Enzymatic Debranching Research Articles

Improvement in crystallization and digestion resistance of debranched amylopectin dextrins using sodium sulfate

In this study, sodium sulfate, a typical kosmotrope salt (100–500 mM), was used to resistant dextrin crystals prepared via enzymatic debranching, and its effects on the crystallization behavior and in vitro digestibility of the dextrin crystals were investigated. Waxy maize starch was enzymatically debranched (50 °C for 24 h) and simultaneously crystallized (50 °C for 2 d) in aqueous solutions containing sodium sulfate (0–500 mM). The yield of dextrin crystals was increased using a sodium sulfate solution (84% at 500 mM sodium sulfate). The dextrin crystals comprise an A-type arrangement of double helices with higher thermal stability than native starch. Based on the DSC, XRD, and in vitro digestibility test, the high thermal stability and crystallinity of the dextrin crystals provide high resistance to digestive enzymes. The addition of sodium sulfate during crystallization increased the crystallinity and thus increased the digestion resistance. According to the in vitro digestibility test, dextrin crystals prepared with 500 mM sodium sulfate contained a resistant fraction of 80.36% in the uncooked state. After cooking (boiling), over half of the dextrin crystals remain resistant to digestive enzymes. This study showed that sodium sulfate, a typical kosmotrope salt, could accelerate the crystallization process of the debranched waxy maize starch chain, increasing yield, thermal stability, crystallinity, and digestion resistance in a short reaction period.

Green banana resistant starch: A promising potential as functional ingredient against certain maladies.

This review covers the significance of green banana resistant starch (RS), a substantial polysaccharide. The food industry has taken an interest in green banana flour due to its 30% availability of resistant starch and its approximately 70% starch content on a dry basis, making its use suitable for food formulations where starch serves as the base. A variety of processing techniques, such as heat-moisture, autoclaving, microwaving, high hydrostatic pressure, extrusion, ultrasound, acid hydrolysis, and enzymatic debranching treatments, have made significant advancements in the preparation of resistant starch. These advancements aim to change the structure, techno-functionality, and subsequently the physiological functions of the resistant starch. Green bananas make up the highest RS as compared to other foods and cereals. Many food processing industries and cuisines now have a positive awareness due to the functional characteristics of green bananas, such as their pasting, thermal, gelatinization, foaming, and textural characteristics. It is also found useful for controlling the rates of cancer, obesity, and diabetic disorders. Moreover, the use of GBRS as prebiotics and probiotics might be significantly proved good for gut health. This study aimed at the awareness of the composition, extraction and application of the green banana resistant starch in the future food products.

Preparation of low estimated glycemic index (eGI) chestnut flours using enzymatic debranching and heat moisture treatment

A low estimated glycemic index (eGI) chestnut flour was prepared using a combination of pullulanase debranching and heat moisture treatment (HMT). The modified samples had a lower eGI range of 49.1–53.0 compared to native chestnut flour (NCF) which had an eGI of 67.3. Furthermore, the dual treatment was found to be more effective than a single treatment. The total starch content decreased from 57.0 to 47.1 g/100 g after debranching and HMT, resulting in an increase in amylose content from 30.6 % to 48.5 %. A 1H NMR (nuclear magnetic resonance) analysis revealed that modified flours showed a reduction in α-1,6-glycosidic bonds. NCF exhibited a C-type crystal structure, while the debranched samples showed B+V-type crystallinity with an increased peak at 20°(2θ). Debranching increased the proportion of short linear chains, which facilitated the formation of amylose-lipid complexes. HMT resulted in partial starch gelatinization and the breakage of amylopectin. These findings emphasize the potential applications of debranching and HMT in producing chestnut flour with a low eGI, which can further be used in functional food products.

Effects of single and dual modifications with debranching and heat-moisture treatments on physicochemical, rheological, and digestibility properties of proso millet starch

The effects of single and dual modifications with heat-moisture treatment (HMT) and enzymatic debranching on the structural, physicochemical, rheological, and in-vitro digestibility properties of proso millet starch (PMS) were comparatively studied. Amylose content increased after gelatinization and all modification treatments. Debranching and dual modification (debranching followed by HMT) decreased the average chain length and swelling power but increased the solubility of PMS. HMT did not change the average chain length and chain length distribution in native and debranched starches but decreased the swelling power and solubility of native starch. The A-type X-ray diffraction pattern of PMS was converted to the A + V type by HMT and to the B + V type by debranching and dual modification. FTIR investigation showed that the short-range molecular order of all modified starches was disrupted. The viscoelasticity and viscosity of all starch pastes decreased in the following order: PMS > HMT starch > debranched starch > dual modified starch. In-vitro starch digestibility showed that HMT increased slowly digestible starch (SDS) to 32.88 %, while debranching increased resistant starch (RS) to 77.14 %. HMT increased RS to 86.17 % in the dual modified starch. Debranching therefore predominates in the dual modification of starch compared to HMT.

Impacts of fatty acid type on binding state, fine structure, and in vitro digestion of debranched starch-fatty acid complexes with different debranching degrees

This study aimed to investigate the effects of fatty acid (FA) type on the binding state, fine structure, and digestibility of debranched maize starch (DMS)-FA complexes with different enzymatic debranching degrees. Maize starch was hydrolyzed by pullulanase for 1 h (DMS1h) and 6 h (DMS6h) and then complexed with seven types of FAs with varying chain lengths and unsaturation degrees, respectively. All the DMS-FA complexes showed V6III-type and B-type crystals. Complex formation greatly increased the relative crystallinity of DMS, but significantly decreased its order degree of short-range structure (p < 0.05). Compared with unsaturated FAs, saturated FAs possessed stronger intermolecular interactions with DMS. DMS6h-FA complexes exhibited a markedly higher complexing degree (p < 0.05) than the corresponding DMS1h-FA complexes. The FA molecules in DMS1h-FA complexes were primarily physically trapped outside the amylose helices, whereas those in DMS6h-FA complexes were mainly weakly bound to the cavity of amylose helices. The resistant starch (RS) content and relative crystallinity of DMS-FA complexes considerably increased with increasing FA chain length. Furthermore, the highest RS content (38.90 %) and relative crystallinity (24.23 %) were observed in DMS6h-FA complexes. The FA unsaturation degree induced little effect on the RS content and long-range structural order of the complexes.

Resistant starch content of dual modification autoclaving‐cooling and pullulanase debranching on various carbohydrate sources: a systematic review

SummaryResistant starch has a low glycaemic index and is commonly used in functional food development. Resistant starch type III can be produced through the combination of enzymatic debranching and the hot and cold cycle method, which is known as autoclaving‐cooling. The study showed that the dual modification of autoclaving‐cooling and pullulanase debranching can increase the resistant starch content, but it also depends on the carbohydrate source and the condition during the modification process. The study aims to determine the effects of pullulanase debranching and autoclaving‐cooling on resistant starch levels through a systematic review approach and produced 17 studies with 124 data. This dual‐modification study showed that 93.5% of data experienced in resistant starch increase while 6.5% experienced a decrease in resistant starch. The factors that should be considered were cooling/heating temperature and cooling/heating time, the number of cycles, the modification process sequence and enzyme concentration as well as temperature and hydrolysis time. Pullulanase debranching and autoclaving‐cooling dual modification with the highest resistant starch content was found on the chestnut starch sample (64.60%) with autoclave heating process (120 °C, 30 min), followed by debranching process (9 U/g, 50 °C, 10 h) and stored at 4 °C for 24 h.

The Effect of Buckwheat Resistant Starch on Intestinal Physiological Function.

Resistant starch appears to have promising effects on hypertension, cardiovascular and enteric illness. The influence of resistant starch on intestinal physiological function has drawn great attention. In this study, we first analyzed the physicochemical characteristics, including the crystalline properties, amylose content, and anti-digestibility among different types of buckwheat-resistant starch. The influence of resistant starch on the physiological functions of the mouse intestinal system, contained defecation, and intestinal microbes were also evaluated. The results showed that the crystalline mold of buckwheat-resistant starch changed from A to B + V after acid hydrolysis treatment (AHT) and autoclaving enzymatic debranching treatment (AEDT). The amylose content in AEDT was higher than in AHT and raw buckwheat. Moreover, the anti-digestibility of AEDT was also stronger than that in AHT and raw buckwheat. The buckwheat-resistant starch can promote bowel intestinal tract movement. The quantity of intestinal microbe was regulated by buckwheat-resistant starch. Our research demonstrates an effective preparation method for improving the quality of buckwheat-resistant starch and found that buckwheat-resistant starch has the role of adjusting the distribution of the intestinal flora and maintaining the health of the body.

Enzymatic debranching is a key determinant of the xylan-degrading activity of family AA9 lytic polysaccharide monooxygenases

BackgroundPrevious studies have revealed that some Auxiliary Activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) oxidize and degrade certain types of xylans when incubated with mixtures of xylan and cellulose. Here, we demonstrate that the xylanolytic activities of two xylan-active LPMOs, TtLPMO9E and TtLPMO9G from Thermothielavioides terrestris, strongly depend on the presence of xylan substitutions.ResultsUsing mixtures of phosphoric acid-swollen cellulose (PASC) and wheat arabinoxylan (WAX), we show that removal of arabinosyl substitutions with a GH62 arabinofuranosidase resulted in better adsorption of xylan to cellulose, and enabled LPMO-catalyzed cleavage of this xylan. Furthermore, experiments with mixtures of PASC and arabinoglucuronoxylan from spruce showed that debranching of xylan with the GH62 arabinofuranosidase and a GH115 glucuronidase promoted LPMO activity. Analyses of mixtures with PASC and (non-arabinosylated) beechwood glucuronoxylan showed that GH115 action promoted LPMO activity also on this xylan. Remarkably, when WAX was incubated with Avicel instead of PASC in the presence of the GH62, both xylan and cellulose degradation by the LPMO9 were impaired, showing that the formation of cellulose–xylan complexes and their susceptibility to LPMO action also depend on the properties of the cellulose. These debranching effects not only relate to modulation of the cellulose–xylan interaction, which influences the conformation and rigidity of the xylan, but likely also affect the LPMO–xylan interaction, because debranching changes the architecture of the xylan surface.ConclusionsOur results shed new light on xylanolytic LPMO9 activity and on the functional interplay and possible synergies between the members of complex lignocellulolytic enzyme cocktails. These findings will be relevant for the development of future lignocellulolytic cocktails and biomaterials.

Characterization of Pickering emulsion by SCFAs-modified debranched starch and a potent for delivering encapsulated bioactive compound

The Pickering emulsion was prepared by short-chain fatty acids (SCFAs) esterified debranched starch. The microstructure, particle size distribution, rheological properties and stability of the emulsions showed that the introduction of acyl groups improved the ability of starch to stabilize the emulsions, in which the butyrylated starch with longer acyl side chains exhibited higher emulsifying ability compared to acetylated and propionylated starches. Pickering emulsions stabilized with butyrylated starch as stabilizer have better stability after 30 days of storage. The particle size distribution of SCFAs-esterified starch emulsions with enzymatic debranching pretreatment was more concentrated and the droplet size was further reduced, which improved the instability factors such as flocculation, agglomeration or Ostwald ripening of emulsions induced by conventional SCFAs-esterified emulsions and further improved the stability of SCFAs-esterified emulsions. More importantly, butyrylated starch (with or without debranched pretreatment) emulsions exhibited smaller and more uniform droplet shapes and higher curcumin encapsulation efficiency (EE%) in SCFAs-esterified starch emulsions, and the EE% of curcumin in debranched butyrylated starch emulsion increasing from 10.04 % in native starch emulsions to 50.70 %.

Preparation and Characterizations of Curcumin Protection and Delivery System Using Linear Dextrin

In this work, linear dextrins (LDs) with the fragment F-40 (DP = 31.44) were fabricated from waxy potato starch through pasteurization and enzymatic debranching by pullulanase and then separated and extracted by ethanol solutions with different concentrations. The LDs were used to encapsulate hydrophobic ligand curcumin to develop a controlled release system that would increase its flavor in food and functions in medicines. The physicochemical properties and the encapsulation mechanism of the inclusion complexes were investigated. It was found that the loading capability for curcumin, the encapsulation rate, and the yield of the complexes depended on the molecular weight of LD. The yield of the LD-Cur complex, its encapsulation rate, and loading of curcumin were 19.86%, 25.81%, and 29.52 μg/mg, respectively, while the yield of the F-40-Cur complex, its inclusion rate, and loading curcumin reached up to 75.98%, 29.97%, and 37.52 μg/mg, respectively. There were both hydrogen bonding and hydrophobic interactions between LD and curcumin, while hydrogen bonding interactions were predominant between F-40 and curcumin. Curcumin was presented in the complex in an amorphous form. The photothermal stability of curcumin increased after being complexed with LD and further enhanced significantly with F-40. The release of curcumin in the intestine was achieved much more effectively.

Debranched waxy maize resistant dextrin: Synthesis, ethanol fractionation, crystallization, and characterization

Waxy maize (Zea mays L.) dextrins (WMD), prepared by enzymatic debranching, were fractionated through precipitation in different concentrations of aqueous ethanol (50 %, 60 %, and 80 %). The fractionated WMDs were then crystallized at 4 °C or 50 °C for 2 days to prepare resistant dextrins (RD). Recovery yield, chain distribution, crystalline structure, thermal transition, and in vitro digestibility of the fractionated/crystallized WMDs were evaluated. Crystallization at 4 °C resulted in higher yields (>90 %) than that at 50 °C, regardless of the fractionation condition. The chain profile of the dextrins recovered at different temperatures appeared similar, but the longer chains had a greater tendency to associate. Crystal arrangement (A- or B-type) depended on the fractionation and crystallization conditions. Most crystals showed a typical B-type arrangement, except for the crystals prepared at 50 °C with 80 % ethanol (A-type). The enzyme resistance ranged from 49.9 % to 92.4 % depending on the fractionation and crystallization conditions.

Amylose Inter-Chain Entanglement and Inter-Chain Overlap Impact Rice Quality.

Retrogradation of cooked rice happens in two ways: one is by the formation of ordered structures, and the other is through intra- and inter-chain entanglement and inter-chain overlap, which in turn are affected by the amylose chain-length distribution. Both entanglement and overlap could affect rice texture. Here, four amylose samples were isolated from starch by precipitation from a dimethyl sulfoxide solution with butan-1-ol and isoamyl alcohol. Following enzymatic debranching, they were then characterized using size-exclusion chromatography. Amylose solutions (10%, m/v) were made by dissolving amylose in 90% (v/v) DMSO. Amylose gels (10%, w/v) were made by dissolving amylose powders into hot water, followed by cooling. The rigidity of the amylose gels and the structural order were measured using a texture analyzer and X-ray diffractometer, respectively. In the amylose solution, for a given mass of polymer in a fixed amount of solvent, the total occupied volume was reduced when the polymer molecular weight was smaller, resulting in less inter-chain overlap and a lower viscosity of the amylose solution. The overall mobility and diffusion of the molecules were inversely related to the square of the molecular weight until the gelation concentration. Thus, amylose gels in which amylose had a lower molecular weight had a greater chance to permeate into other molecules, which counterintuitively led to more inter-chain entanglement and more rigid amylose gels during retrogradation. This information could help rice breeders improve rice quality by using the molecular structure of starch as a guide.

Fabrication and characterization of Pickering high internal phase emulsions stabilized by debranched starch-capric acid complex nanoparticles

High internal phase emulsions (HIPEs) stabilized by debranched starch-capric acid (DBS-CA) complex nanoparticles were fabricated and their performance was evaluated. DBS-CA was prepared through enzymatic debranching and solid encapsulation methods, and displayed V-type crystalline structure. Contact angle measurements show enhanced hydrophobicity of DBS-CA compared to native starch. The DBS-CA nanoparticles have an average size of 463.77 nm and tended to be aggregating as analyzed by scanning electron microscope and dynamic light scattering particle size analysis. When used as a particulate emulsifier, DBS-CA could stabilize HIPEs with oil volume fraction as high as 80%. The HIPEs showed pH-dependent properties; good storage stability and mechanical strength were achieved within pH range from 3 to 11, especially under alkaline conditions. It was proposed that smaller particle size and higher surface charging were responsible for the more tightly connected gel structure and thus their performance. This study demonstrates a novel approach to fabricate food-grade Pickering HIPEs, which may have many promising potential applications in the food industry.

Effects of extrusion and enzymatic debranching on the structural characteristics and digestibility of corn and potato starches

Amylose content has a profound impact on the contents of slowly digestible starch and resistant starch. Enzymatic debranching is a safe method to increase the amylose content, however, the lower substrate concentration and high viscosity of fully gelatinized starch limit the efficiency and yield of this method. This paper aims to explore the effects of extrusion and enzymatic debranching on increasing the amylose content thereby increasing slowly digestible starch and resistant starch contents. Different starch concentrations (10%, 15%, and 20%) of extruded corn starch (ECS) and extruded potato starch (EPS) were used to debranch. Both debranched ECS and debranched EPS showed high amylose content of approximately 90%, indicating that all samples with different starch concentrations achieved high-efficiency enzymatic debranching. The high-performance liquid chromatograph results indicated that the samples were mainly short amylose. The samples exhibit a typical B-type crystalline structure and the relative crystallinity of them exceeds 37%. The short amylose exhibited rapid rearrangement ability, with the gelatinization temperature range of rescanning determined as 80–125 °C, this will facilitate the formation of slowly digestible starch and resistant starch. The slowly digestible starch and resistant starch contents of the samples (debranched ECS and debranched EPS) were between 23% and 30% and between 31% and 37%, respectively. These results indicate that the extrusion and enzymatic debranching of a high substrate concentration can efficiently increase the amylose content, thereby significantly reducing the digestibility of starch, and has broad prospects of the actual production of slowly digestible starch and resistant starch.

Novel insight into valorization of potato peel biomass into type III resistant starch and maltooligosaccharide molecules

Food processing activities in the potato industry generate potato peel in huge quantities. Potato peel residual biomass is rich in biomolecules with high added value. In the present study, the carbohydrate macromolecule, starch, was extracted from the potato peel, followed by enzymatic debranching by employing type 1 pullulanase, Pul M . The retrogradation of debranched starch resulted in the production of type III resistant starch (RS3). The structural details of the granular and debranched starch samples were investigated by scanning electron microscopy. The yield of about 45% RS3 was achieved from the caloric starch obtained from potato peel. The remaining digestible proportion of the peel starch was used for making the preparation of maltooligosaccharides. The chemical composition of native and resistant starch samples was studied by FTIR. XRD profiling confirmed increased crystallinity of peel starch derived RS3 as compared to the native starch. Furthermore, RS3 exhibited higher thermal stability and remarkable resistance against α -amylase digestion than its native counterpart. The bioprocess developed in the present study can be used as an alternative technology to valorize potato peel into functional bio-products. • Granular starch was extracted from potato peel biomass. • Debranching and retrogradation of peel starch resulted biosynthesis of type III resistant (RS3). • The yield of about 45% RS3 was achieved from the peel starch. • RS3 exhibited increased crystallinity, and higher thermal stability. • RS3 showed remarkable resistance against α -amylase digestion.

Starch structure-property relations in Australian wild rices compared to domesticated rices

There are many genetic differences between Australian wild rices (AWRs) and domesticated rices (DRs), causing differences in starch molecular structure and starch-related functional properties; these are examined here for polished AWRs and polished DRs. Starch structural parameters for amylopectin and amylose were obtained using size-exclusion chromatography, with and without enzymatic debranching. Thermal properties of starch, in-vitro digestibility and texture of three AWRs were measured and compared to those of typical DRs. The results showed that AWR starches had (a) higher amylose content than most DRs, resulting in a higher gelatinization temperature, (b) fewer amylopectin short chains, causing a higher gelatinization enthalpy, and (c) more amylose shorter chains and more amylopectin longer chains, both causing a slower in-vitro digestion rate. The textural characteristics of AWRs are not significantly different from those of DRs. These findings suggest that AWRs are a potential source of nutritionally-desirable but palatable slowly-digestible starch.

Identification of Structure-Controlling Rice Biosynthesis Enzymes.

The main enzymes controlling the chain-length distributions (CLDs) of starches are starch synthases (SSs), starch branching enzymes (SBEs), and debranching enzymes (DBEs), which have various isoforms, denoted as SSI, SSII-1, etc. Different isozymes dominate the CLD in different ranges of degrees of polymerization (DPs). Models have been developed for the CLDs in terms of the activities of isoforms of these enzymes, in terms of two parameters: βi, which is the ratio of the activity of SBE to that of SS in set i, and hi, which is the relative activity of SS in that set. These provide good fits to data but without specifying which isozymes are in set i. Here, CLDs for amylopectin and amylose synthesis in rice endosperm are explored. Molecular weight distributions of the different chains formed in 87 rice varieties were obtained using size-exclusion chromatography following enzymatic debranching (converting a complex branched macromolecule to linear polymers), and fitted by the biosynthesis-based models. The mutants of each isoform among tested rice varieties were identified by amino-acid mutations in coding sequences based on the extraction and analysis of whole gene sequences. The significant differences between mutant groups of different isoforms indicate that SSI, SSII-3, SSIII-1, SSIII-2, and SBEI as well as GBSSI (an isozyme of granule-bound starch synthase) belong to the enzymes sets that control amylose biosynthesis. Further, GBSSI is in the enzyme sets that control amylopectin chains. This enables specification of all isozymes and the DP range, which they dominate, over the entire DP range. As the CLD controls many functional properties of rice, this can help breeders target and develop improved rice species.

Structural characteristics and prebiotic activities of resistant starch from Solanum tuberosum: Kufri Bahar, a popular Indian tuber variety

The present study reports the effect of enzymatic debranching and heating-cooling treatment on morphology, crystallinity and thermal properties of resistant starch preparations from Solanum tuberosum, variety Kufri Bahar. Further, the proliferative and protective effect of resistant starch on viability of probiotic cultures in fermented milk during storage at refrigeration was also investigated. The results indicated that enzymatic treatment resulted in more crystalline form of resistant starch granules with an increased thermal stability. A strain specific prebiotic activity of potato resistant starch was observed with a higher proliferative activity towards Lactobacillus paracasei CD4 among the tested probiotic strains. In synbiotic fermented milk preparation, the supplementation of enzymatic treated resistant starch increased the viability of L. paracasei CD4 under refrigeration up to 14 days as compared to control (p < 0.05).

Using a novel hyperthermophilic amylopullulanase to simplify resistant starch preparation from rice starches

• A hyperthermophilic amylopullulanase is recombinantly expressed in E. coli. • The enzyme is able to efficiently improve resistant starch yield from rice starches. • Pregelatinization of starches and enzymatic hydrolysis were combined into one step. A glycoside hydrolase family 57 protein, designated APU_Aquae, from hyperthermophilic bacterium Aquifex aeolicus was expressed in Escherichia coli . APU_Aquae was biochemically defined as a novel bifunctional amylopullulanase, which exhibited efficient activity towards rice starches. It is extremely thermostable with half-life of 7 h at 100 °C. Therefore, APU_Aquae was used to simplify the process of enzymatic debranching treatment in resistant starch type III (RS3) preparation from rice starches. Starch pregelatinization and enzymatic hydrolysis were combined into one step. It took only 7 h, which was much shorter than that of other enzymatic treatments. RS3 content of treated rice starches was 40.5 ± 0.7%. APU_Aquae treatment resulted in more medium branch chains B1 with degree of polymerization (DP) values 13–24, which might be one main reason to promote the RS3 formation. This study provides the new design for the industrial RS3 production.

Effects on the structure and properties of native corn starch modified by enzymatic debranching (ED), microwave assisted esterification with citric acid (MCAE) and by the dual ED/MCAE treatment

Native corn starch was modified by enzymatic debranching (ED), microwave assisted citric acid esterification (MCAE), and by dual ED/MCAE. The structure and properties of native starch (NS), and the resulting debranched starch (DS), microwave assisted citric acid esterified starch (MCS), and microwave assisted citric acid debranched starch (MCDS) were determined and compared. Both the morphology and crystalline regions of the modified starches were changed by ED and MCAE. ED increased significantly the amylose content and transparency, but decreased the in vitro enzymatic digestibility, freeze thaw stability and relative crystallinity of DS compared to those of NS. MCAE produced a decrease in amylose content, transparency, in vitro enzymatic digestibility, and relative crystallinity, but increased the freeze-thaw stability of MCS compared to NS, and of MCDS compared to DS. The A-type crystalline structure of NS and DS was changed to B-type crystalline structure after MCAE treatment, and a new FTIR characteristic band at 1735 cm−1 was observed for MCS and MCDS. This work provides insights for producing esterified corn starches by a combined enzyme, microwave and organic acid novel technology.