Enzymatic Hydrolysis Of Starch Research Articles

GC–MS analysis, molecular docking, and pharmacokinetic studies on Dalbergia sissoo barks extracts for compounds with anti-diabetic potential

Diabetes is a metabolic condition defined by abnormal blood sugar levels. Targeting starch-hydrolyzing enzymes and Dipeptidyl Peptidase 4 (DPP-4) expressed on the surface of numerous cells is one of the key strategies to lower the risk of Type-2 diabetes mellitus (T2DM). Dalbergia sissoo Roxb. bark (DSB) extracts have been reported to have anti-diabetic properties. This study intended to scientifically validate use of alcoholic and hydro-alcoholic extracts of DSB for T2DM by conducting preliminary phytochemical investigations, characterising potential phytochemicals using Fourier transform infrared (FT-IR) spectroscopy and Gas chromatography-mass spectrometry (GC–MS) analysis followed by comprehensive in-silico analysis. A qualitative phytochemical evaluation indicated the presence of alkaloids, phenolics, glycosides, conjugated acids and flavonoids. Ethanolic extracts showed highest total phenolic content (TPC) (127.072 ± 14.08031 μg GAE/g dry extract) and total flavonoid content (106.911 ± 5.84516 μg QE /g dry extract). Further FT-IR spectroscopy also revealed typical band values associated with phenol, alcohol, alkene, alkane and conjugated acid functional groups. The GC–MS analysis identified 139 compounds, 18 of which had anti-diabetic potential. In-silico ADMET analysis of potential compounds revealed 15 compounds that followed Lipinski’s rule and demonstrated drug-like properties, as well as good oral bioavailability. Molecular docking was utilised to analyse their potential to interact with three targets: α-amylase, α-glucosidase, and DPP-4, which are crucial in managing diabetes-related problems. Molecular Docking analysis and membrane permeability test utilising the PerMM platform revealed that compounds in the extracts, such as Soyasapogenol B and Corydine, had better interactions and permeability across the plasma membrane than standard drugs in use. Molecular dynamics simulations also showed that selected compounds remained stable upon interaction with α-amylase. Overall, using the in-silico approaches it was predicted that DSB extracts contain potential phytochemicals with diverse anti-diabetic properties. It further needs to be investigated for possible development as formulation or drug of choice for treating T2DM.

Maltose gradient-induced biosensor-based high-throughput screening for directed evolution of maltogenic amylase from Bacillus stearothermophilus

Maltogenic amylase is a starch-hydrolyzing enzyme commonly used in bread baking and high-concentration maltose syrup production. However, low catalytic activity limits its industrial application. Improving catalytic activity based on molecular modification and directed evolution requires a High-Throughput Screening (HTS) method. In this study, a maltose gradient-induced (MaGI) biosensor was designed and applied for the directed evolution of maltogenic amylase AmyM, showing a good positive correlation between enzyme activity and fluorescence. The MaGI biosensor detected maltose and maltogenic amylase activity efficiently and specifically. Two mutants, Q440N and S442N/Q661L, were identified through the screening of 3000 mutants using the MaGI biosensor, showing a significant increase in catalytic activity of 35.56 % and 24.51 %, respectively, compared to the wild-type. Meanwhile, the t1/2 of Q440N and S442N/Q661L at 60 °C increased by 58.53 % and 66.66 %, respectively. In industrial applications, the enhancement of catalytic activity and stability is conducive to improving production efficiency and reducing costs. MD simulation has found that when modifying multidomain enzymes, distal mutations can enhance catalytic activity. In conclusion, the developed MaGI biosensor is a promising tool for high-throughput and specific detection of maltose.

Optimized ceramic membrane-based method for efficient and acid-free rice protein preparation from alkaline extracts

Rice protein, stands out as a high-quality plant-based protein, suitable for dietary supplementation and food processing. Traditional methods, involving alkaline extraction and acid precipitation, are challenged by the extensive use of chemicals and the difficulty in desalination. This study proposed a novel separation method that utilizes ceramic membranes for direct alkali filtration, eliminating the formation of salts by acid neutralization. The membrane pore size and the operating parameters such as transmembrane pressure, cross-flow velocity, and pH were optimized to enable a high flux (>80 L m−2·h−1) and favorable protein rejection (>95 %) while permitting the removal of excess alkali. Additionally, the effects of non-protein substances, e.g., starch, present in the alkaline extract on the separation performance were explored. Subsequently, an optimized strategy for starch removal was proposed, involving the concentration of the alkali extract using an ultrafiltration membrane, followed by enzymatic hydrolysis of starch and diafiltration to eliminate the residuals of starch hydrolysates and alkali. Under stabilization of protein particles by carboxymethyl cellulose, rice protein with a uniform particle size of 25 nm and a purity of over 80 % was produced. The direct removal of alkali from alkali extracts of rice by ceramic membranes presents a promising strategy for producing rice protein with high purity and reduced aggregation degree, enabling better functionalities such as solubility than the conventional alkali-extraction and acid-precipitation method.

Synergistic action of novel maltohexaose-forming amylase and branching enzyme improves the enzymatic conversion of starch to specific maltooligosaccharide

As attractive functional ingredients, maltooligosaccharides (MOS) are typically prepared by controlled enzymatic hydrolysis of starch. However, the random attack mode of amylase often leads to discrete product distribution, thereby reducing yields and purities. In this study, a novel glycoside hydrolase family 13 amylase AmyEs from marine myxobacteria Enhygromyxa salina was identified efficient maltohexaose (G6)-forming ability (40 %, w/w). By deciphering external chain length, we found that the high density of α-1,6-branching points benefits the G6 formation of AmyEs with high purity (71–82 %), indicating the substrate selectivity of AmyEs toward high-branched starch. Based on this, asynchronous conversion strategy was designed to enhance specific MOS yield from corn starch by exploiting branching enzymes and AmyEs, and the purity and yield of G6 respectively increased by 9.5 % and 5 % compared to single AmyEs treatment. Our results demonstrate that combinatorial catalysis of MOS-forming amylases and branching enzymes provides a favorable industrial preparation of specific MOS.

Comparison of vegetable powders as ingredients of flatbreads: technological and nutritional properties

SummaryHealth‐driven innovation is transforming bakery products, particularly with non‐conventional ingredients. This study aimed to produce healthy and technologically acceptable flatbreads, exploring the inclusion (2%) of vegetable powders (black and green olives, orange peel, lemon, tomato, beetroot, carrot, onion, artichoke, spinach, chard, kale and pak choi). Spinach and chard increased the mineral content (1.42 and 1.30 g/ 100 g) respect to control (1.01 g/100 g). Fibre content ranged from 9.33 to 11.18 g/100 g when added onion, chard, pak choi, tomato or artichoke. Beetroot was the most effective changing the colour (ΔE* 43.09), while tomato reduced the hardness (4.06 vs 5.29 N in the control). Lemon and tomato were effective reducing the extent of starch enzymatic hydrolysis by 65% and 34%, respectively. Vegetable powders can be innovative, natural, sustainable and healthy ingredients in the breadmaking of flatbreads. The incorporation of these non‐conventional ingredients opens new opportunities for the bakery industry.

The multifactorial phenomenon of enzymatic hydrolysis resistance in unripe banana flour and its starch: A concise review.

Unripe banana flour starch possesses a high degree of resistance to enzymatic hydrolysis, a unique and desirable property that could be exploited in the development of functional food products to regulate blood sugar levels and promote digestive health. However, due to a multifactorial phenomenon in the banana flour matrix-from the molecular to the micro level-there is no consensus regarding the complex mechanisms behind the slow enzymatic hydrolysis of unripe banana flour starch. This work therefore explores factors that influence the enzymatic hydrolysis resistance of raw and modified banana flour and its starch including the proportion and distribution of the amorphous and crystalline phases of the starch granules; granule morphology; amylose-amylopectin ratio; as well as the presence of nonstarch components such as proteins, lipids, and phenolic compounds. Our findings revealed that the relative contributions of these factors to banana starch hydrolytic resistance are apparently dependent on the native or processed state of the starch as well as the cultivar type. The interrelatability of these factors in ensuring amylolytic resistance of unripe banana flour starch was further highlighted as another reason for the multifactorial phenomenon. Knowledge of these factors and their contributions to enzymatic hydrolysis resistance individually and interconnectedly will provide insights into enhanced ways of extraction, processing, and utilization of unripe banana flour and its starch.

Leaf starch degradation by β-amylase ZmBAM8 influences drought tolerance in maize

As a typical C4 plant and important crop worldwide, maize is susceptible to drought. In maize, transitory starch (TS) turnover occurs in the vascular bundle sheath of leaves, differing from that in Arabidopsis (a C3 plant). This process, particularly its role in drought tolerance and the key starch-hydrolyzing enzymes involved, is not fully understood. We discovered that the expression of the β-amylase (BAM) gene ZmBAM8 is highly upregulated in the drought-tolerant inbred line Chang7-2t. Inspired by this finding, we systematically investigated TS degradation in maize lines, including Chang7-2t, Chang7-2, B104, and ZmBAM8 overexpression (OE) and knockout (KO) lines. We found that ZmBAM8 was significantly induced in the vascular bundle sheath by drought, osmotic stress, and abscisic acid. The stress-induced gene expression and chloroplast localization of ZmBAM8 align with the tissue and subcellular sites where TS turnover occurs. The recombinant ZmBAM8 was capable of effectively hydrolyzing leaf starch. Under drought conditions, the leaf starch in ZmBAM8-OE plants substantially decreased under light, while that in ZmBAM8-KO plants did not decrease. Compared with ZmBAM8-KO plants, ZmBAM8-OE plants exhibited increased drought tolerance. Our study provides insights into the significance of leaf starch degradation in C4 crops and contributes to the development of drought-resistant maize.

Analysis of the mechanism of resistance to enzymatic hydrolysis of RS-5 resistant starch

RS-5 refers to the resistant starch formed by complexation of starch molecules with other molecules. In this study, the molecular mechanism of RS-5 was analysed. First, it was found, when α-amylase acted on the starch-lipid complexes, the glucose residues involved in complexation cannot be hydrolyzed by α-amylase, while the glucose residues not directly involved in complexation can be hydrolyzed. Second, lipid molecules are not necessary for the formation of RS-5 and can be replaced with small peptides or decanal molecules. Considering the multiple health hazards that may result from excessive lipid intake, small peptides composed of essential amino acids may be more desirable materials for RS-5 preparation. Third, starch-lipid complexes had strong interactions with α-amylase, which provides evidence in support of the sliding continuum hydrolysis hypothesis of α-amylase. These results revealed the mechanism of RS-5 at the molecular level, which provides a reference for the production and research of RS-5.

Brewing Mainly from Stale Bread: A Pale Ale Case Study

Contemporary environmental concerns have led to the prioritization of sustainable production and material circularity, no matter what the industrial field of activity. Bread waste is a major element of overall food waste since, worldwide, bread remains a widespread staple food. A considerable proportion of bread consumption is of fresh, baked bread, consumed daily, generating substantial amounts of stale bread. Therefore, efforts to reintroduce this waste into the food value chain can make a significant contribution to reaching zero food waste, which is a major target in European countries. Possible ways to produce new raw materials through starch enzymatic hydrolysis include brewing, which is an activity in which incorporating stale bread is of great interest. Mashing parameters in brewing processing are the main focus of this study, primarily the time and temperature required to acquire optimal enzymatic activity for starch-efficient hydrolysis. Extending the mashing time to 290 min, within a temperature range of 45–75 °C, allowed us to replace 50% of the required malt with stale bread, thus obtaining a successful pale ale beer. The incorporation of stale bread in a 50:50 ratio did not affect the overall character of the beer, although the alcohol levels stood around 2% below a standard beer’s average level. Depending on the brewer’s final goal, this lighter kind of beer may be well-aligned with new consumer trends supporting more sustainable and lower-alcohol beverages.

Digestion and importance of starch in ruminants

Ruminants have a unique digestive physiology that heavily relies on microbial fermentation specifically in the rumen. This review explores the complex mechanisms of starch digestion in ruminants and highlights its crucial role in shaping their nutritional landscape. The rumen, a complex microbial ecosystem, is the primary site for starch digestion. Enzymatic hydrolysis and microbial fermentation of starch in this compartment produce important byproducts, such as volatile fatty acids (VFAs) and microbial proteins. These byproducts are crucial sources of energy and protein, which affect the overall metabolic dynamics of ruminants. It is essential to have a comprehensive understanding of the factors that influence starch digestion rates to optimize ruminant nutrition. This review examines the various factors involved in starch digestion, including feed composition, microbial populations, and enzymatic activity, and how they contribute to the digestive process. Additionally, identifying and characterizing starch fractions in concentrated feed sources is crucial for formulating well-balanced rations. In conclusion, this review synthesizes current knowledge on starch digestion in ruminants, offering insights into the complexities of the process. The collected information not only contributes to our academic understanding but also has practical implications for optimizing feeding strategies, enhancing nutrient utilization, and promoting the overall well-being of ruminants.

Abstract 1805 A Computational Study of Enzymatic Starch-Hydrolysis by a Soy Bean Amylase

Abstract 1805 A Computational Study of Enzymatic Starch-Hydrolysis by a Soy Bean Amylase

THE INFLUENCE OF STARCH PRODUCTS ON THE RHEOLOGI-CAL, PHYSICAL AND CHEMICAL PROPERTIES OF YOGURT

Expanding the assortment of low-fat yogurt with natural structuring sweeteners and regulated quality indicators is an actual direction of scientific research. The purpose of the study is to study the patterns of influence of the products of enzymatic hydrolysis of corn starch with different dextrose equivalents on the rheological, physical and chemical characteristics of yogurt. Тhe functional and technological properties of starch products of different chemical composition were studied – maltodextrin MD-10, glucose syrup IG-42 and glucose-fructose syrup GFS-42. The possibility of completely replacing dry skimmed milk and sugar in the composition of yogurt with a fat content of 1% drinkable with an equivalent content of dry substances of maltodextrin MD-10 and glucose syrup IG-42, which ensure the formation of rheological characteristics inherent high-quality yogurt. Yogurt with sucrose, which inhibits the organization of macromolecular chains of milk proteins, has the lowest thixotropic ability, and yogurt with maltodextrin MD-10 and glucose syrup IG-42 has the highest. The predominance of higher sugars in the composition of MD-10 maltodextrin lengthens, and the increased content of monosaccharides in GFS-42 accelerates the duration of fermentation of milk mixtures in the technological process of production of yogurt with a fat content of 1.0%. Monosaccharides in the composition of GFS-42 significantly affect the activity of water and at the same time reduce the moisture-retaining capacity of the sour-milk yogurt curd. The degree of influence on the rheological characteristics and moisture-binding capacity of yogurt increases with a decrease in the dextrose equivalent of starch products in the following sequence: GFS-42 → IG-42 → MD-10. So, it is the dextrose equivalent of starch products that is an objective criterion of their technological activity as part of yogurt. The results of the study will allow purposeful management of the process of forming low-fat yogurt quality indicators.

Modeling and simulation of an integrated bioethanol-hydrogen production process

Hydrogen (H2) production from renewable feedstocks such as bioethanol represents one of the sustainable and environmentally benign methods of making the proposed utilization of H2 as energy carrier of the future a reality. However, successful implementation of this technology depends on several factors which include understanding of the technicality, as well as process design and modelling. Generally, this area of research has not received adequate attentions. In this article, an integrated process for producing H2 through starch hydrolysis, fermentation, and ethanol steam reforming (ESTR) process has been proposed. Enzymatic hydrolysis of starch was performed to obtain the fermentable sugar (hydrolysate). In addition, batch fermentation experiments were carried out using a laboratory scale fermenter with yeast (Saccharomyces cerevisiae) culture adapted for anaerobic conversion of the hydrolysate to bioethanol. Subsequently, kinetic data obtained from these experiments was employed to guide thermodynamic modeling of fermentation process which was coupled with the ESTR model and was implemented in the Aspen HYSYS simulator. The results ultimately revealed pertinent information which could find applications in the implementation of the proposed integrated process for bioethanol and H2 production.

Comprehensive Identification of the β-Amylase (BAM) Gene Family in Response to Cold Stress in White Clover.

White clover (Trifolium repens L.) is an allopolyploid plant and an excellent perennial legume forage. However, white clover is subjected to various stresses during its growth, with cold stress being one of the major limiting factors affecting its growth and development. Beta-amylase (BAM) is an important starch-hydrolyzing enzyme that plays a significant role in starch degradation and responses to environmental stress. In this study, 21 members of the BAM gene family were identified in the white clover genome. A phylogenetic analysis using BAMs from Arabidopsis divided TrBAMs into four groups based on sequence similarity. Through analysis of conserved motifs, gene duplication, synteny analysis, and cis-acting elements, a deeper understanding of the structure and evolution of TrBAMs in white clover was gained. Additionally, a gene regulatory network (GRN) containing TrBAMs was constructed; gene ontology (GO) annotation analysis revealed close interactions between TrBAMs and AMY (α-amylase) and DPE (4-alpha-glucanotransferase). To determine the function of TrBAMs under various tissues and stresses, RNA-seq datasets were analyzed, showing that most TrBAMs were significantly upregulated in response to biotic and abiotic stresses and the highest expression in leaves. These results were validated through qRT-PCR experiments, indicating their involvement in multiple gene regulatory pathways responding to cold stress. This study provides new insights into the structure, evolution, and function of the white clover BAM gene family, laying the foundation for further exploration of the functional mechanisms through which TrBAMs respond to cold stress.

Effect of electrochemically activated aqueous solution on the yield of reducing agents during enzymatic hydrolysis of green buckwheat starch

The present study was conducted to investigate the effect of complex amylolytic preparations on green buckwheat starch in the environment of oxidized and reduced fractions of electrochemically activated solutions. The novelty of the work lies in the study of the effect of catholyte and anolyte obtained from tap water on the accumulation of reducing substances in the process of enzymatic hydrolysis of green buckwheat starch with the use of preparations AmyloLux ATS and Maltogenase 2X L. The practical significance of the work is related to the possibility of improving the technology of grain drinks or preparation of buckwheat malt in the production of gluten-free beer. It was found that enzymatic hydrolysis of green buckwheat starch with the use of complex amylolytic preparations proceeded most intensively when using the oxidized anodic fraction of electrochemically activated solutions. The level of reducing substances in the anolyte environment exceeded the analogous index in the samples based on water and catholyte by 1.2-1.3 times, already after 30 minutes and before the end of the process.

Enzymatic hydrolysis of starch in electrochemically activated aqueous solution

In this work, the hydrolysis of starch in an enzyme solution was studied, where softened water or fractions of a metastable electrochemically activated aqueous solution (ECAS) were used as a solvent. Extracts obtained after hydrolysis of food starch grains were analyzed using optical density spectrometry of the sample and micro-weighing with a quartz resonator of the dry residue contained in an aqueous solution. It is shown that the enzyme solution on the reduced fraction of ECAS (catholyte) contains the least amount of extracted substances, but it contains the highest concentration of oligosaccharides. This fact may mean the presence of a more efficient cleavage by the enzyme of water-insoluble polysaccharides to low molecular weight derivatives in the medium of the electrolyte. As a result of this synergistic effect, a relatively high content of oligosaccharides is observed even at a low level of primary hydrolysis on the surface of the starch grain. This assumption was investigated on modified starch soluble in water. For the compared aqueous solutions of the enzyme preparation, which initially contain the same concentration of amylodextrins, faster hydrolysis is observed in the enzyme medium on softened water.

A Fractional‐Order Kinetics Approach for Modeling Enzymatic Starch Multiscale Digestion

AbstractThe characterization of the enzymatic hydrolysis of starch is a prerequisite for assessing the impact of starchy food in the gastrointestinal tract. The issue is relevant given the necessity of tailoring food products with a prescribed capacity of glucose production. Several empirical functions have been proposed for modeling starch digestograms. The first‐order exponential and the Weibull models are among the most used functions. Although tight numerical fitting of the experimental data may be obtained with such functions, a reliable interpretation of the underlying parameters is commonly a drawback. This work explores the use of fractional‐order kinetics for the modeling of starch digestograms. The aims are to show that: a) the fractional‐order framework offers a framework for capturing multiscale patterns observed, and b) the Weibull's and Peleg's models are particular cases of a more general function expressed as a generalized (Mittag‐Leffler) exponential function. The results showed that the Mittag–Leffler exponential function provided the best‐fitting results. However, the gain relative to Weibull's and fractional Peleg's functions may be small. It is concluded that Weibull's function approximates a more general function (the Mittag‐Leffler function), and as such its parameters find an easier interpretation in terms of the hydrolysis kinetics.

Encapsulation of Amyloglucosidase in Chitosan-SDS Coacervates as a Means to Control Starch Hydrolysis in Plant-Based Beverages

Starch enzymatic hydrolysis is a key technology for preparing plant-based dairy alternative beverages, preferred for its low energy consumption, high product yield, and quality. However, the high-temperature requirement to terminate reactions and challenges in continuous operations hinder enzyme applicability. Therefore, encapsulation techniques have been explored to address these limitations, enhancing the enzyme’s stability and facilitating process control. This study developed a novel amyloglucosidase encapsulation system based on chitosan-SDS hydrogel capsules produced by simple coacervation. The enzymatic activity of free and immobilised amyloglucosidase was assessed using corn starch and maltose as substrates. The encapsulation process successfully yielded self-supporting structured and spherical capsules, achieving an immobilisation yield of 71% and an enzyme efficiency of 83%. The residual enzymatic activity after 28 days of storage remained at 76%. A feedback inhibition investigation was performed by varying the concentration of corn starch, and favourable applicability of encapsulated amyloglucosidase for continuous processes was found. The encapsulation process offered several advantages, such as a straightforward separation step to halt enzymatic reactions and no need for high temperatures that may affect food product attributes. Lastly, the amyloglucosidase-chitosan-SDS coacervates show promise in improving enzyme stability, facilitating hydrolysis control, and expanding enzyme utilisation in dairy alternative plant beverage manufacture.

Binding interactions between protein and polyphenol decreases inhibitory activity of the polyphenol against α-amylase: A new insight into the effect of dietary components on starch-hydrolyzing enzyme inhibition

The influence of three proteins on α-amylase inhibition of by tannic acid (TA, a typical phenolic competitive inhibitor) were studied. It was found that bovine serum albumin (BSA) and isolated whey protein (IWP) significantly decreased the inhibitory activity of TA against α-amylase with the decreasing effect in the order of BSA > IWP, while egg albumin (EA) hardly affected the enzyme inhibition. TA interacted with both α-amylase and the proteins through non-covalent forces, including hydrogen bondings and π-stackings. The binding affinity of TA to three proteins followed the order of BSA > IWP > EA. Therefore, the similar order in the influence of three proteins on TA-amylase binding interactions was observed, resulting in the respective strongest and weakest decreasing effects of BSA and EA on α-amylase inhibition by TA. Interestingly, the proteins maintained the quenching effect of TA on α-amylase fluorescence. These results indicated that there formed a respective protein-TA-amylase ternary complex, in which TA-protein binding interactions maintained π-stacking between TA and α-amylase but weakened hydrogen bondings between TA and the enzyme active site. Therefore, the competitive inhibition intensity of TA was reduced by BSA and IWP. Conclusively, proteins should be considered as an influencing factor of dietary polyphenols regarding the regulation of starch digestion.

Characterisation of the Enzymatically Extracted Oat Protein Concentrate after Defatting and Its Applicability for Wet Extrusion.

An oat protein concentrate (OC1) was isolated from oat flour through starch enzymatic hydrolysis, by subsequent defatting by ethanol and supercritical fluid extraction (SFE) reaching protein concentrations of 78% and 77% by weight in dry matter, respectively. The protein characterisation and functional properties of the defatted oat protein concentrates were evaluated, compared and discussed. The solubility of defatted oat protein was minor in all ranges of measured pH (3-9), and foamability reached up to 27%. Further, an oat protein concentrate defatted by ethanol (ODE1) was extruded by a single screw extruder. The obtained extrudate was evaluated by scanning electron microscope (SEM), texture and colour analysers. The extrudate's surface was well formed, smooth, and lacking a tendency to form a fibrillar structure. Textural analysis revealed a non-unform structure (fracturability 8.8-20.9 kg, hardness 26.3-44.1 kg) of the oat protein extrudate.