Prevention of quercetin precipitation in red wines: a promising enzymatic solution

Flavonols are known for causing undesirable deposits in both red and white wines. Among flavonols, quercetin is widely considered the principal factor determining this phenomenon. One of the most accredited hypotheses claims that glycosylated derivatives of quercetin undergo hydrolysis of the glycosylic bond during the fermentation and the wine ageing, releasing quercetin aglycone, which is much less soluble in water solution and causes the precipitation. Our work describes the dynamics of quercetin-derived deposition in Chianti wines and purposes a new method, based on the enzymatic quercetin glycoside hydrolysis of the glycosidic bond, to prevent the unpleasant deposit formation during the wine ageing. In our study, forty-four monovarietal wines obtained from 7 different Italian grape varieties were compared in the content of total quercetin-3-glycosides (rutin, quercetin-3-glucuronide, quercetin-3-glucoside) and quercetin aglycone. The data confirmed the literature revealing Sangiovese as the richest in quercetin. We tested then, in a Sangiovese wine, four fining agents (PVPP, PVPP/PVI, bentonite and a vegetal protein) for quercetin removal, showing that only the PVPP had a modest aglycone removal activity. Then, the kinetics of deposit formation was studied in three Chianti wines which differed in the initial content of quercetin aglycone. This investigation highlighted that the chemical equilibrium of quercetin changes over time as the turbidity slowly increases, as previously documented. The comparison of the three dynamics also permitted us to conclude that different wines show a different ability to keep in solution quercetin. Finally, a new approach for deposit prevention was studied by a precocious Chianti wine treatment with a pectolytic enzyme having secondary glycosidase activity. This enzyme significantly accelerated the hydrolysis of glycosylated quercetins into their aglycone, which could enhance the deposition before bottling, without serious wine colour depletion. Our study represents the first evidence of the promising potential of using the pectolytic enzyme with secondary glycosidase activity to prevent quercetin deposit during Chianti ageing, in a way that is compatible with organic wine production.

Polyphenols Content and In Vitro α-Glycosidase Activity of Different Italian Monofloral Honeys, and Their Effect on Selected Pathogenic and Probiotic Bacteria

We evaluated the polyphenol content and the α-glucosidase activity exhibited by different monofloral honeys of Italian origin. Their capacity to act on different pathogenic (Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus) as well as probiotic bacteria (Lacticaseibacillus casei, Lactobacillus acidophilus, Lactiplantibacillus plantarum, Lactobacillus gasseri, and Lacticaseibacillus rhamnosus) was also assessed. Total polyphenols varied between 110.46 μg/g of fresh product (rhododendron honey) and 552.29 μg/g of fresh product (strawberry tree honey). Such result did not correspond to a parallel inhibitory α-glycosidase activity that, in each case was never higher than 33 μg/mL. Honeys were differently capable to fight the biofilm formation of the pathogens (inhibition up to 93.27%); they inhibited the in vitro adhesive process (inhibition up to 84.27%), and acted on mature biofilm (with values up to 76.64%). Their effect on bacterial metabolism was different too. Honeys were ineffective to inhibit E. coli mature biofilm nor to act on its metabolism. The action of the honey on probiotic strains seemed almost always stimulate their growth. Thus, these monofloral honeys might exhibit effects on human health and act positively as prebiotics.

Coexistence of Lateriporus teres (Cestoda: Dilepididae) and Polymorphus phippsi (Palaeacanthocephala: Polymorphidae) in the intestine of common eider

The purpose of the research is studying the peculiarities of parasite infection of the cestodes Lateriporus teres Krabbe, 1869 (Cestoda: Dilepididae) and acanthocephalans Polymorphus phippsi Kostylew, 1922 (Palaeacanthocephala: Polymorphidae) during coexistence in the intestine of the common eider, particularly, the localization of these parasites, the activity of protein and carbohydrate metabolism enzymes on the tegument of the worms, the degree of parasite influence on the host digestion, as well as to assess total digestive activity in the gastrointestinal tract of birds infected with these helminths.Materials and methods. Using methods of biochemical analysis, the activity of digestive enzymes (protease and glycosidase) and the intensity of a digestion with the participation of these enzymes along the intestine of the common eider were determined. The digestive activity of enzymes in the body of helminthes was measured, and the intensity of membrane digestion on the surface of their tegument was estimated.Results and discussion. During coexistence, L. teres were observed mainly in the proximal parts of the intestine of the birds, P. phippsi in the distal parts. Membrane digestion, involving the action of proteases and glycosidases, occurred on the surface of the tegument of both cestodes and acanthocephalans. The protein metabolism intensity in both helminth species was nearly the same, but the glycosidase activity was higher on the tegument of L. teres. The glycosidase activity in the body of the acanthocephalans exceeded that in the cestodes strobile six times. In the intestine parts inhabited by L. teres, both protease and glycosidase activity decreased. In the intestine parts where P. phippsi parasitized, protease activity increased in the intestinal mucosa of the common eider. The total activity of protease and glycosidase along the entire length of the intestine in common eider infected with L. teres and P. phippsi was lower compared to the uninfected birds.

A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule

Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans. From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

A Glycan FRET Assay for Detection and Characterization of Catalytic Antibodies to the Cryptococcus neoformans Capsule

<p>Classical antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of <i>Cryptococcus neoformans</i>. From this, we designed and synthesized two glycan based Förster Resonance Energy Transfer (FRET) probes, which we used to discover antibodies with innate glycosidase activity and analyse their enzyme kinetics, including mAb 2H1, a polysaccharide lyase, and the most efficient glycosidase to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Further the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labelled by hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing <i>C. neoformans </i>cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages — suggesting the anti-phagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.</p>

A Glycan FRET Assay for Detection and Characterization of Catalytic Antibodies to the Cryptococcus neoformans Capsule

<p>Classical antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of <i>Cryptococcus neoformans</i>. From this, we designed and synthesized two glycan based Förster Resonance Energy Transfer (FRET) probes, which we used to discover antibodies with innate glycosidase activity and analyse their enzyme kinetics, including mAb 2H1, a polysaccharide lyase, and the most efficient glycosidase to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Further the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labelled by hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing <i>C. neoformans </i>cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages — suggesting the anti-phagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.</p>

Enhanced Functionality of Canavalia gladiata Hull by Fermentation with Lactic Acid Bacteria

Objectives Immature Canavalia Gladiata Hull (ICGH) has been used traditionally as food but the research data on its functionality is insufficient. Antioxidant and anti-inflammation activity of ICGH and fermentation was performed using lactic acid bacteria (LAB) with glycosidase activity to confirm whether the bioconversion affect the functionality. Methods LAB was screened from Kimchi for the glycosidase activity. Antioxidant activity was evaluated by ABTS, DPPH, cellular antioxidant activity (CAA), and nitrite oxide (NO) production was evaluated with RAW264.7 cell treated with lipopolysaccharide (LPS). Active component in ICGH was analyzed using HPLC-PDA, MS/MS, and NMR. Results LABs could grow in medium with ICGH as a sole nutrient source. After fermentation the contents of lactic acid was increased as sugar contents declined and asparagine and γ-aminobutyrate(GABA) were detected as main metabolite, and some flavonoids were identified. After fermentation, higher free radical scavenging activity was confirmed in ABTS and DPPH assay and the tendency to decrease in reactive oxygen species and nitrite production was found in a dose dependent manner. Conclusions The fermentation of ICGH with two selected strains enhanced the antioxidant activity and decreased NO production by increase in the bioavailability of active compounds in ICGH. Funding Sources National Institute of Agricultural Sciences.

Novel heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

ABSTRACTGlycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ~5500 reported crystallographic structures of ~1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.