Dietary 5,6,7-Trihydroxy-flavonoid Aglycones and 1-Deoxynojirimycin Synergistically Inhibit the Recombinant Maltase-Glucoamylase Subunit of α-Glucosidase and Lower Postprandial Blood Glucose.

1-Deoxynojirimycin (1-DNJ) is the major effective component of mulberry leaves, exhibiting inhibitory activity against α-glucosidase. However, due to the low content of 1-DNJ in mulberry products, its level cannot meet the lowest dose to exhibit its activity. In this study, a combination of dietary 5,6,7-trihydroxy-flavonoid aglycones with 1-DNJ showed synergistic inhibitory activity against maltase of mice α-glucosidase and recombinant C- and N-termini of maltase-glucoamylase (MGAM) and baicalein with 1-DNJ exhibited the strongest synergistic effect. The synergistic effect of the combination was also confirmed by the maltose tolerance test in vivo. Enzyme kinetics, molecular docking, fluorescence spectrum, and circular dichroism spectrometry studies indicated that the major mechanism of the synergism is that baicalein was a positive allosteric inhibitor and bound to the noncompetitive site of MGAM, causing an increase of the binding affinity of 1-DNJ to MGAM. Our results might provide a theoretical basis for the design of dietary supplements containing mulberry products.

Porous starches modified with double enzymes: Structure and adsorption properties.

To explore an effective enzyme combination instead of a common enzyme method, sequential α-amylase and glucoamylase, a method of sequential glycosyltransferase and branching enzyme was chosen to compare the macroscopic features, structure characteristics, porosity characteristics and adsorption quantity of potato, corn, wheat and sweet potato starches. The results indicated that after enzyme treatment, the relative crystallinity of potato, corn, wheat and sweet potato starches increased. Moreover, amylose levels decreased, while pore size and volume, and specific surface area increased after sequential glycosyltransferase and branching enzyme. In terms of pore size, sequential α-amylase and glucoamylase produced abundant mesopores (2-50 nm), whereas sequential glycosyltransferase and branching enzyme developed much more macropores (>50 nm). The adsorption quantities of the starch obtained with sequential glycosyltransferase and branching enzyme were about 2 folds higher than that of the starch obtained with sequential α-amylase and glucoamylase. Therefore, the sequential glycosyltransferase and branching enzyme may be an ideal method to create porous starch as a desirable green adsorbent for industries.

Effect of thermal processing for rutin preservation on the properties of phenolics & starch in Tartary buckwheat achenes.

The endogenous rutin-degrading enzymes (RDEs) in Tartary buckwheat (TB) considerably limit the development of TB functional foods. In this study, three thermal treatments, including superheated steam (SS), saturated steam (ST), and far-infrared drying (FID), were used to inactivate RDEs in TB. Results showed that SS and ST could efficiently inactivate RDEs, whereas FID could not. The extractable rutin contents in TB were increased by 52.3% and 12.3% by SS and ST, respectively, with 90 s of treatment time. Furthermore, the properties of phenolics and starch were used to evaluate the influence of thermal inactivation on TB. Results showed that the soluble phenolic compounds contents in TB were significantly improved (p < 0.05) by SS. The bound phenolic compounds contents were decreased after SS and ST treatments. The change in antioxidant properties was consistent with that of phenolics and flavonoids. Besides, the starch in SS- and ST-treated TB achenes had higher relative crystallinity, setback, transition temperatures, rapidly digestible starch, and slowly digestible starch contents, but a lower ratio of 1047 cm-1/1022 cm-1, peak viscosity, breakdown, gelatinization enthalpy, and resistant starch contents, than native TB starch. In conclusion, SS was a better method for the inactivation of RDEs than ST.

Revisiting Mechanisms Underlying Digestion of Starches.

The factors that determine the digestion rate of starches were revealed using different forms of starches and a mixture of α-amylase and amyloglucosidase. Gelatinized starch samples with a degree of gelatinization (DG) from 12.2 to 100% for potato starch and from 7.1 to 100% for lotus seed starch were obtained. With an increasing DG, the short- and long-range molecular orders of both starches were disrupted progressively. The first-order digestion rate constant (k) of both starches increased with an increasing DG, although the positive linear relationships between DG and k differed (R2 = 0.87 for potato starch, and R2 = 0.74 for lotus seed starch). The mean fluorescence intensity showed a positive linear correlation with DG, which was strong for potato starch (R2 = 0.99) and relatively weaker for lotus seed starch (R2 = 0.54). These results indicated that DG is a major determinant for the digestion rate of potato starch and lotus seed starch and that the access/binding of enzymes to starch was the main rate-limiting factor for digestion of starches.

Optimization and scale up of itaconic acid production from potato starch waste in stirred tank bioreactor.

Present study used Aspergillus terreus strain C1 isolated from mangrove soil for itaconic acid (IA) production from potato starch waste. Fermentation parameters were optimized by classical one factor approach and statistical experimental designs, such as Plackett-Burman and response surface designs. Anionic deionization of potato waste was found to be a very effective, economic, and easy way of improving IA production. The increase in IA production by deionization was found to correlate with removal of phosphate. In our knowledge, this is the first report on application of deionization of potato waste to enhance IA production. Other parameters like inoculum development conditions, pH, presence of peptone and certain salts in the medium also significantly affected IA production. IA production by strain C1 increased 143-fold during optimization when compared with the starting condition. The optimized IA level (35.75 g/L) was very close to the maximum production predicted by RSM (38.88 g/L). Bench scale production of IA was further optimized in 3-L stirred tank reactor by varying parameters like agitation and aeration rate. The maximum IA production of 29.69 g/L was obtained under the agitation speed of 200 rpm and aeration rate of 0.25 vvm. To the best of our knowledge, it is the first report on IA production from potato starch waste at bioreactor level. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2774, 2019.

Enzyme-assisted development of biofunctional polyphenol-enriched buckwheat protein: physicochemical properties, in vitro digestibility, and antioxidant activity.

BACKGROUND: During the last decade buckwheat was reported to have positive health effects. The present study investigated a high-polyphenol buckwheat protein (Fagopyrum esculentum Moench) prepared by enzyme-assisted processing, together with its physicochemical properties, in vitro digestibility, and antioxidant activity. RESULTS: Buckwheat protein prepared from the synergistic enzymatic action of α-amylase and amyloglucosidase (E-BWP) had much higher polyphenol content than buckwheat protein prepared by isoelectric precipitation (I-BWP) or salt extraction (S-BWP). Rutin degraded during the process, giving quercetin. The protein constituents and amino acid composition of E-BWP were very similar to those of native buckwheat and were able to meet the WHO/FAO requirements for both children and adults. During in vitro digestion, E-BWP showed anti-digestive behavior with a nitrogen release that was lower than that of I-BWP or S-BWP. The positive effect of the polyphenol content of E-BWP resulted in a higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) content and greater reducing activity. CONCLUSION: Buckwheat protein with high polyphenol content was successfully developed by enzyme-assisted processing. It had a well-balanced amino acid profile, antidigestive behavior, and high antioxidant activities. The results suggest that enzyme-assisted processing is promising in the production of polyphenol-enriched cereal protein, contributing higher functionality with good nutritional and antioxidant properties. © 2018 Society of Chemical Industry.

Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus.

Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0-7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.

In vitro digestibility and changes in physicochemical and structural properties of common buckwheat starch affected by high hydrostatic pressure.

High hydrostatic pressure (HHP), a non-thermal processing technology, was applied at 120, 240, 360, 480, and 600MPa to assess its effect on the in vitro digestibility, physicochemical, and structural properties of common buckwheat starch (CBS). HHP treatment resulted in CBS granules with more rough surfaces. With the increasing pressure level, amylose content, pasting temperature, and thermal stability substantially increased and relative crystallinity, hardness, swelling power, and viscosity decreased. At 120-480MPa, HHP did not affect the 'A'-type crystalline pattern of CBS. However, at 600MPa, HHP contributed to a similar 'B'-type pattern. Compared with native starch, HHP-modified CBS samples had lower in vitro hydrolysis, reduced content of rapidly digestible starch, and increased levels of slowly digestible starch and resistant starch. These results revealed that the in vitro digestibility, physicochemical, and structural properties of CBS are effectively modified by HHP.

Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase.

This work illustrates the preparation of magnetic macromolecular glucoamylase CLEAs using dialdehydic pectin, as a cross linker instead of traditional glutaraldehyde. The effect of precipitators type and amount, cross linker concentration, cross linking time and amount of amino functionalized magnetic nanoparticles (AFMNs) on glucoamylase activity was studied. Glucoamylase magnetic macromolecular CLEAs prepared by precipitation in presence of AFMNs by ammonium sulfate were subsequently cross linked by dialdehydic pectin. After cross-linked by pectin, 95.4% activity recovery was achieved in magnetic macromolecular CLEAs, whereas in case of glutaraldehyde cross linker, 85.3% activity recovery was achieved. Magnetic macromolecular CLEAs showed 2.91 and 1.27 folds higher thermal stability as compared to free and magnetic glutaraldehyde CLEAs. In kinetics study, magnetic macromolecular CLEAs retained same Km values, whereas magnetic glutaraldehyde CLEAs showed higher Km value than free enzyme. The porous structure of magnetic macromolecular CLEAs was not only enhanced mass transfer toward macromolecular substrates, but also showed compression resistance for 5 consecutive cycles which was checked in terms of effectiveness factor. At the end, in reusability study; magnetic macromolecular CLEAs were retained 84% activity after 10(th) cycle without leaching of enzyme which is 22% higher than traditional magnetic CLEAs.

Selected properties of acetylated adipate of retrograded starch.

Native potato starch (NS) and retrograded starch (R - obtained via freezing and defrosting of a starch paste) were used to prepare starch acetates: NS-A and R-A, and then acetylated distarch adipates: NS-ADA and R-ADA. The chemically-modified preparations produced from retrograded starch (R-A; R-ADA) were characterized by a higher degree of esterification compared to the modified preparations produced under the same conditions from native potato starch (NS-A; NS-ADA). Starch resistance to amylolysis was observed to increase (to 30-40 g/100 g) as a result of starch retrogradation and acetylation. Starch cross-linking had a significant impact on the increased viscosity of the paste in the entire course of pasting characteristics and on the increased values of rheological coefficients determined from the equations describing flow curves. The produced preparation of acetylated retrograded starch cross-linked with adipic acid (R-ADA) may be deemed an RS3/4 preparation to be used as a food thickening agent.

Co-conjugation vis-à-vis individual conjugation of α-amylase and glucoamylase for hydrolysis of starch.

Two enzymes, α-amylase and glucoamylase have been individually and co-conjugated to pectin by covalent binding. Both the enzyme systems showed better thermal and pH stability over the free enzyme system with the complete retention of original activities. Mixture of individually conjugated enzymes showed lower inactivation rate constant with longer half life than the co-conjugated enzyme system. Individually conjugated enzymes showed an increase of 56.48 kJ/mole and 38.22 kJ/mole in activation energy for denaturation than the free enzymes and co-conjugated enzymes, respectively. Km as well as Vmax of individually and co-conjugated enzymes was found to be higher than the free enzymes. SDS-polyacrylamide gel electrophoresis confirmed the formation of conjugate and co-conjugate as evident by increased molecular weight. Both the enzyme systems were used for starch hydrolysis where individually conjugated enzymes showed highest release of glucose at 60 °C and pH 5.0 as compared to free and co-conjugated enzyme.

Effect of preparation method on the properties of potato starch acetates with an equal degree of substitution.

Acetylated retrograded starch is one of the forms of resistant starch (RS3/4). Apart from the known resistance to amylolysis, it is characterized by the capability to form viscous pastes. Properties of this type of acetates are mainly determined by the degree of substitution and raw material used for esterification. The objective of this study was to produce starch acetates with a degree of substitution DS=0.1 from native potato starch and retrograded potato starch, and to compare selected properties of the resultant preparations. Retrograded starch was produced by freezing pastes with concentrations of 1, 4, 10, 18 or 30 g/100 g. Starch acetates with a degree of substitution DS∼0.1 were produced from native or retrograded starch through acetylation with various doses of acetic acid anhydride (6.5-26.0 cm(3)/100 g of starch). The preparations produced were characterized by various properties. A positive correlation was observed between resistance to amylolysis and the number of acetyl groups at C2 and C3 the produced starch acetates.

Optimization of enzymatic hydrolysis of cassava to obtain fermentable sugars.

This work evaluates the enzymatic hydrolysis of starch from cassava using pectinase, α-amylase, and amyloglucosidase. A central composite rotational design (CCRD) was carried out to evaluate the effects of amyloglucosidase, pectinase, reaction time, and solid to liquid ratio. All the experiments were carried out in a bioreactor with working volume of 2 L. Approximately 98% efficiency hydrolysis was obtained, resulting in a concentration of total reducing sugar released of 160 g/L. It was concluded that pectinase improved the hydrolysis of starch from cassava. Reaction time was found to be significant until 7 h of reaction. A solid to liquid ratio of 1.0 was considered suitable for hydrolysis of starch from cassava. Amyloglucosidase was a significant variable in the process: after its addition to the reaction media, a 30%-50% increase in the amount of total reducing sugar released was observed. At optimal conditions the maximum productivity obtained was 22.9 g/(L·h).

An extracellular glucoamylase produced by endophytic fungus EF6.

A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1% soluble starch. The enzyme was purified and characterized. Purification steps involved (NH4)2SO4 precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained was 9.15%. The enzyme is monomeric with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of 62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60 degrees C. After 30 min for incubation, glucoamylase was found to be stable lower than 50 degrees C. The activity decrease rapidly when residual activity was retained about 45% at 55 degrees C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0-7.0 at 50 degrees C. The activity of glucoamylase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg2+. Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes the hydrolysis of soluble starch and maltose as the substrate, the enzyme had Km values of 2.63, and 1.88 mg/ml and Vmax, values of 1.25, and 2.54 U/min/mg protein, and Vmax/Km values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQVV DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-alpha-D-glucan glucanohydrolase).

Isolation and characterization of glucoamylase from a wastewater treatment yeast Hansenula fabianii J640, and construction of expression vector.

Hansenula fabianii J640 highly expresses an extracellular glucoamylase (GA). Here, we purified the GA and showed that it has pH and temperature optima of 5.0 and 50 °C, respectively, stable at temperatures up to 50 °C, and is inhibited by Ag(2+), Hg(2+), and Cu(2+). The gene was found in an expression library with anti-GA antibodies. A cDNA was found to encode 491 amino acids, including a putative signal peptide of 21 amino acids. Because of the gene's high expression, we used its promoter and terminator regions to improve a previously developed H. fabianii J640 expression system.

Continuous production of ethanol from starch using glucoamylase and yeast co-immobilized in pectin gel.

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica-enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica-enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 x 10(-4) cm/s.

[Immobilized glucoamylase: A biocatalyst of dextrin hydrolysis].

Heterogeneous biocatalysts of starch conversion based on glucoamylase and carbon-containing carriers were obtained, and their biocatalytic properties in enzymatic hydrolysis of corn dextrins were studied. It was shown that the morphology of the surface carbon layer of carriers markedly affected the properties of biocatalysts. Glucoamylase that was immobilized by adsorption on the surface of carriers covered with a layer of catalytic fibrous or pyrolytic carbon had the maximum enzymatic activity and stability, whereas the biocatalysts prepared on the basis of carriers that had no carbon layer or were covered with graphite-like surface carbon had a low activity and stability.

Production of amylases by Aspergillus tamarii

A strain of Aspergillus tamarii, a filamentous fungus isolated from soil, was able to produce both Ó-amylase and glucoamylase activities in mineral media supplemented with 1(per cent) (w/v) starch or maltose as the carbon source. Static cultivation led to significantly higher yields than those obtained using shaking culture. The production of amylases was tolerant to a wide range of initial culture pH values (from 4 to 10) and temperature (from 25 to 42degree C). Two amylases, once Ó-amylase and one glucoamylase, were separated by ion exchange chromatography. Both partially purified enzymes had optimal activities at pH values between 4.5 and 6.0 and were stable under acid conditions (pH 4.0-7.0). The enzymes exhibited optimal activities at temperatures between 50(degree) and 60(degree) C and wete stable for more than ten hours at 55(degree) C.

Italian buckwheat (Fagopyrum esculentum) starch: physico-chemical and functional characterization and in vitro digestibility.

A study on the physico-chemical properties and structure together with the evaluation of starch digestibility was carried out on starch isolated from buckwheat (Fagopyrum esculentum) cultivated in different Italian areas. Results showed that buckwheat samples analysed were different among them and from wheat starch used as reference. Buckwheat granules were polygonal in shape and had a smaller diameter than the wheat starch granule. The starch obtained from buckwheat had a higher swelling power than the wheat one, probably as a consequence of the wheaker but more extensive bonding forces in the granule. During cooling, buckwheat samples showed a good paste stability.