Pullulanase M1 (Klebsiella planticola)

Glycogen branching enzymes (GBEs; EC 2.4.1.18) are essential for forming α-1,6-O-glycosidic branches in starch modification and glycogen biosynthesis. They are classified into glycoside hydrolase (GH) families 13 and 57. GH13 GBEs are further divided into subfamilies GH13_9, containing predominantly sequences from bacteria, and GH13_8, comprising sequences from both bacteria and eukaryotes. So far, only three eukaryotic GH13_8 enzymes have been studied in detail while no crystal structures or functional activities of prokaryotic GH13_8 GBEs have been reported. In this study, the GH13_8 and GH13_9 GBE of Anaerococcus prevotii (Ap) were studied in detail. It was shown for the first time that this prokaryotic GH13_8 GBE is active on amylose and creates α-1,6-O-glycosidic linked branches. In contrast to GH13_9 GBEs, the ApGBE13_8 is active on very short oligosaccharides ranging from DP2 to DP5 (degree of polymerization) transferring glucose or maltose. The preference for short oligosaccharides might be correlated with the presence of two short beta stranded loops at position 131 and 509. These loops may function like a ‘door,’ dynamically adjusting to the donor chain, affecting branch length and cleavage specificity. These findings reveal ApGBE13_8's distinct function, advance GH13_8 research, and suggest potential applications for GH13_8 GBEs in starch modification.

The effects of speed (250–450 rpm) and time (10-50 min) on milling energy (E), particle size distribution (PSD), crystallinity degree (CD), damaged starch (DS), microstructure, hydration and pasting properties of flours obtained in a planetary ball mill (PBM) were investigated. As increasing milling severity quinoa flours showed polydispersed PSD (peaks at 417, 40 and 2 μm) with a shift towards smaller size and greater dispersion (Span: 3.0-5.2) as well as particles with rounded edges and polished surfaces (SEM images). The relationship E − D50 were satisfactorily predicted by the Walker's equation. The hydration tests as function of temperature and milling conditions revealed a complex flour behavior due to differences in PSD, composition, and DS content. The increasing milling energy caused a decrease in peak viscosity (PV), trough viscosity (TV) and final viscosity (FV) of up to 36%, 29%, and 25%, respectively. Significant correlations between flour attributes were found (DS-CD, r = −0.83, p < 0.01; PV-D50, r = 0.92, p < 0.01; TV-D50, r = 0.94, p < 0.01; FV-D50, r = 0.90, p < 0.01) denoted the increase of starch degradation as milling severity rises. These results can be used to improve the manufacture and the selection criteria of quinoa flour with specific functional attributes.

Potato noodles are a popular food due to their unique texture and taste, but native potato starch often fails to meet consumer demands for precise textural outcomes. The effect of blending small granule (waxy amaranth, non-waxy oat and quinoa) starch with potato starch on the properties of noodles was investigated to enhance quality of noodles. Morphological results demonstrated that small granule starch filled gaps between potato starch granules, some of which gelatinized incompletely. Meanwhile, XRD and FTIR analysis indicated that more ordered structures and hydrogen bonding among starch granules increased with addition of small granule starch. The addition of oat or quinoa starch increased gel elasticity, decreased viscosity of the pastes, and increased the tensile strength of noodles, while addition of 30 % and 45 % waxy amaranth starch did not increase G′ value of gel or tensile strength of noodles. These results indicated that amylose molecules played an important role during retrogradation, and may intertwine and interact with each other to enhance the network structure of starch gel in potato starch blended with oat or quinoa starch. This study provides a natural way to modify potato starch for desirable textural properties of noodle product.

Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality.

Octenyl succinate anhydride (OSA) modified high amylose maize starch (HAMS) showed limited emulsion stability due to the OSA groups were located on the surface of starch granules. However, amylose is enriched in the internal region of HAMS and provides the reaction site for OSA groups. In this study, five types of starches with different amylose content were prepared using optimized reaction conditions. Our data showed that amylose content (AC) was the most important parameter controlling the degree of substitution (DS), in comparison with temperature, pH, starch concentration and time. DS reached a maximum (2.1%) at AC of 58%. Importantly, OSA groups were mainly located in the internal regions of HAMS under the optimized reaction conditions. This was supported by (1) the decrease of the crystallinity, (2) the weaker fluorescence intensity of the starch fluorophore probe 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) in the internal regions of HAMS, and (3) unchanged signals from Fourier transform infrared spectroscopy which characterized the granular surface structure ordering. This new OSA reaction pattern permits HAMS to be used in stabilizing Pickering emulsions This is the first report that OSA groups located in the internal regions of HAMS granules.

Glycogen branching enzymes (GBEs) have been used to generate new branches in starches for producing slowly digestible starches. The aim of this study was to expand the knowledge about the mode of action of these enzymes by identifying structural aspects of starchy substrates affecting the products generated by different GBEs. The structures obtained from incubating five GBEs (three from glycoside hydrolase family (GH) 13 and two from GH57) on five different substrates exhibited minor but statistically significant correlations between the amount of longer chains (degree of polymerization (DP) 9-24) of the product and both the amylose content and the degree of branching of the substrate (Pearson correlation coefficient of ≤−0.773 and ≥0.786, respectively). GH57 GBEs mainly generated large products with long branches (100-700 kDa and DP 11-16) whereas GH13 GBEs produced smaller products with shorter branches (6-150 kDa and DP 3-10).

Starch molecules are first degraded to slowly digestible α-limit dextrins (α-LDx) and rapidly hydrolyzable linear malto-oligosaccharides (LMOs) by salivary and pancreatic α-amylases. In this study, we designed a slowly digestible highly branched α-LDx with maximized α-1,6 linkages using 4,6-α-glucanotransferase (4,6-αGT), which creates a short length of α-1,4 side chains with increasing branching points. The results showed that a short length of external chains mainly composed of 1–8 glucosyl units was newly synthesized in different amylose contents of corn starches, and the α-1,6 linkage ratio of branched α-LDx after the chromatographical purification was significantly increased from 4.6% to 22.1%. Both in vitro and in vivo studies confirmed that enzymatically modified α-LDx had improved slowly digestible properties and extended glycemic responses. Therefore, 4,6-αGT treatment enhanced the slowly digestible properties of highly branched α-LDx and promises usefulness as a functional ingredient to attenuate postprandial glucose homeostasis.

Designing a highly concentrated (45 %, w/w) starch liquefaction process is a green method to enhance the productivity of starch syrup and related fermentation products. Previous studies mainly focused on handling highly concentrated normal corn starch slurries, but the production efficiency and product performance cannot perfectly match the conventional liquefaction process (30 %, w/w). In the present research, four starches from various botanical sources were selected with an objective to accelerate highly concentrated starch liquefaction process. The results demonstrated that with potato starch or tapioca starch as a substrate, liquefaction process was more feasible as observed from the obvious reduction in paste viscosity and acceleration in amylolysis. To clarify the mechanism of these differences, changes in the fine structure during liquefaction were further characterized. The long external chains (16.2 glucose units on average) in potato starch and long internal chains (5.1 glucose units on average) in tapioca starch, which indicated high proportion of consecutive α-1,4 linkages, seemed more susceptible to enzymatic attack under highly concentrated substrate condition. This caused rapid degradation of starch molecules. The liquefied products were suitable for glucose syrup production. By comparison, normal corn starch and waxy corn starch, which contain relatively shorter linear fragments, were less accessible to α-amylase. This suppressed liquefaction process led to the survival of large molecules, thereby being unsuitable for subsequent saccharification process. The results suggest that selecting an appropriate substrate is an effective strategy to accelerate highly concentrated starch liquefaction and improve product performance.

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.

Pure granular amylose (AM) and pure granular amylopectin (waxy) starch (AP) granules have the high nutritional value in food industry. Effects of microwave treatment (400 W/g DW, 1-8 min) on the structure and properties of transgenic AM granules and AP granules were investigated in direct comparison. Microwave treatment, especially during the first 3 min, decreased the molecular weight of molecules in both the AM and the AP samples. The crystallinity of AM starch initially increased from 15.6% to 20.6%, which was associated with the formation of new V_h-type crystals. After that, crystallinity decreased alongside to 11.3% with the complete disruption of B-type crystals. In contrast, the crystallinity of AP starch initially decreased from 18.9% to 10.8% followed by an increase to 20.0%. Upon prolonged treatment of AM granules, the resistant starch and water solubility was significantly increased. Our data demonstrate notable different microwave-dependent reorganization patterns for pure granular AM and AP molecules as native granular systems, which is helpful to the improvement of functionality of these two starches.