Amyloglucosidase Activity Research Articles

Immobilizing amyloglucosidase on inorganic hybrid nanoflowers to prepare time–temperature integrators for chilled pork quality monitoring

Time-temperature integrators (TTIs) based on amyloglucosidase@Cu3(PO4)2 nanoflowers (AMG@NFs) were developed to monitor the freshness of chilled pork. AMG@NFs were synthesized through biomineralization, resulting in enhanced activity and stability of amyloglucosidase. The TTI prototypes were constructed by hydrolyzing maltodextrin with AMG@NFs. The hue of the TTIs varied from burgundy to colorless, and the discoloration kinetics were investigated. The deterioration process of chilled pork was explored, and the activation energy (Ea) was calculated as 67.32 ± 5.13 kJ/mol. To optimize costs and match TTIs with food, 6#TTI was selected to predict the quality of chilled pork. The dynamic temperature test revealed that the cumulative effective temperatures of chilled pork and 6#TTI were 289.34 K and 290.05 K, respectively, which indicated that 6#TTI was highly reliable and suitable for monitoring the actual chilled pork system. This study offers a new approach for real-time and accurate visual monitoring of chilled pork quality.

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.

Accumulation of complex oligosaccharides and CAZymes activity under acid conditions constitute the Thatcher+Lr9 defence responses to Puccinia triticina

Puccinia triticina (Pt) is an important pathogen of wheat. While breeding programmes develop resistant wheat cultivars to mitigate the effects of such rust-causing pathogens, the emergence of new rust races with wider virulence mandates the implementation of other control strategies. Our study investigated whether acidic pH conditions affected selected Carbohydrate-Active Enzymes (CAZymes) in Pt-inoculated Thatcher + Lr9 (IR) wheat compared to those found in the Thatcher (IS) wheat. The β-glucosidase and amyloglucosidase activity levels significantly increased in IR compared to the control from 1 to 14 days post-inoculation (dpi). In contrast, activity levels of invertase did not change in the IR wheat relative to the control at 1 and 7 dpi, but were significantly reduced in the IR plants at 14 dpi. The IS had higher activity of all three hexose-producing enzymes under acidic conditions. These enzyme activities could be increased in the IS to produce hexose sugars required by Pt to develop and advance infection. The phenotypic analysis supported this view because leaf rust disease symptoms were only visible in the IS plants. For cell wall loosening-related enzymes, the IR displayed higher activity of exoglucanase, xylanase and peroxidase enzymes compared to IS. The liquid chromatography–mass spectrometry analysis showed IR had higher concentrations of complex oligosaccharides compared to the IS. Thus, we concluded that the higher exoglucanase, xylanase and peroxidase activity could be involved in cell wall loosening under acidic conditions, while oligosaccharides could be building-blocks for synthesizing cell wall barriers that apprehend Pt growth in inoculated Thatcher + Lr9.

Effects of rice protein, soy isolate protein, and whey concentrate protein on the digestibility and physicochemical properties of extruded rice starch.

Protein, as the second major component in starchy foods, is crucial for its influence on the physicochemical properties and digestibility of starch. However, the effect of different sources of protein on starch digestibility is still unclear. In this paper, the effects of different sources of proteins (rice protein: RP, soybean isolate protein: SPI, and whey concentrate protein: WPC) on structural features, digestibility, and enzyme activity of extruded rice starch were investigated. The addition of all three proteins reduced the starch digestibility of extrudates. Native SPI and WPC suppressed amyloglucosidase activity, and all three proteins exhibited stronger amyloglucosidase inhibition when hydrolyzed. The rheological properties and Fourier transform infrared spectroscopy results revealed the exogenous proteins and starch interacted through non-covalent bonds and improved the ordered structures in the extrudates. The extrusion process also facilitated the formation of a V-type structure. The sum of SDS and RS content of extrudates was negatively correlated with the content of leached amylose and positively correlated with the ratio of 1047/1022cm-1 . These findings suggest that the inclusion of exogenous proteins during extrusion can affect starch digestibility through mechanisms such as the interaction with starch molecules, as well as the inhibition of amylase activity. PRACTICAL APPLICATION: This result indicated that the addition of protein during extrusion not only increased the nutritional value of the extrudate, but also decreased the starch digestibility. Extrusion technology can efficiently produce extruded products with protein, expanding further applications of protein in food and providing new healthy staple food options for special populations, such as diabetic and overweight people.

Substrate concentration: A more serious consideration than the amount of 5-hydroxymethylfurfural in acid-catalyzed hydrolysis during bioethanol production from starch biomass

5-hydroxymethylfurfural (5-HMF) yield during bioethanol production from starch was determined using spectrophotometry and chromatography. Increasing acid concentration and time favored 5-HMF production with HCl while yield decreased after 45-minute hydrolysis time for HNO3 and H2SO4 hydrolyzed samples. Impacts of glucose (substrate) concentration and produced 5-HMF on bioethanol yield were studied with different sulphuric acid concentrations and different α-amylase and amyloglucosidase activities. A central composite rotational design was utilized to determine the conditions of hydrolysis for optimum glucose production. The results showed that maximum glucose yield occurred at 0.5 M acid concentration and 45-minute hydrolysis time, while maximum yield was achieved at 120 and 280 units of α-amylase and amyloglucosidase activities respectively. It was shown that 5-HMF did not exhibit much inhibition on ethanol yield at low acid concentrations but became pronounced at higher acid concentrations, while high glucose concentrations had a pronounced negative effect on ethanol yield and fermentation efficiency.

Influence of native cellulose, microcrystalline cellulose and soluble cellodextrin on inhibition of starch digestibility

Cellulose is a major component of dietary fiber and it is proved to influence starch digestibility. The effects of native cellulose (NC), microcrystalline cellulose (MC), soluble cellodextrin (SC) on starch digestion have not been clearly elucidated. In this study, three types of cellulose with representative molecular weights (NC, 422500 Da; MC, 27750 Da; SC, 2202 Da) were prepared and their effects on starch digestion, glucose diffusion, α-amylase and amyloglucosidase activity were compared. The results suggested SC inhibited starch digestibility to a greater degree than those of NC and MC. When addition of SC reached 3 %, rapidly digestible starch proportion decreased from 31.2 % to 11.3 % and resistant starch proportion increased from 15.0 % to 58.0 %. Notably, hindrance effects of SC on glucose diffusion were higher than those of NC and MC. Moreover, SC reduced activity of α-amylase and amyloglucosidase to a larger extent than those of MC and NC. With the effect of starch digestion inhibition, NC, MC and SC could be utilized as functional food ingredients. Especially, the soluble property and the highest starch digestion inhibition ability of SC favors its application in food industry.

Effect of physical and thermal pretreatments on enzymatic activity in the production of microporous cassava starch

Cassava starch is modified to increase porosity and lacerations that are limited when only enzymatic treatments are used. This study proposes to improve enzymatic activity of α-amylase and amyloglucosidase on the polymer chains of cassava starch by implementing physical and thermal pretreatments below the gelatinization temperature and before the hydrolytic process. The pretreatments increased the biocatalytic action of the enzymes, causing significant changes in the morphology of the granules, and superficial lacerations were found in samples of starches pretreated with ultrasound (UTS) or annealing and ultra-rapid freezing (ANN-C). At the structural level, the modified starches revealed substantial changes as the infrared spectra reflected a displacement of the absorption bands in the region from 900 to 1100 cm-1. This is associated with an alteration and reorganization of the amorphous and crystalline zones of the granules and is consistent with a decrease in amylose content (from 19.53% to 17.64%) and an increase in the crystallinity index. The thermal behavior of the starches was also modified by increasing the peak temperature (from 68.22°C to 75.38°C) and reducing the gelatinization enthalpy (from 19.34 to 15.79 J/g). UTS and ANN-C pretreatments significantly improved the mesoporous and hydrophilic properties of the modified cassava starches.

Effects of whey protein on the in vitro digestibility and physicochemical properties of potato starch

The aim of this study was to examine the effect of whey protein isolate (WPI) on the digestibility and physicochemical properties of potato starch (PS) after heat treatment. WPI reduced the digestibility of PS and increased the order and aggregation structure of gelatinized PS. Examination of the rheological properties of the PS-WPI mixed system before and after adding different chemicals (sodium chloride, urea, and sodium dodecyl sulfate) indicated an involvement of hydrogen bonds and hydrophobic interactions in the PS-WPI gelatinization system. The pasting properties, swelling power, and thermal properties indicated that WPI suppressed the swelling and gelatinization of PS. The addition of WPI reduced the amylose leaching rate from the starch granules, indicating that the presence of exogenous protein could prevent amylose diffusion from the starch granules. Native WPI and its hydrolysate also inhibited amyloglucosidase activity. These findings indicated that the mechanism by which WPI reduces PS digestion involves hydrophobic interactions and hydrogen bonding between WPI and PS, as well as enzyme activity inhibition.

Understanding the mechanisms of whey protein isolate mitigating the digestibility of corn starch by in vitro simulated digestion

Exogenous proteins in starchy foods may alter the digestibility of the starch. In this study, the effect of whey protein isolate (WPI) with different ratios on the digestibility of co-gelatinized corn starch (CS) was studied, and the possible mechanisms were investigated. The rapidly digestible starch (RDS) content was reduced from 66.89% to 59.01% and the resistant starch (RS) content was increased from 14.47% to 22.78% when 4.5% WPI was added during co-gelatinization. This exogenous protein also suppressed the reduction of the average molecular weight of starch during digestion. WPI had no effect on CS digestibility when starch and protein were gelatinized separately and mixed during digestion. The inhibition of starch digestion by WPI was closely related to the protein-starch interaction. Rheological property analysis indicated strong hydrogen bonding interactions were formed between WPI and CS during co-gelatinization. The physical barrier of WPI also had a certain inhibitory effect on the digestion of starch, while WPI only had a transient effect on the viscosity of the digestive juice. Meanwhile, WPI restricted the hydrolysis of CS by inhibiting the activities of α-amylase and amyloglucosidase, and the inhibition of amyloglucosidase activity had a special effect on the rapid digestion of starch at the early stage.

In vitro α amylase and amyloglucosidase inhibitory activities of selected underutilized cereals, yams and root crops

Diabetes is one of the most challenging global health problems. Currently, reducing postprandial blood glucose concentration by inhibiting carbohydrate hydrolyzing enzymes is recognized as an effective treatment against diabetes. In this study, in vitro α-amylase and amyloglucosidase inhibitory effect of selected, underutilized and locally grown cereals (P. miliaceum, P. scrobiculatum and S. italica), yams (D. alata and D. esculenta) and root crops (L. spinosa and N. nucifera) were evaluated related to their dietary fiber contents. Results showed that the inhibitory effect of selected food crops has a positive correlation with their dietary fiber contents. L. spinosa with higher dietary fiber content generally had higher α-amylase (78.0%) and amyloglucosidase (71.28%) enzyme inhibitory percentages among yams and root crops. Significantly highest (p<0.05) dietary fiber content was in S. italic among three cereal varieties and it showed the highest inhibition activity against α-amylase (67.8%) and amyloglucosidase (59.40%) activity. These tested food crops are considerable sources of dietary fiber and are having α-amylase and amyloglucosidase enzyme inhibitory actions against starch hydrolysis.

USE OF COMPOSITE AGRO-SUBSTRATES FOR AMYLOGLUCOSIDASE SYNTHESIS AND CHARACTERIZATION BY Aspergillus niger OTF AND Aspergillus flavus CLOR1 USING SOLID STATE FERMENTATION

Amyloglucosidase catalyses the hydrolysisof glycosidic residues from non-reducing ends of amylose and amylopectin in starch components to produce β-D-glucose for industrial processes. The utilization of composite agro-substrates for amyloglucosidase production byAspergillus niger OTF and A. flavus CLOR1 using solid state fermentation and characterization of the enzyme were investigated. The substrates include rice bran (R), soybeans flour (S), palm kernel cake (P), wheat bran (W) and oyster shell (O) at various compositions (Medium I – VIII) were supplied with adequate moisture content for a solid culture medium of fermentation.They were inoculated into with the isolates and incubated at 30 °C for 120 h respectively. Cultural conditions such as temperature, pH and storage stability were optimized. Medium I (WSPO; 100:2:3:1, w/w) yielded amyloglucosidase activity 83.92 Ug-1and Medium V (WSP; 100:2:3, w/w) resulted into activity 67.64 Ug-1 at 48 h optimal time. There was decline in activity after 48 h in all samples but with maximum activity at pH 5. Aspergillus niger OTF showed maximum amyloglucosidase activity23.5 Ug-1 as compared with A. flavus CLOR1 20.07 Ug-1 at 30 ºC. The residual activities in term of storage stability were 43.9% and 44.9% at 4 °C and 68. 6% and 27.8% at 28 °C at week 2 respectively which later declined over a period of time. This study revealed that higher glucoamylase activity was significantly enhanced using these inexpensive and readily available agro-residues by both strains of isolates in solid culture medium. Thus, these residues could be a potential and economic source for the production of amyloglucosidase.

Magneto-Controlled Enzyme Activity with Locally Produced pH Changes.

Biocatalytic activity of amyloglucosidase (AMG), immobilized on superparamagnetic nanoparticles, is dynamically and reversibly activated or inhibited by applying a magnetic field. The magnetic field triggers aggregation/deaggregation of magnetic particles that are also functionalized with urease or esterase enzymes. These enzymes produce a local pH change in the vicinity of the particles changing the AMG activity.

Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization

In this study, amyloglucosidase was immobilized within agar-agar through entrapment technique for the hydrolysis of soluble starch. Enzymatic activities of soluble and entrapped amyloglucosidase were compared using soluble starch as a substrate. Partially purified enzyme was immobilized and maximum immobilization yield (80%) was attained at 40 gL-1 of agar-agar. Enzyme catalysis reaction time shifted from 5.0 min to 10 min after immobilization. Similarly, a five-degree shift in temperature (60 °C–65 °C) and a 0.5 unit increase in pH (pH-5.0 to pH-5.5) were also observed. Substrate saturation kinetics revealed that Km of entrapped amyloglucosidase increased from 1.41 mg ml−1 (soluble enzyme) to 3.39 mg ml−1 (immobilized enzyme) whereas, Vmax decreased from 947 kU mg−1 (soluble enzyme) to 698 kU mg−1 (immobilized enzyme). Entrapped amyloglucosidase also exhibited significant catalytic performance during thermal and storage stability when compared with soluble enzyme. Reusability of entrapped amyloglucosidase for hydrolysis of soluble starch demonstrated its recycling efficiency up to six cycles which is an exceptional characteristic for continuous bioprocessing of soluble starch into glucose.

English

Little or no attention has so far been paid to using sweet sorghum malt for commercial beer brewing. Thus, three sweet sorghum varieties (SSV) and four sorghum varieties (SV) were analyzed for brewing quality. Variations were observed in their thousand grain weights (22.8 to 58.7 g), grain moisture contents (12.5 to 20.5%), germination energies (99.0 to 100%) and germination capacities (99.7 to 100%). After 4-day germination, radicle lengths of seedlings were 2 to 5 fold of plumules. Remarkable variations existed in their water sensitivity (7.1 to 27.6%) and grain protein contents (7.0 to 11.8%). Malts moisture contents (8.6 to 13.8%), malting losses (16.3 to 26.0%) and malts protein contents (12.2 to 19.5%) differed among cultivars. Cold water extracts (CWE) (3.8 to 8.8%) and hot water extracts (HWE) (8.8 to 17.5%) varied with cultivars. HWE were 1.5 to 3-fold of CWE. Diastatic powers (DP) were substantially higher in SSV (123.7 to 136.7&ordm;) compared to SV (111.8 to 117&ordm;). Amyloglucosidase (AMG) activities were detected in SSV malts but not in SV. &alpha;-Amylases activities in both SSV and SV malts were about 2 to 4-fold of &beta;-amylases. Generally, DP in SV malts = &alpha;-amylase + &beta;-amylase activities. But DP in SSV malts &gt;&alpha;-amylase + &beta;-amylase + AMG activities, thus, suggesting synergism between the enzymes. SSV showed similar wort yields with SV. Reducing sugars in wort of SSV (12.3 to 15.6 mg/ml) were higher than those of SV (6.2 to 10.5 mg/ml). Malts and worts analyses suggest that SSV have greater beer brewing potentials than SV. Key words: Diastatic power, amylase, amyloglucosidase, sweet sorghum, malt, wort. &nbsp

A multi-pumping flow analysis system for β-galactosidase activity assays

A fully mechanized Arduino-controlled multi-pumping flow analysis system and procedure for the determination of β-galactosidase activity are proposed. The applied bioanalytical method is based on the determination of p-nitrophenol formed in the course of enzyme-catalyzed hydrolysis of p-nitrophenyl-galactopyranosides. The photometric detection is performed using dedicated flow-through optoelectronic detector made of paired light emitting diodes. The developed bioanalytical system was applied for evaluation of optimal enzyme detection conditions (pH, temperature and reaction time), selection of appropriate substrate for the assays, comparison of enzymes of different origins (isoenzymes), detection of β-galactosidase inhibitor and finally to the determination of enzyme activity in some dietary supplements dedicated for people suffering from lactose intolerance. Depending on measurements conditions the developed bioanalytical system allows determination of β-galactosidase in the wide range of activity (up to 15 U/mL at detection limit ca 0.01 U/mL) with high sample flowthroughput (up to 30 detections per hour). Additionally, the potential utility of the developed analytical system for amyloglucosidase activity assays has been demonstrated.

Flexible and expeditious assay for quantitative monitoring of alpha-amylase and amyloglucosidase activities

The monitoring of the activity of alpha-amylase and amyloglucosidase is an important tool for studying their role in the hydrolysis of starch. Here we introduced an improved method capable to measure the activity of alpha-amylase and amyloglucosidase from different sources based on a quantitative starch-iodine assay. The developments of the assay sought the consistent preparation of the reagents, the rescale of the assay and the adjustment of the sensitivity. This was complemented by a glucose yield assay for amyloglucosidase that allowed a secondary source of information when insoluble starches were studied. The proposed method showed high precision in long-term use (RSD < 6.3%). Furthermore, all experimental conditions can be adapted according to the equipment available at each laboratory, transforming this method in a broadband analytical tool for screening alpha-amylase and amyloglucosidase activities.•Tailorable assay based on the starch-iodine staining for the determination of alpha-amylase and amyloglucosidase activities.•Enhanced consistence of reagent preparation.•High intra-day and inter-day reproducibility.

Agarose Hydrogel Beads: An Effective Approach to Improve the Catalytic Activity, Stability and Reusability of Fungal Amyloglucosidase of GH15 Family

This study deals with the immobilization of amyloglucosidase within agarose using method of entrapment. Enzyme was produced from Aspergillus fumigatus KIBGE-IB33 and then partially purified using 40% ammonium sulphate saturation. Using 40 gl−1 concentration of agarose and adjusting 3.0 mm size of hydrogels, maximum entrapment yield (78%) was obtained. The kinetic behavior was slightly changed after immobilization as reaction time and reaction temperature increases from 5.0 min (soluble) to 10.0 min (immobilized) and 60 °C (soluble) to 65 °C (immobilized), respectively while, pH optima remained same (pH 5.0). Substrate saturation kinetics revealed that Km was increased from 1.47 to 4.215 mg ml−1 while, the value of Vmax decreased from 947 to 611 kU mg−1 for soluble and entrapped amyloglucosidase, respectively. The stability profile of amyloglucosidase also significantly improved after entrapment in agarose hydrogels at 50, 60 and 70 °C for 120 min with retention of 77, 59 and 25% residual activity, respectively. Furthermore, the t1/2 of soluble and immobilized amyloglucosidase at 60 °C was 167 and 375 min respectively. Due to increase in reusability for various subsequent cycles of entrapped amyloglucosidase, about 8.73 mg ml−1 increase in glucose production was observed as compared to soluble enzyme.

Digestion rate of tapioca starch was lowed through molecular rearrangement catalyzed by 1,4-α-glucan branching enzyme

The 1,4-α-glucan branching enzyme (GBE) was used to modify native starch. GBE treatment resulted in the molecular rearrangement of tapioca starch, leading to an increase in the α-1,6 linkage content, the weight ratio of the short-chain fraction to the long-chain fraction (SF:LF), the content of cyclic molecules, and a decrease in the average ECL. GBE treatment also suppressed the digestion rate of starch. The magnitude of these changes increased in a time-dependent manner. After a 10-h GBE treatment, the rapidly digestible starch (RDS) content of starch was decreased by 14.5%. The RDS content was negatively related with the α-1,6 glycosidic linkage content (R2 = 0.8982) and the SF:LF ratio (R2 = 0.9646) but positively related with the average ECL of starch (R2 = 0.9406). Cyclic structures may also have suppressed the hydrolytic activity of pancreatin and amyloglucosidase. These joint actions of the molecular rearrangements catalyzed by GBE, not just the increase in α-1,6 linkage content, provides a mechanistic basis for the digestion rate reduction.

Purification and Characterization of Amyloglucosidase Enzyme from the Thermophilic &lt;i&gt;Endomycopsis fibuligera&lt;/i&gt; Using Sago Starch as a Substrate

An investigation on purification and characterization of amyloglucosidase enzyme from Endomycopsis fibuligera by fermentation of sago starch has been carried out. This enzyme is inductive and can be produced by fermenting sago starch in a medium containing E. fibuligera. Crude enzyme was obtained by centrifuging the medium cultures containing E. fibuligera at 6,000 rpm for 20 min and then adding with 0.15 M acetate buffer (pH 5.0). Enzyme activity was determined using Somogyi-Nelson method by quantifying the released glucose from amyloglucosidase catalysis of starch (0.2%) as substrate. Prepurification process was conducted by ammonium sulphate fractionation and it showed that the ammonium sulphate fractionation with the degree of saturation of 40-60% produced a maximum activity of enzyme. Purification by DEAE-Cellulose and Sephadex G-75 column chromatography produced three and one fractions with purifity 17.4 and 22.5 times, respectively, compared to the crude extract enzyme. Characterization of this enzyme showed the optimum condition at pH 5.0 and 55 °C with 0.2% starch as substrate. The amyloglucosidase activities was strongly increased by addition of Co2+ and Mn2+ ions, whereas the activities were weakly decreased by addition of K+, Mg2+, and Fe3+ ions.

The glycogen of Galdieria sulphuraria as alternative to starch for the production of slowly digestible and resistant glucose polymers

Highly branched glucose polymers produced from starch are applied in various products, such as peritoneal dialysis solutions and sports drinks. Due to its insoluble, granular nature, the use of native starch as substrate requires an energy consuming pre-treatment to achieve solubilization at the expense of process costs. Glycogen, like starch, is also a natural glucose polymer that shows more favorable features, since it is readily soluble in cold water and more accessible by enzymes. The extremophilic red microalga Galdieria sulphuraria accumulates large amounts of a small, highly branched glycogen that could represent a good alternative to starch as substrate for the production of highly branched glucose polymers. In the present work, we analyzed the structure-properties relationship of this glycogen in its native form and after treatment with amyloglucosidase and compared it to highly branched polymers produced from potato starch. Glycogen showed lower susceptibility to digestive enzymes and significantly decreased viscosity in solution compared to polymers derived from starch, properties conferred by its shorter side chains and higher branch density. The action of amyloglucosidase on native glycogen was somewhat limited due to the high branch density but resulted in the production of a hyperbranched polymer that was virtually resistant to digestive enzymes.