Generation of short-chained granular corn starch by maltogenic α-amylase and transglucosidase treatment.

We describe a method for permitting efficient modification by transglucosidase (TGA), from glycoside hydrolase family 31 (GH31), sequentially after the pre-treatment by maltogenic α-amylases (MA) from GH13. TGA treatment without MA pre-treatment had negligible effects on native starch, while TGA treatment with MA pre-treatment resulted in porous granules and increased permeability to enzymes. MA→TGA treatments lead to decreased molecular size of amylopectin molecules, increased α-1,6 branching, and increased amounts of amylopectin chains with the degree of polymerization (DP)<10 and decreased amounts of DP 10-28 after debranching. Wide-angle X-ray scattering (WAXS) data showed a general decrease in crystallinity except for a long term (20 h) TGA post-treatment which increased the relative crystallinity back to normal. MA→TGA treatment significantly lowered the starch retrogradation of starch and retarded the increase of storage- and loss moduli during storage. This work demonstrates the potential of sequential addition of starch active enzymes to obtain granular starch with improved functionality.

Optimization of Catechin and Proanthocyanidin Recovery from Grape Seeds Using Microwave-Assisted Extraction.

Grape seed extract (GSE) is a rich source of condensed flavonoid tannins, also called proanthocyanidins (PACs). The high molecular weight of polymeric PAC limits their biological activity due to poor bioavailability. The present study was undertaken to explore the potential applicability of microwave-assisted extraction (MAE) to convert GSE-PAC into monomeric catechins. A central composite design (CCD) was used to optimize the processing conditions for the MAE. The maximum total yield of monomeric catechins (catechin, epicatechin, and epicatechin gallate) and PAC were 8.2 mg/g dry weight (DW) and 56.4 mg catechin equivalence (CE)/g DW, respectively. The optimized MAE condition was 94% ethanol, 170 °C temperature, and a duration of 55 min. Compared to the results for PACs extracted via conventional extraction (Con) (94% ethanol; shaking at 25 °C for 55 min), MAE yielded 3.9-fold more monomeric catechins and 5.5-fold more PACs. The MAE showed higher antioxidant capacity and α-glucosidase inhibitory activity than that of the conventional extract, suggesting the potential use of the MAE products of grape seeds as a functional food ingredient and nutraceutical.

Phenolic compounds extraction in enzymatic macerations of grape skins identified as low-level extractable total anthocyanin content.

Anthocyanins in wine principally depend on grape skin extractable anthocyanin content, that is, the amount of anthocyanins present in grape skin that are released to wine during the maceration stage. This amount of extractable anthocyanins is closely linked to the cell wall degradation of skin cells. Indeed, among other methodologies, the maceration in presence of different enzymes can be used to increase cell wall degradation, and therefore, the amount of anthocyanins extracted from grape skins to wine. Vitis vinifera L. cv. Tempranillo and Syrah red grapes have been identified as samples with low anthocyanin extraction potential by near infrared hyperspectral imaging. Grape skins have been macerated in the presence of cellulase, glucosidase, and pectinase. Then, color of the supernatants and phenolic compounds extracted from grape skins (total phenols, total flavanols, and total and individual anthocyanins) has been determined. Cellulase and glucosidase have shown a positive effect in the extraction of phenolic compounds from these grapes. Macerations carried out in the presence of cellulase have produced supernatants with a more intense color (lower lightness and higher chroma values), and a higher extraction of flavanols and anthocyanins than the respective control essays. However, pectinase treatments have produced the opposite effect, which could be partially explained by an eventual interaction between the cell wall polysaccharides liberated by pectinase and the phenolic compounds extracted. Synergy effects do not appear between cellulase and glucosidase. Moreover, the negative effect of the addition of pectinase might be due to the interactions between the cell wall material liberated by pectinase and the phenolic compounds extracted. PRACTICAL APPLICATION: In the present study, grape samples with a low anthocyanin extraction potential have been identified, and these samples have been macerated in the presence of different enzymes. The applied enzymes were three of the most common enzymes that are applied in the wine industry. Individual enzymes and mixtures have been applied to Syrah and Tempranillo grape skin samples and the results have been compared to control macerations. Knowledge in this topic will help the production of quality wines.

Molecular Mechanisms of Interactions between Monolayered Transition Metal Dichalcogenides and Biological Molecules.

Single layered two-dimensional (2D) materials such as transition metal dichalcogenides (TMDs) show great potential in many microelectronic or nanoelectronic applications. For example, because of extremely high sensitivity, TMD-based biosensors have become promising candidates for next-generation label-free detection. However, very few studies have been conducted on understanding the fundamental interactions between TMDs and other molecules including biological molecules, making the rational design of TMD-based sensors (including biosensors) difficult. This study focuses on the investigations of the fundamental interactions between proteins and two widely researched single-layered TMDs, MoS2, and WS2 using a combined study with linear vibrational spectroscopy attenuated total reflectance FTIR and nonlinear vibrational spectroscopy sum frequency generation vibrational spectroscopy, supplemented by molecular dynamics simulations. It was concluded that a large surface hydrophobic region in a relatively flat location on the protein surface is required for the protein to adsorb onto a monolayered MoS2 or WS2 surface with preferred orientation. No disulfide bond formation between cysteine groups on the protein and MoS2 or WS2 was found. The conclusions are general and can be used as guiding principles to engineer proteins to attach to TMDs. The approach adopted here is also applicable to study interactions between other 2D materials and biomolecules.

Synergistic effects of branching enzyme and transglucosidase on the modification of potato starch granules.

Potato starch displayed high viscosity, low hydroscopicity and dispersity, and acid susceptibility leading to the limited application of potato starch. To expand the potato starch utility with appropriate processing characteristics, potato starch granules were modified with branching enzyme (BE) and transglucosidase (TG). The results indicated that the susceptibility of potato starch granules to TG was higher than BE. Moreover, the two enzymes showed the synergistic effect in enzymatic modification of potato starch granules. They cooperatively attacked the external and interior of potato starch granules. The crystal forms of potato starch changed from B to C-type after double enzyme treatments, and enzyme-treated starches exhibited homogeneous crystal distribution. Compared to BE or TG alone, the combined action of BE and TG increased significantly the ratio of α-1,6-glycosidic linkage and the amounts of short chains of potato starch, which led to the significant reduction in degree of crystallinity, viscosity, gelatinization temperature and enthalpy, and a remarkable increase in solubility. Especially, the physicochemical characteristics of modified starch largely depended on the treatment time of TG. Thus, through the combination of BE and TG, the appropriate treatment time of TG may be chosen to improve the physicochemical properties of potato starch in processed starch-based products.

New Acylglycosides Flavones from Fuzhuan Brick Tea and Simulation Analysis of Their Bioactive Effects.

Four novel acylglycosides flavones (AGFs) including two quercetin acylglycosides and two kaempferol acylglycosides were isolated from Fuzhuan brick tea (FBT) as follows: quercetin 3-O-[α-l-rhamnopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [ß-d-glucopyranosyl (1→3)-α-l-rhamnopyranosyl (1→6)]-ß-d-galactoside was named as camelliquercetiside E (1), quercetin 3-O-[α-l-rhamnopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [α-l-rhamnopyranosyl (1→6)]-ß-d-galactoside was named as camelliquercetiside F (2), kaempferol 3-O-[α-l-arabinopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [ß-d-glucopyranosyl (1→3)-α-l-rhamnopyranosyl (1→6)]-ß-d-glucoside was named as camellikaempferoside D (3), kaempferol 3-O-[α-l-arabinopyranosyl (1→3)] [2-O''-(E)-p-coumaroyl] [α-l-rhamnopyranosyl (1→6)]-ß-d-glucoside was named as camellikaempferoside E (4). Chemical structures of AGFs were identified by time-of-flight mass (TOF-MS) and NMR spectrometers (¹H NMR, 13C NMR, ¹H-¹H COSY, HMBC and HSQC), and the MS² fragmentation pathway of AGFs was further investigated. The inhibitory abilities of AGFs and their proposed metabolites on α-glucosidase and HMG-CoA reductase were analyzed by molecular docking simulation, and the results suggested that inhibitory activities of AGFs were significantly affected by acyl structure, number of glycosyl and conformation, and part of them had strong inhibitory activities on α-glucosidase and HMG-CoA reductase, suggesting that AGFs and their metabolites might be important ingredients that participate in the regulation of hypoglycemic and hypolipidemic effects. The results provided new AGFs and research directions for the practical study of FBT health functions in future.

Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in Escherichia coli.

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable ß-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.

Carbon-degrading enzyme activities stimulated by increased nutrient availability in Arctic tundra soils.

Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N · m(-2) · year(-1) and 5 g P · m(-2) · year(-1). Within the 2006 site, two fertilizer regimes were established--one in which plots received 5 g N · m(-2) · year(-1) and 2.5 g P · m(-2) · year(-1) and one in which plots received 10 g N · m(-2) · year(-1) and 5 g P · m(-2) · year(-1). The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems.

Enzymatic transformation of nonfood biomass to starch.

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world's future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture's environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

Prebiotics and bioactive natural substances induce changes of composition and metabolic activities of the colonic microflora in cancerous rats.

Prebiotics are defined as selectively fermented food ingredients that induce specific changes in the composition and/or activity in the gastrointestinal microbiota beneficial to the host well-being and health. The aim of the presented experiment was to investigate the effect of a prebiotic applied alone or in combination with Hyppocastani extractum siccum, and Lini oleum virginale in rats with dimethylhydrazine induced colon cancer. Wistar albino rats were fed high fat diet supplemented with the prebiotic alone or in combination with Horse chestnut and flaxseed oil. The activity of faecal glycolytic enzymes, lipid parameters, bile acids, short chain fatty acids and counts of coliforms and lactobacilli were determined. Treatment with the prebiotic alone and in combination with selected substances significantly decreased the activity of glycolytic bacterial enzyme ß-glucuronidase (P<0.001) and increased activities of ß-galactosidase and ß-glucosidase. Bile acids concentration was significantly decreased (P<0.01) except for the combination of the prebiotic with Horse chestnut. The prebiotic alone decreased the lipid parameters (P<0.001) and enhanced production of short chain fatty acids. Application of prebiotic and bioactive natural substances significantly reduced number of coliforms (P<0.05). Prebiotic alone significantly increased the count of lactobacilli (P<0.05). These results show that prebiotics have a protective effect and may be the useful for colon cancer prevention and treatment.

Ginsenoside compound K production from ginseng root extract by a thermostable beta-glycosidase from Sulfolobus solfataricus.

Ginsenoside compound K was produced from ginseng root extract using a thermostable recombinant beta-glycosidase from Sulfolobus solfataricus. The pH and temperature for maximum production were 5.5 and 90 degrees C. The half-lives of the enzyme were 66, 30, and 1.7 h at 80, 85, and 90 degrees C respectively. The ginsenoside-hydrolyzing activity (Rd>Rb(1)>Rc>Rb(2)) and compound K-producing activity (Rd>Rc>Rb(1)>Rb(2)) of the beta-glycosidase were also determined. At pH 5.5 at 85 degrees C, 1.63 mg/ml of compound K was produced within 12 h by 40 U/ml of enzyme from 1.9 mg/ml of ginsenoside Rb(1), 0.52 mg/ml of ginsenoside Rb(2), 0.92 mg/ml of ginsenoside Rc, and 0.23 mg/ml of ginsenoside Rd in a 10% (w/v) ginseng root extract via two transformation pathways, Rb(1) or Rb(2) --> Rd --> F(2) --> compound K, and Rc --> compound Mc --> compound K.

Production of highly purified hydroxytyrosol from Olea europaea leaf extract biotransformed by hyperthermophilic beta-glycosidase.

A large amount of highly purified hydroxytyrosol (91-94% in weight) is obtained in short time by a simple biotransformation of Olea europaea leaf extract by a partially purified hyperthermophilic beta-glycosidase immobilized on chitosan support. The biotransformation conditions have been modulated for increasing the hydroxytyrosol yield, whilst chitosan and chitin matrices are used as adsorbent materials in liquid phase hydroxytyrosol extraction from the biotransformed mixtures. Natural and non-toxic hydroxytyrosol has been by this way produced from a vegetal source, and this compound appeared for the first time highly purified by natural and biocompatible safe biopolymers in comparison to previous results. Moreover, the GC analyses have displayed that the eluates from a two-step bioreactor have qualitative composition very similar to that of the extra-virgin olive oil polar fraction. The proposed bioreactor could also find application in the utilization of olive mill waste waters (OMWW), medium rich in large amounts of oleuropein, which can be converted in pharmacologically active compounds.

Sweet pregnane glycosides from Telosma procumbens.

An intensely sweet polyoxypregnane glycoside, telosmoside A15 (15), was isolated from an Asian Asclepiadaceae plant, Telosma procumbens, collected in Vietnam. This is the first time a sweet pregnane glycoside has been found, and its sweetness intensity is 1000 times greater than that of sucrose. From the same plant, 17 other new glycosides were isolated, having the same aglycone; they are named telosmosides A1-A14 (1-14) and A16-A18 (16-18). Some of these glycosides are also sweet, but others are tasteless or bitter. Chemical structures of the 18 glycosides were determined, and the structure-taste relationship was discussed.

Cardenolide 16'-O-glucohydrolase from Digitalis lanata. Purification and characterization.

A three-step chromatographic procedure was developed for purification of cardenolide 16'-O-glucohydrolase (CGH) from Digitalis lanata Ehrh. leaves, including Phenyl-Sepharose hydrophobic interaction chromatography followed by SP-Sepharose cation exchange and Q-Sepharose anion-exchange chromatography. Starting with acetone dry powder the purification resulted in an 760-fold enrichment of CGH. Molecular weight, substrate specificity, pH optimum and temperature stability of CGH were determined. Antibodies against CGH were prepared in rabbits. The SDS gel electrophoresis of protein extracts from leaves of D. lanata and other D. species showed bands at 70 kDa and 36 kDa reacting with the antibodies. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegraded CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywoodii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments obtained were sequenced. One fragment had the typical amino-acid sequence of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x). Cardenolide 16'-O-glucohydrolase, like the other members of this enzyme family, appeared to have a glutamic acid residue directly involved in glycosidic bond cleavage as a nucleophile.