESULTS: In this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants. CONCLUSIONS: This is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Structure and property engineering of α-D-glucans synthesized by dextransucrase mutants.

Seven dextran types, displaying from 3 to 20% α(1→3) glycosidic linkages, were synthesized in vitro from sucrose by mutants of dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, obtained by combinatorial engineering. The structural and physicochemical properties of these original biopolymers were characterized. When asymmetrical flow field flow fractionation coupled with multiangle laser light scattering was used, it was determined that weight average molar masses and radii of gyration ranged from 0.76 to 6.02 × 10(8) g·mol(-1) and from 55 to 206 nm, respectively. The ν(G) values reveal that dextrans Gcn6 and Gcn7, which contain 15 and 20% α(1→3) linkages, are highly branched and contain long ramifications, while Gcn1 is rather linear with only 3% α(1→3) linkages. Others display intermediate molecular structures. Rheological investigation shows that all of these polymers present a classical non-Newtonian pseudoplastic behavior. However, Gcn_DvΔ4N, Gcn2, Gcn3, and Gcn7 form weak gels, while others display a viscoelastic behavior that is typical of entangled polymer solutions. Finally, glass transition temperature T(g) was measured by differential scanning calorimetry. Interestingly, the T(g) of Gcn1 and Gcn5 are equal to 19.0 and 29.8 °C, respectively. Because of this low T(g), these two original dextrans are able to form rubber and flexible films at ambient temperature without any plasticizer addition. The mechanical parameters determined for Gcn1 films from tensile tests are very promising in comparison to the films obtained with other polysaccharides extracted from plants, algae or microbial fermentation. These results lead the way to using these dextrans as innovative biosourced materials.

Effects of grinding processes on enzymatic degradation of wheat straw.

The effectiveness of wheat straw fine to ultra-fine grindings at pilot scale was studied. The produced powders were characterised by their particle-size distribution (laser diffraction), crystallinity (WAXS) and enzymatic degradability (Trichoderma reesei enzymatic cocktail). A large range of wheat-straw powders was produced: from coarse (median particle size ∼800 µm) to fine particles (∼50 µm) using sieve-based grindings, then ultra-fine particles ∼20 µm by jet milling and ∼10 µm by ball milling. The wheat straw degradability was enhanced by the decrease of particle size until a limit: ∼100 µm, up to 36% total carbohydrate and 40% glucose hydrolysis yields. Ball milling samples overcame this limit up to 46% total carbohydrate and 72% glucose yields as a consequence of cellulose crystallinity reduction (from 22% to 13%). Ball milling appeared to be an effective pretreatment with similar glucose yield and superior carbohydrate yield compared to steam explosion pretreatment.

Hydrolysis of concentrated raw starch: A new very efficient α-amylase from Anoxybacillus flavothermus.

A new α-amylase from Anoxybacillus flavothermus (AFA) was found to be effective in hydrolyzing raw starch in production of glucose syrup at temperatures below the starch gelatinization temperature. AFA is very efficient, leading to 77% hydrolysis of a 31% raw starch suspension. The final hydrolysis degree is reached in 2-3h at starch concentrations lower than 15% and 8-24h at higher concentrations. AFA is also very efficient in hydrolyzing the crystalline domains in the starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in agreement with the observed preferential hydrolysis of amylopectin. Amylose-lipid complexes are degraded in a second step, yielding amylose fragments which then re-associate into B-type crystalline structures forming the final α-amylase resistant fraction. The mode of action of AFA and the factors limiting complete hydrolysis are discussed in details.

Effects of granule-bound starch synthase I-defective mutation on the morphology and structure of pyrenoidal starch in Chlamydomonas.

Lowering of the CO2 concentration in the environment induces development of a pyrenoidal starch sheath, as well as that of pyrenoid and CO2-concentrating mechanisms, in many microalgae. In the green algae Chlamydomonas and Chlorella, activity of granule-bound starch synthase (GBSS) concomitantly increases under these conditions. In this study, effects of the GBSS-defective mutation (sta2) on the development of pyrenoidal starch were investigated in Chlamydomonas. Stroma starch- and pyrenoid starch-enriched samples were obtained from log-phase cells grown with air containing 5% CO2 (high-CO2 conditions favouring stromal starch synthesis) and from those transferred to low-CO2 conditions (air level, 0.04% CO2, favouring pyrenoidal starch synthesis) for 6h, respectively. In the wild type, total starch content per culture volume did not increase during the low-CO2 conditions, in spite of the development of pyrenoidal starch, suggesting that degradation of some part of stroma starch and synthesis of pyrenoid starch simultaneously occur under these conditions. Even in the GBSS-deficient mutants, pyrenoid and pyrenoid starch enlarged after lowering of the CO2 concentration. However, the morphology of the pyrenoid starch was thinner and more fragile than the wild type, suggesting that GBSS does affect the morphology of pyrenoidal starch. Surprisingly normal GBSS activity is shown to be required to obtain the high A-type crystallinity levels that we now report for pyrenoidal starch. A model is presented explaining how GBSS-induced starch granule fusion may facilitate the formation of the pyrenoidal starch sheath.

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp.

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.