Enzymatic Debranching Research Articles

Characterization of the baking-induced changes in starch molecular and crystalline structures in sugar-snap cookies

This study aims to understand the starch molecular structural changes from baking sugar-snap cookies. Changes in the whole-molecule size distribution and chain-length distribution of the parent wheat flour and from final cookie products were measured by size-exclusion chromatography with and without enzymatic debranching, and the results fitted by two biosynthesis-based models. Fraction crystallinity was also analyzed. After cooking, there was a significant decrease in average molecular sizes of amylopectin and in the average lengths of amylose chains, and some starch granules lost birefringence. However, the chain-length distributions of amylopectin showed no noticeable difference, resulting in little change in relative crystallinity and gelatinization temperatures. Both the short-range ordered structure and the periodic lamellar structure were disrupted. This study provides new insight into starch structural changes in sugar-snap cookies after baking, which play an important role in determining final cookie quality. For example, a decrease in size of amylose chains influences cookie sensory properties, and thus can be used as an additional tool for choice of grains.

The Effect of Processing Conditions on Production of Resistant Starch Type III (RS3) from Breadfruit Starch

Resistant starchtype III (RS3) possess various beneficial physiological effects as well as functionality in food containing resistant starch (RS). In this study, RS3 is produced from breadfruit (Artocarpus altilis) starch using different processing conditions involving disruption of starch granules, enzymatic debranching of starch polymer, starch retrogradation and drying. Then, the morphology and size of the starch granules were observed using scanning electron microscope (SEM). A sample with the highest RS content (54.59%), denoted as P8 was produced when the breadfruit starch was suspended in distilled water, gelatinised by heating at 90°C for 30 minutes, followed by starch debranching with 20 New Pullulanase Unit Novo (NPUN) enzyme per g starch at 60°C for 24 hours. The suspension was then autoclaved at 121°C for 1 hour and cold stored at 4°C for 24 hours. Further treatment of P8 sample with 0.5 M HCl acid at 60°C for 24 hours, produced HCl-breadfruit RS3 with 57.86% of RS content. It was observed that hydrolysis of breadfruit RS3 sample with HCl produced smoother starch granules with more crystalline region and proportionally increased the RS content when compared to other treated sample. This study revealed that different processing conditions were significantly influenced the percentage of yield and properties of RS type III produced from breadfruit starch.

Production and Characterization of Type III Resistant Starch from Native Wheat Starch Using Thermal and Enzymatic Modifications

The most widely employed methodologies for type III (retrograded) resistant starch formation is a cyclic heating-cooling treatment or its combination with a previous enzymatic debranching step. In this work, two retrogradation temperatures, 0 and 4 °C, were evaluated for obtaining type III resistant starch from non-debranched and pullulanase-debranched wheat starch. The starches were characterized by their thermal and pasting behavior. Also, microstructural and crystalline aspects were related to the production process and the nutritional properties of the products. The most influencing factor for resistant starch formation was the application of the pullulanase-debranching step previous to the cyclic treatment. This led to a higher starch retrogradation and a more compact microstructure, which decreased the thickening capacity and the digestibility of the starches. Then, even though all the obtained starches exhibited lower digestibility, the pullulanase-debranched ones presented the lowest estimated glycemic index (ca. 34%), showing the highest yield of resistant starch formation. Finally, this work presents a concise protocol for obtaining type III resistant starch enriched powder with low digestibility that could be used in the formulation of different healthy foods such as dairy products, cereal bars, and even drinks.

Preparation of thermally stable and digestive enzyme resistant flour directly from Japonica broken rice by combination of steam infusion, enzymatic debranching and heat moisture treatment

Through a series of gelatinization, enzymatic debranching and heat moisture treatment, unmilled japonica broken rice was applied to prepare thermally stable and digestive enzyme resistant flour. The optimal conditions of gelatinization temperature, enzyme/substrate ratio, and debranching duration were determined by the three-factor three-level response surface methodology (RSM). Raw flour and products processed in each step were collected to test and compare the hydration ability, reheated solubility, pasting property, starch classification (RDS, SDS, RS) and in vitro digestibility. The results of RSM showed that E/S ratio was the main factor influencing the RS formation, while influence aroused by hydrolysis time and gelatinization temperature were minor. Gelatinization broke down the intermolecular bonds of starch molecules, allowed the hydrogen bonding sites to engage more water, and improved the availability of digestion. However, debranching provided an opportunity for the alignment of short-chain and long-chain amylose molecules. Therefore, RS increased to 42%, and digestibility decreased accordingly. HMT could further enhance the molecular rearrangement, while the RS was increased to as high as 55% and the final product met the low GI criteria.

Effects of Nonstarch Genetic Modifications on Starch Structure and Properties.

This paper examines if, in maize, starch structure and starch-dependent properties might be altered by pleiotropic effects arising from genetic modifications that are not directly related to starch synthesis. The molecular structure, specifically the starch chain-length distributions (CLDs), of two maize lines transformed with Bar (bialaphos resistance) and Cry1c genes (an artificial gene, encoding proteinaceous insecticidal δ-endotoxins) were compared to those of their control lines. The two transgenes are responsible for herbicidal resistance and insect tolerance, respectively. The starch CLDs were measured by enzymatic debranching and measuring the molecular weight distributions of the resulting linear chains. It was found that although all the lines had similar amylose contents, the CLDs of both amylopectin and amylose for Cry1c were noticeably different from the others, having more short amylopectin and long amylose chains. These CLDs are known to affect functional properties, and indeed it was found that the Cry1c transgenic lines showed a lower gelatinization temperature and faster digestion rate than the control or Bar lines. However, a slower digestion rate is nutritionally desirable. Thus, pleiotropic effects from genetic modifications can indirectly but significantly affect the starch synthesis pathway and thus change functional properties of significance for human health.

Competition between Granule Bound Starch Synthase and Starch Branching Enzyme in Starch Biosynthesis

BackgroundStarch branching enzymes (SBE) and granule-bound starch synthase (GBSS) are two important enzymes for starch biosynthesis. SBE mainly contributes to the formation of side branches, and GBSS mainly contributes for the synthesis of amylose molecules. However, there are still gaps in the understanding of possible interactions between SBE and GBSS.ResultsNineteen natural rice varieties with amylose contents up to 28% were used. The molecular structure, in the form of the chain-length distribution (CLDs, the distribution of the number of monomer units in each branch) was measured after enzymatic debranching, using fluorophore-assisted carbohydrate electrophoresis for amylopectin and size- exclusion chromatography for amylose. The resulting distributions were fitted to two mathematical models based on the underlying biosynthetic processes, which express the CLDs in terms of parameters reflecting relevant enzyme activities.ConclusionsFinding statistically valid correlations between the values of these parameters showed that GBSSI and SBEI compete for substrates during rice starch biosynthesis, and synthesis of amylose short chains involves several enzymes including GBSSI, SBE and SSS (soluble starch synthase). Since the amylose CLD is important for a number of functional properties such as digestion rate, this knowledge is potentially useful for developing varieties with improved functional properties.

Purification of Branched Dextrin from Nägeli Amylodextrin by Ethanol Precipitation and Characterization of Its Aggregation Property in Methanol-Water.

Ethanol precipitation process for purification of branched dextrin (BD) in Nägeli amylodextrin from waxy rice starch was developed. Temperature and ethanol concentration for precipitation were main parameters affecting the recovery and purity of BD, and the purification condition at 4 °C and 10 % (v/v) ethanol in water was adopted. After four-time precipitation, the BD recovery was 34.6 %, whereas the purity improved from 78.5 % at the initial to 94.5 % at the four-time purified BD (BD4). BD4 mainly showed a chain length distribution between 18 to 35 with a mode length of 25, which shifted after enzymatic debranching with isoamylase to that between 9 and 20 with a mode length of 14. Each purified BD was solubilized in water, and each solution was mixed with methanol-water at 25 °C to a final methanol concentration of 16 M. The flakes of BD precipitated with 16 M methanol exhibited an A-type crystal structure by an X-ray diffraction analysis, and the speed generation of white flakes in 16 M methanol dramatically increased as the purification time increased. The effect of addition of highly branched cyclic dextrin (HBCD) or sodium tetraborate on BD aggregation in 16 M methanol was also investigated, where the former retarded aggregation but the latter had no effect on the velocity. Thus, the purified BD enables rapid characterization of aggregation of double helix structures of A-type crystal structure, and screening of compounds which could affect the phenomena for prediction of potentials in starch modification as well.

Starch branching enzymes contributing to amylose and amylopectin fine structure in wheat

Starch branching enzymes (SBEs) play a major role in determining starch molecular structure in cereal endosperm. This study investigates how SBEIIs contribute to the chain-length distributions (CLDs) of both amylopectin and amylose, obtained by enzymatic debranching of native starch. Wheat starches with low (37%) to high (93%) amylose content were obtained through altering SBEII in planta. Multiple components were detected in both amylose and amylopectin CLDs. Model fitting of these CLDs reveals a quantitative association between the enzyme activities in amylopectin and amylose. SBEIIa modifies shorter branches (degree of polymerization DP ≲ 12) in amylopectin and longer amylose chains with a CLD peak at ˜3000 DP. SBEIIb acts on longer branches (DP≲ 32) in amylopectin, while its effect on amylose fine structure is not significant. Using both the amylose and amylopectin models to analyze the CLD reveals connections between amylose and amylopectin in wheat starch biosynthesis.

Effects of amylose content and enzymatic debranching on the properties of maize starch-glycerol monolaurate complexes

The effects of pullulanase debranching on the properties of maize starch (waxy, normal and high amylose)-glycerol monolaurate (GML) complexes were studied. Pullulanase pretreatment produced more starch chains with favourable lengths (DP ≥ 25) to encourage inclusion complex formation. The debranched starch showed higher amylose content (from 11.2 to 58.6%) than the native starch (from 0.68 to 53.4%). The V-type characteristic diffraction peaks (7.5°, 13.1° and 20.2° 2θ) became more obvious after the starch received the debranching treatment. Thermal properties indicated that the melting enthalpy of the debranched starch-GML complexes (from 6.45 to 10.13 J/g) was higher than those of the untreated complexes (from 0 to 8.91 J/g). The swelling power values and degree of hydrolysis of the starches decreased with the increase in amylose content. In vitro digestibility analysis suggested that the hydrolysis of the starch-GML samples was further restricted by applying the debranching treatment to the starch.

Preparation and characterization of inclusion complexes between debranched maize starches and conjugated linoleic acid

Normal and high amylose maize starches were enzymatically debranched at different degrees, and then used for the preparation of inclusion complexes with conjugated linoleic acid (CLA). The recovery yield of CLA, percent ratio of CLA in the complexes to its initial amount, was increased by debranching, reaching 65% with a debranched high amylose starch (24 h). High amylose starch was more effective in the formation of CLA complexes with greater stability and crystallinity than normal starch. Both V6I and II crystals were formed by the complexation with CLA, and more V6II crystal was formed as the debranching and complexation times increased. However, an excessive complexation reaction for 24 h at 90 °C resulted in decreasing the CLA recovery, especially when normal maize starch was used. X-ray diffraction analysis revealed that B type crystals induced by starch recrystallization were also present during the complex formation. The enzymatic debranching facilitated the formation of crystalline complexes with CLA having a high thermal stability. The enzymatic debranching of starch enhanced the formation of crystalline complexes with CLA and availability of linear starch chains to form the complexes with CLA.

Self-assembly kinetics of debranched short-chain glucans from waxy maize starch to form spherical microparticles and its applications.

Starch microparticles (SMPs) of well-defined size and morphology were synthesized through pullulanase-mediated debranching of waxy maize starch followed by spontaneous re-assembly of the resulting short-chain glucan molecules in aqueous solution. Enzymatic debranching of amylopectins from native starch generated two major fractions corresponding to a smaller glucan and partially digested larger amylopectin molecules. The ratio of short-chain glucan (SCG) over partially digested amylopectin (PDAp) turned out to be the deterministic factors for the size and crystallinity of SMPs, of which the ratio could be controlled by the concentration of debranching enzyme. The PDAp fraction was closely associated with the creation of nuclei, determining the growth kinetics of SMPs which led to the formation of SMPs with a diameter ranging from 0.52.5 μm. In addition, we demonstrated that iron oxide nanoparticles (IONPs) were successfully incorporated into the starch microstructure by introducing them during the self-assembly reaction, conferring desired functionality onto the final SMPs. The incorporated IONPs rendered the SMPs an excellent magnetic sensitivity, which were successfully applied for the separation and concentration of target bacteria upon conjugation of specific antibody on the surface of SMPs. The simple processes and biocompatible nature of starch would make this approach attractive for many applications in the area of food, medicine and other related materials sciences.

Enzymatic debranching of starches from different botanical sources for complex formation with stearic acid

Starches from different botanical sources (corn, potato, and tapioca) were modified enzymatically using pullulanase, and the resultant debranched starches were used to formulate inclusion complexes with stearic acid. Effects of the debranching and botanical source of starch on the degree of complex formation, thermal and crystalline characteristics of the complexes were analyzed. The amount of stearic acid in the complexes, recovered as precipitates from aqueous reaction mixtures, was high in the order of tapioca, potato and corn starches. By debranching for 12 h, tapioca starch could form the complex with stearic acid at a recovery yield of 71%, which was the maximum among the values obtained with other starches. Further, all the starch/stearic acid complexes exhibited a combination of V- and B-type polymorphs under X-ray diffraction analyses. From a thermal transition analyses, both type I and II crystals were observed in the inclusion complexes. Melting enthalpy of the inclusion complexes improved by debranching, indicating that the complexes were thermally stabilized. Overall data revealed that the enzymatic debranching of starch effectively improves the complexation, crystallinity, dispersion ability and stability of starch-stearic acid complex.

Structure Dependent-Immunomodulation by Sugar Beet Arabinans via a SYK Tyrosine Kinase-Dependent Signaling Pathway.

There is much interest in the immunomodulatory properties of dietary fibers but their activity may be influenced by contamination with microbial-associated molecular patterns (MAMPs) such as lipopolysaccharide (LPS) and lipoteichoic acids, which are difficult to remove completely from biological samples. Bone marrow-derived dendritic cells (BMDCs) from TLR2x4 double-KO mice were shown to be a reliable approach to analyse the immunomodulatory properties of a diverse range of dietary fibers, by avoiding immune cell activation due to contaminating MAMPs. Several of the 44 tested dietary fiber preparations induced cytokine responses in BMDCs from TLR2x4 double-KO mice. The particulate fractions of linear arabinan (LA) and branched arabinan (BA) from sugar beet pectin were shown to be strongly immune stimulatory with LA being more immune stimulatory than BA. Enzymatic debranching of BA increased its immune stimulatory activity, possibly due to increased particle formation by the alignment of debranched linear arabinan. Mechanistic studies showed that the immunostimulatory activity of LA and BA was independent of the Dectin-1 recognition but Syk kinase-dependent.

Surface-Engineered Starch Magnetic Microparticles for Highly Effective Separation of a Broad Range of Bacteria

Polymeric magnetic particles (PMPs) have become a powerful tool for the separation and concentration of microorganisms from a heterogeneous liquid matrix. The functionalization of PMPs with polycationic polymers, such as chitosan, provides an effective means of capturing a broad spectrum of pathogenic bacteria through the intrinsic nature of chitosan interacting with the surface components of bacteria. Here, we report a fairly simple approach for the preparation of starch magnetic microparticles (SMMPs) through molecular rearrangement of short-chain glucans (SCGs) produced by enzymatic debranching of waxy maize starch. The surfaces of SMMPs were readily functionalized with chitosan through electrostatic interaction and hydrogen bonding. The chitosan-functionalized SMMPs (CS@SMMPs) showed high capture efficiency (>90%) for both Gram-positive and Gram-negative bacteria. To further investigate the mechanisms of chitosan–bacteria interaction, we employed model bacteria with different surface compositions. The...

Molecular Rearrangement of Glucans from Natural Starch To Form Size-Controlled Functional Magnetic Polymer Beads.

Herein, we report a fairly simple and environmentally friendly approach for the fabrication of starch-based magnetic polymer beads (SMPBs) with uniform shape and size through spontaneous rearrangement of short-chain glucan (SCG) produced by enzymatic debranching of waxy maize starch. The paramagnetic materials, dextran-coated iron oxide nanoparticles (Dex@IONPs), were readily incorporated into the starch microstructure and rendered a superparamagnetic property to the SMPBs. The morphology and size of resulting SMPBs turned out to be modulated by Dex@IONPs in a concentration-dependent manner, of which Dex@IONPs was assumed to be acting as a seed inducing the epitaxial crystallization of SCG and further transforming it into homogeneous microparticles. The surface of SMPBs was readily functionalized with an antibody through a one-step reaction using a linker protein. The immuno-SMPBs showed great capture efficiency (>90%) for target bacteria. The colloidal stability and favorable surface environment for biomolecules are believed to be responsible for the high capture efficiency and specificity of the SMPBs. Furthermore, the captured bacteria along with antibody and linker protein were effectively eluted from the surface of SMPBs by adding free maltose, making this new material suitable for various chromatographic applications.

Starch film-coated microparticles for oral colon-specific drug delivery

The aim of this study was to prepare and characterize a novel type of starch-coated microparticles (MPs) allowing site-specific delivery of bioactives to the colon. An oral colon-specific controlled-release system was developed in the form of MPs coated with a resistant starch (RS2/RS3) film (RS@MPs) through an aqueous suspension coating process. The RS2 was chosen from a high-amylose cornstarch with 88.5% digestion resistibility. The RS3 was prepared by a high-temperature/pressure (HTP) treatment, with the following of enzymatic debranching, and retrogradation, resulting in a dramatic increase in enzymatic resistance (RS3 content: 76.6%). RS@MPs showed 40.7% of 5-aminosalicylic acid release within 8 h. The in vivo study of fluorescein-loaded RS@MPs indicated the high acidic and enzymatic resistibility of RS@MPs and a restrained release in the upper GIT. Therefore, RS@MPs has revealed to be a high potential system for accurately targeting bioactive compound delivery to the colon.

Complex formation between starch and stearic acid: Effect of enzymatic debranching for starch

Effect of debranching for a high-amylose starch (∼70% amylose) on its V-complex formation with stearic acid was examined. Gel-permeation chromatograms showed that amylopectin was degraded to smaller molecules as the debranching time increased from 6 to 24 h. Increased formation of debranched starch/stearic acid complexes (recovery yield of stearic acid from 45.17 to 89.31% and starch from 39.92 to 55.43%) was observed with increased debranching time (from 6 to 24 h) and complexation time (from 6 to 24 h). The X-ray diffraction patterns of the debranched starch/stearic acid complexes displayed a mixture of B-type and V-type patterns, with 2θ peaks at 7.6°, 13.1°, 17.2°, 20°, 21.6°, and 23.4°. The melting temperature and enthalpy changes of the debranched starch/stearic acid complexes were gradually enhanced with increasing debranching time. These results suggest that starch can be modified by debranching to produce a significant amount of debranched starch/stearic acid complexes.

Effect of diurnal photosynthetic activity on the fine structure of amylopectin from normal and waxy barley starch

The impact of diurnal photosynthetic activity on the fine structure of the amylopectin fraction of starch synthesized by normal barley (NBS) and waxy barley (WBS), the latter completely devoid of amylose biosynthesis, was determined following the cultivation under normal diurnal or constant light growing conditions. The amylopectin fine structures were analysed by characterizing its unit chain length profiles after enzymatic debranching as well as its φ,β-limit dextrins and its clusters and building blocks after their partial and complete hydrolysis with α-amylase from Bacillus amyloliquefaciens, respectively. Regardless of lighting conditions, no structural effects were found when comparing both the amylopectin side-chain distribution and the internal chain fragments of these amylopectins. However, the diurnally grown NBS and WBS both showed larger amylopectin clusters and these had lower branching density and longer average chain lengths than clusters derived from plants grown under constant light conditions. Amylopectin clusters from diurnally grown plants also consisted of a greater number of building blocks, and shorter inter-block chain lengths compared to clusters derived from plants grown under constant light. Our data demonstrate that the diurnal light regime influences the fine structure of the amylopectin component both in amylose and non-amylose starch granules.

Acid-thinned corn starch-impact of modification parameters on molecular characteristics and functional properties

Acid-thinned corn starches were prepared systematically by varying acid concentrations (0.09–0.72 M HCl) and hydrolysis times (4 and 24 h). The relationships between modification parameters, molecular starch structure, and functional properties were investigated. The carbohydrate solubilization was significantly enhanced with increasing acid concentration only in the long-term process. SEM micrographs revealed the preservation of the granular structure and the thermal properties changed only moderately applying different hydrolysis conditions. Molecular characterization using SEC-MALS (size exclusion chromatography-multi angle laser light scattering) showed a strong degradation of amylopectin after 4 h of hydrolysis. Enzymatic debranching prior to the analysis and separation of the amylose peak using deconvolution enabled the calculation of MMD curves of the acid-thinned amylose fraction. The data gave evidence of extensive molecular degradation of the amylose with increasing acid concentration when hydrolyzed for 24 h. The hot paste viscosity decreased with decreasing Mw and the sol-to-gel-transition temperature was found to be strongly dependent on both, the Mw of the starch as well as the amylose fraction. The correlation between molecular properties and gel strength gave evidence of an optimum degree of hydrolysis. Acid-thinning of the starch to Mw of about 12 · 106–8 · 106 g · mol−1 and 3 · 105–2.5 · 105 g · mol−1 for the amylose fraction, respectively, resulted in the highest gel strength within the present study. However, differences in gel strength had only marginal impact on the gel elasticity. The peak separation permitted a comprehensive and detailed characterization of the amylose fraction. The obtained data proved the exposed role of the amylose in the gelation process of acid-thinned starches and their gel firmness. Correlations between the modification process, the molecular starch structure, and resulting functional properties were found.

Molecular characterization of acid‐thinned wheat, potato and pea starches and correlation to gel properties

Molecular parameters and gel properties of systematically prepared acid‐thinned starch products (wheat, potato, and pea) were investigated. The impacts of acid type (HCl, H2SO4), acid concentration (0.36 and 0.72 N), and hydrolysis time (4 and 24 h) were found to be statistically significant on weight‐average molar mass (Mw) of the starch as revealed by analysis of variance (ANOVA). Especially for potato starch samples, the blue value (BV) was found to increase after short‐time hydrolysis and decrease after longer modification, indicating a reduced amount of polysaccharide chains able to complex with iodine, which was found for all products. Simultaneously, the wavelength of the absorption maximum (λmax) decreased systematically with an increasing degree of modification, giving evidence for decreasing length of linear polymer chains. Enzymatic debranching of amylopectin and dextrins and molecular characterization by means of size exclusion chromatography‐multi angle laser light scattering (SEC‐MALS) enabled the detection of Mw and the amount of amylose fraction. Generally, declining Mw of amylose was accompanied by a mass‐specific loss. Starch pastes with very good solution states were prepared in general. Pastes of the acid‐thinned starches showed dominant viscous behavior, whereat storage and loss modulus decreased with increasing degradation. Additionally, a significant reduction of the sol‐to‐gel‐transition temperature was found applying oscillation measurements. Increasing gel strength of the starch samples was detected due to acid‐thinning up to a certain degree of modification and optimal molecular parameters of the starch as well as the amylose fraction were determined resulting best strength properties. The gel clarity was reduced due to acid modification in general.