Pullulanase pretreatment of highly concentrated maltodextrin solution improves maltose yield during ß-amylase-catalyzed saccharification.

Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with ß-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of ß-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for ß-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.

Enhancement of ß-Cyclodextrin Production Using a Glycogen Debranching Enzyme from Saccharolobus solfataricus STB09.

Efficient production of cyclodextrins (CDs) has always been challenging. CDs are primarily produced from starch via cyclodextrin glycosyltransferase (CGTase), which acts on α-1,4 glucosidic bonds; however, α-1,6 glucosidic bonds in starch suppress the enzymatic production of CDs. In this study, a glycogen debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE) was utilized to promote the production of ß-CD by hydrolyzing α-1,6 glucosidic bonds. The addition of SsGDE (750 U/g of starch) at the liquefaction stage remarkably improved the ß-CD yield, with a 43.9% increase. Further mechanism exploration revealed that SsGDE addition could hydrolyze specific branches with less generation of byproducts, thereby promoting CD production. The chain segments of a degree of polymerization ≥13 produced by SsGDE debranching could also be utilized by ß-CGTase to convert into CDs. Overall, these findings proposed a new approach of combining SsGDE with ß-CGTase to enhance the CD yield.

Effects of hydrocolloids on mechanical properties, viscoelastic and microstructural properties of starch-based modeling clay.

The effects of various hydrocolloids (guar gum, xanthan gum, and carboxymethyl cellulose) on the texture, rheology, and microstructural properties of modeling clay prepared with cassava starch were investigated. Notably, incorporation of 3 % guar gum and 4 % xanthan gum into starch-based modeling clay resulted in enhancements of 94.12 % and 77.47 % in cohesiveness, and 64.70 % and 66.20 % in extensibility, respectively. For starch-based modeling clay with added guar gum and xanthan gum, compared to formulations without hydrocolloids, the linear viscoelastic range exceeded 0.04 %, and the frequency dependence of both maximum creep compliance (Jmax) and storage modulus (G') was significantly reduced. This indicates a more stable network structure and enhanced resistance to deformation. Results from Fourier Transform Infrared (FTIR) spectroscopy and X-ray diffraction (XRD) confirmed that the physical interactions between starch and various hydrocolloids, along with the addition of these hydrocolloids, inhibited the degradation effect of thermomechanical processing on the crystalline structure of starch. With the addition of guar gum, it is observed that a continuous and dense network structure forms within the starch-based modeling clay, and starch particles are distributed uniformly. In conclusion, hydrocolloids enhances the properties of starch-based modeling clay, introducing an innovative solution to the modeling clay sector.

Effect of solution on starch structure: New separation approach of amylopectin fraction from gelatinized native corn starch.

The complete dissolution of starch without degradation are necessary prerequisites for starch fractionation to obtain amylose or amylopectin (AP). With the recent, continuous progress in finding efficient and eco-friendly starch-dissolving solutions, applying new solvents for starch fractionation is important. In this study, the effects of dimethyl sulfoxide (DMSO), NaOH, and CaCl2 solutions on starch structure and AP product parameters during starch fractionation were compared with respect to the starch deconstruction effect. This study proved that the CaCl2 solution could effectively dissolve corn starch (50 °C, solubility of 98.96 %), and promote the regeneration of starch into uniform and fine particles. Furthermore, the three solvents (DMSO, NaOH, and CaCl2) changed the crystal structure of corn starch, but they were all non-derivatizing solvents. The effect of the CaCl2 solution on the molecular structure of corn starch was the least significant of the three solvents. Finally, the extraction rate of AP from the CaCl2 solution reached 69.45 %. In conclusion, this study presents a novel and effective method for AP extraction.

The substitution sites of hydroxyl and galloyl groups determine the inhibitory activity of human pancreatic α-amylase in twelve tea polyphenol monomers.

Tea polyphenols have been reported as potential α-amylase inhibitors. However, the quantitative structure-activity relationship (QSAR) between tea polyphenols and human pancreas α-amylase (HPA) is not well understood. Herein, the inhibitory effect of twelve tea polyphenol monomers on HPA was investigated in terms of inhibitory activity, as well as QSAR analysis and interaction mechanism. The results revealed that the HPA inhibitory activity of theaflavins (TFs), especially theaflavin-3'-gallate (TF-3'-G, IC50: 0.313 mg/mL), was much stronger than that of catechins (IC50: 18.387-458.932 mg/mL). The QSAR analysis demonstrated that the determinant for the inhibitory activity of HPA was not the number of hydroxyl and galloyl groups in tea polyphenol monomers, while the substitution sites of these groups potentially might play a more important role in modulating the inhibitory activity. The inhibition kinetics and molecular docking revealed that TF-3'-G as a mixed-type inhibitor had the lowest inhibition constant and bound to the active sites of HPA with the lowest binding energy (-7.74 kcal/mol). These findings could provide valuable insights into the structures-activity relationships between tea polyphenols and the HPA inhibitors.

Optimization and analysis of dual-enzymatic synthesis for the production of linear glucan.

Linear α-glucan (LG), a linear polymer linked by α-1,4 bonds, has received increasing attention for its potential applications in synthetic polymer production. Notably, the functionality of LG is strongly influenced by its degree of polymerization (DP). In this study, SP and GP were successfully constructed and expressed. The reaction of enzymatic co-polymerization into LG was investigated. The preferred reaction was carried out at 37 °C and pH 7.4 for 72 h, with a maximum conversion rate of 25 %. Afterwards, two approaches were used to modulate the molecular structures of LGs. Firstly, LGs with distinct molecular weights ranging from 1062.33 ± 16.04 g/mol to 5679 ± 80.29 g/mol were obtained by adjusting the substrate/primer ratio during the LG synthesis process. Secondly, two distinct products could be produced by altering the enzyme addition method: short-chain LG with a DP < 10 (64.34 ± 0.54 %) or long-chain LG with a DP > 45 (45.57 ± 2.18 %). Additionally, theoretical synthesis model was constructed which subdivided the reaction into three stages to evaluate this dual-enzyme cooperative system. These findings have significant implications in promoting the application of LG in the fields of biomedicine and material science.

A Comprehensive Review of the Effects of Glycemic Carbohydrates on the Neurocognitive Functions Based on Gut Microenvironment Regulation and Glycemic Fluctuation Control.

Improper glycemic carbohydrates (GCs) consumption can be a potential risk factor for metabolic diseases such as obesity and diabetes, which may lead to cognitive impairment. Although several potential mechanisms have been studied, the biological relationship between carbohydrate consumption and neurocognitive impairment is still uncertain. In this review, the main effects and mechanisms of GCs' digestive characteristics on cognitive functions are comprehensively elucidated. Additionally, healthier carbohydrate selection, a reliable research model, and future directions are discussed. Individuals in their early and late lives and patients with metabolic diseases are highly susceptible to dietary-induced cognitive impairment. It is well known that gut function is closely related to dietary patterns. Unhealthy carbohydrate diet-induced gut microenvironment disorders negatively impact cognitive functions through the gut-brain axis. Moreover, severe glycemic fluctuations, due to rapidly digestible carbohydrate consumption or metabolic diseases, can impair neurocognitive functions by disrupting glucose metabolism, dysregulating calcium homeostasis, oxidative stress, inflammatory responses, and accumulating advanced glycation end products. Unstable glycemic status can lead to more severe neurological impairment than persistent hyperglycemia. Slow-digested or resistant carbohydrates might contribute to better neurocognitive functions due to stable glycemic response and healthier gut functions than fully gelatinized starch and nutritive sugars.

Catalytic activity enhancement of 1,4-α-glucan branching enzyme by N-terminal modification.

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) has garnered considerable attention for its ability to increase the degree of branching of starch and retard starch digestion, which has great industrial applications. Previous studies have reported that the N-terminal domain plays an important role in the expression and stability of GBEs. To further increase the catalytic ability of Gt-GBE, we constructed five mutants in the N-terminal domain: L19R, L19K, L25R, L25K, and L25A. Specific activities of L25R and L25A were increased by 28.46% and 23.46%, respectively, versus the wild-type Gt-GBE. In addition, the α-1,6-glycosidic linkage ratios of maltodextrin samples treated with L25R and L25A increased to 5.71%, which were significantly increased by 19.96% compared with that of the wild-type Gt-GBE. The results of this study suggest that the N-terminal domain selective modification can improve enzyme catalytic activity, thus further increasing the commercial application of enzymes in food and pharmaceutical industries.

Conserved residues at the family and subfamily levels determine enzyme activity and substrate binding in glycoside hydrolase family 13.

Site-directed mutagenesis is a valuable strategy for modifying enzymes, but the lack of understanding of conserved residues regulating glycosidase function hinders enzyme design. We analyzed 1662 enzyme sequences to identify conserved amino acids in maltohexaose-forming amylase at both family and subfamily levels. Several conserved residues at the family level (G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360) were found, mutations of which resulted in reduced enzyme activity or inactivation. At the subfamily level, several conserved residues (L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346) were identified, which primarily facilitate substrate binding in the enzyme's active site, as shown by molecular dynamics and kinetic assays. Our findings provide critical insights into conserved residues essential for catalysis and can inform targeted enzyme design in protein engineering.

Bifunctional Mutant of Oligo-1,6-glucosidase for the Production of Glucose from Glucose Mother Liquor.

Glucose mother liquor (GML) is a byproduct of the glucose (G1) crystallization process. However, the presence of maltooligosaccharides and isomaltooligosaccharides within GML imposes limitations on its reutilization. Furthermore, the high concentration of G1 in GML leads to product inhibition of G1-producing enzymes. To overcome these challenges, a variant enzyme called V219A was developed through genetic mutation. The V219A exhibits the ability to hydrolyze both maltooligosaccharides and isomaltooligosaccharides. Product inhibition kinetics showed that the IC50 value of V219A was 7 times higher than that of the wild type. Upon subjecting primary, secondary, and tertiary GML to treatment with V219A, the G1 content exhibited notable increases, reaching 96.88, 95.70, and 90.46%, respectively. These significant findings not only establish an innovative and environmentally conscious approach for G1 production from GML but also provide a promising strategy for enzyme construction that caters to the demands of industrial-scale production.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes.

Debranching enzymes (DBEs) directly hydrolyze α-1,6-glucosidic linkages in glycogen, starch, and related polysaccharides, making them important in the starch processing industry. However, the ambiguous substrate specificity usually restricts synergistic catalysis with other amylases for improving starch utilization. Herein, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) and two isoamylases from Pseudomonas amyloderamosa (PaISO) and Chlamydomonas reinhardtii (CrISO) were used to investigate the molecular mechanism of substrate specificity. Along with the structure-based computational analysis, the aromatic residues in the substrate-binding region of DBEs played an important role in binding substrates. The aromatic residues in SsGDE appeared clustered, contributing to a small substrate-binding region. In contrast, the aromatic residues in isoamylase were distributed dispersedly, forming a large active site. The distinct characteristics of substrate-binding regions in SsGDE and isoamylase might explain their substrate preferences for maltodextrin and amylopectin, respectively. By modulating the substrate-binding region of SsGDE, variants Y323F and V375F were obtained with significantly enhanced activities, and the activities of Y323F and V375F increased by 30 and 60% for amylopectin, and 20 and 23% for DE4 maltodextrin, respectively. This study revealed the molecular mechanisms underlying the substrate specificity for SsGDE and isoamylases, providing a route for engineering enzymes to achieve higher catalytic performance.

A new micro-agar dilution method to determine the minimum inhibitory concentration of essential oils against microorganisms.

Essential oils (EOs) from plants have attracted wide attention due to their unique flavors and antimicrobial, insecticidal, antioxidant, and anti-inflammatory properties. Antimicrobial activity, the main reason for their widespread use in the food industry, can be determined in vitro by the minimum inhibitory concentration (MIC), which is a key step to evaluate the antimicrobial potential of EOs. However, EOs are lipophilic and insoluble, resulting in the difficulty of accurately measuring their MIC values. In this study, in addition to analyzing the problems in the MIC determination of EOs using the common micro-broth dilution method or the agar method, a new solution called micro-agar dilution method was proposed. As the miniaturization of agar dilution method, this novel method could reduce the dosage of EOs by 16.3 times and medium/PBS by 3.3 times, respectively, and showed better reliability and accuracy than the typical methods. Additionally, the limit of solvents should be evaluated before use if used. Notably, this research could also provide a guide for the MIC determination of other insoluble antibacterial substances.

Physicochemical characterizations, α-amylase inhibitory activities and inhibitory mechanisms of five bacterial exopolysaccharides.

Inhibiting pancreatic α-amylase activity can decrease the release rate of glucose, thereby delaying postprandial blood glucose. This study aimed to investigate the physicochemical properties and porcine pancreatic α-amylase (PPA) inhibitory activities of five bacterial exopolysaccharides (EPSs). We also aimed to analyze the differences of their inhibitory activities, exploring the inhibition mechanism between EPSs and PPA. Five EPSs had a low molecular weight (55-66 kDa), which were mainly composed of mannose and glucose with total content exceeding 86 %. The IC50 values of five EPSs (0.162-0.431 mg/mL) were significantly lower than that of acarbose (0.763 mg/mL), indicating that the inhibitory effects of five EPSs on PPA were stronger than acarbose, especially the EPS from Bacillus subtilis STB22 (BS-EPS). Moreover, BS-EPS was a mixed-type inhibitor, whereas other EPSs were noncompetitive inhibitors of PPA. Five EPSs quenched the fluorophore of PPA by the mixed quenching or apparent static quenching. Interestingly, BS-EPS showed stronger binding affinity to PPA than other EPSs. It can be speculated that EPSs with low molecular weight, high carboxylic acid content, and α-glycosidic bond exhibited high PPA inhibitory activity. These results suggest that BS-EPS can effectively inhibit PPA activity and has potential applications in reducing postprandial hyperglycemia.

Designing a Specific Pretreatment on Corn Starch to Facilitate Enzymatic Rearrangement of Glycosidic Bonds for Efficiently Reducing Starch Digestibility.

Bacterial 1,4-α-glucan branching enzymes (GBEs) provide a viable strategy for glycosidic bond rearrangement in starch and regulation of its digestion rate. However, the exponential increase in paste viscosity during starch gelatinization has a detrimental effect on the catalytic action of GBEs, thereby limiting productivity and product performance. Here, we designed an enzymatic treatment on corn starch granules by the GBE from Rhodothermus obamensis STB05 (Ro-GBE) prior to the glycosidic bond rearrangement of gelatinized starch catalyzed using the GBE from Geobacillus thermoglucosidans STB02 (Gt-GBE). Specifically, a moderate amount of Ro-GBE was required for the pretreatment stage. The dual GBE modification process enabled the treatment of more concentrated starch slurry (up to 20%, w/w) and effectively reduced starch digestibility. The resulting product contained a rapidly digestible starch fraction of 66.0%, which was 11.4% lower than that observed in the single Gt-GBE-modified product. The mechanistic investigation showed that the Ro-GBE treatment promoted swelling and gelatinization of starch granules, reduced starch paste viscosity, and increased the mobility of water molecules in the starch paste. It also created a preferable substrate for Gt-GBE. These changes improved the transglycosylation efficiency of Gt-GBE. These findings provide useful guidance for designing an efficient process to regulate starch digestibility.

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.

Fabrication of octenyl succinic anhydride starch grafted with folic acid and its loading potential for doxorubicin hydrochloride.

In this study, octenyl succinic anhydride (OSA) starch with different folic acid (FA) grafting time was prepared and the degree of FA substitution at different grafting time was determined. The results of XPS quantitatively reflected the surface elemental composition of OSA starch grafted with FA. FTIR spectra further confirmed the successful introduction of FA on OSA starch granules. SEM images showed that the surface roughness of OSA starch granules was more obvious with higher FA grafting time. The particle size, zeta potential, and swelling properties were determined to study the effect of FA on the structure of OSA starch. TGA indicated that FA effectively enhanced the thermal stability of OSA starch at high temperature. The crystalline form of the OSA starch gradually transformed from A type to a hybrid A and V-type with the progress of FA grafting reaction. In addition, the anti-digestive properties of OSA starch were enhanced after grafting FA. Using doxorubicin hydrochloride (DOX) as the model drug, the loading efficiency of OSA starch grafted with FA for DOX reached 87.71 %. These results provide novel insights into OSA starch grafted with FA as potential strategy for loading DOX.

Conserved residues Glu and Phe at substrate binding groove of α-1,6-glucanases modulate branch of the product.

Understanding what amino acids in α-1,6-glucanases target α-1,6 glycosidic bonds of polysaccharides is timely and important for generating products with branch structure. With this objective, we investigated 330 sequences from seven subfamilies to excavate amino acids for recognition or catalysis of α-1,6 glycosidic bonds. Computational analysis identified two amino acids, E343 and W521, trigger α-1,6 glycosidic bond specificity of enzymes. To explore the effect of E343 and W521 on the product structure, several engineered mutants were studied in our research. Product structural analysis showed that the ratio of amylose and amylopectin is obviously different. The catalytic mechanism revealed that the bulky aromatic side chain is a trigger that controls the ratio of branch glucans. The E148 acts as a proton donor to regulate the generation of branched structures in the product during transglycosidation of the glucan branching enzyme (GBE).

Designing liquefaction and saccharification processes of highly concentrated starch slurry: Challenges and recent advances.

Starch-based sugars are an important group of starch derivatives used in food, medicine, chemistry, and other fields. The production of starch sugars involves starch liquefaction and saccharification processes. The production cost of starch sugars can be reduced by increasing the initial concentration of starch slurry. However, the usage of the highly concentrated starch slurry is characterized by challenges such as low reaction efficiency and poor product performance during the liquefaction and saccharification processes. In this study, we endeavored to provide a reference guide for improving high-concentration starch sugar production. Thus, we reviewed the effects of substrate concentration on the starch sugar production process and summarized several potential strategies. These regulation strategies, such as physical field pretreatment, complex enzyme-assisted, and temperature control, can significantly increase the starch concentration and mitigate the challenges of using highly concentrated starch slurry. We believe that highly concentrated starch sugar production will achieve a qualitative leap in the future. This review provides theoretical guidance and highlights the importance of high concentration in starch-based sugar production. Further studies are needed to explore the fine structure and enzyme attack mode during the liquefaction and saccharification processes to regulate the production of more targeted products.

Highland Barley Starch: Structures, Properties, and Applications.

Highland barley (HB) is a nutritious crop with excellent health benefits, and shows promise as an economically important crop with diverse applications. Starch is the main component of HB and has great application potential owing to its unique structural and functional properties. This review details the latest status of research on the isolation, chemical composition, structure, properties, and applications of highland barley starch (HBS). Suggestions regarding how to better comprehend and utilize starches are proposed. The amylopectin content of HBS ranged from 74% to 78%, and can reach 100% in some varieties. Milling and air classification of barley, followed by wet extraction, can yield high-purity HBS. The surface of HBS granules is smooth, and most are oval and disc-shaped. Normal, waxy, and high-amylose HBS have an A-type crystalline. Due to its superb freeze-thaw stability, outstanding stability, and high solubility, HBS is widely used in the food and non-food industries. The digestibility of starch in different HB whole grain products varies widely. Therefore, the suitable HB variety can be selected to achieve the desired glycemic index. Further physicochemical modifications can be applied to expand the variability in starch structures and properties. The findings provide a thorough reference for future research on the utilization of HBS.

A review of controlled release from cyclodextrins: release methods, release systems and application.

The controlled release of guest molecules from cyclodextrin (CD) inclusion complexes is very important for specific industrial applications in foods, medicine, cosmetics, textiles, agriculture, environmental protection, and chemical materials. The term "controlled release" encompasses several related methods, including those referred to as immediate release, sustained release and targeted release. Many different CD-based controlled release systems are currently used in practical applications. CD inclusion complexes, CD coupling, supramolecular hydrogels, and supramolecular micelles are among the most common. This review systematically introduces the principles and applications of CD-based controlled release systems, providing a theoretical basis for improving the bioavailability of effective substances and broadening their range of application.