Interactions of Galloylated Polyphenols with a Simple Gram-Negative Bacterial Membrane Lipid Model.

Differential scanning calorimetry (DSC) was used to explore the interactions of isolated polyphenolic compounds, including (-)-epigallocatechin gallate ((-)-EGCg), tellimagrandins I and II (Tel-I and Tel-II), and 1,2,3,4,6-penta-O-galloyl-d-glucose (PGG), with a model Gram-negative bacterial membrane with a view to investigating their antimicrobial properties. The model membranes comprised 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), fabricated to mimic the domain formation observed in natural membranes, as well as ideally mixed lipid vesicles for the interaction with (-)-EGCg. Polyphenols induced changes in lipid mixing/de-mixing depending on the method of vesicle preparation, as was clearly evidenced by alterations in the lipid transition temperatures. There was a distinct affinity of the polyphenols for the DPPG lipid component, which was attributed to the electrostatic interactions between the polyphenolic galloyl moieties and the lipid headgroups. These interactions were found to operate through either the stabilization of the lipid headgroups by the polyphenols or the insertion of the polyphenols into the membrane itself. Structural attributes of the polyphenols, including the number of galloyl groups, the hydrophobicity quantified by partition coefficients (logP), and structural flexibility, exhibited a correlation with the temperature transitions observed in the DSC measurements. This study furthers our understanding of the intricate interplay between the structural features of polyphenolic compounds and their interactions with model bacterial membrane vesicles towards the exploitation of polyphenols as antimicrobials.

First Record of Aedes Japonicus in St. Tammany Parish, Louisiana.

Information of species presence and abundance is useful for taking a targeted approach to controlling populations of medically important and nuisance mosquito species. In April 2023, staff at St. Tammany Parish Mosquito Abatement District (STPMAD), in Slidell, Louisiana, identified Aedes japonicus (Theobald, 1901) mosquitoes from a larval sample brought in from the field for identification. Although invasive Aedes species like Aedes albopictus are commonly found in Louisiana, this is the first record of Ae. japonicus in St. Tammany Parish.

FIRST RECORD OF AEDES JAPONICUS IN ST. TAMMANY PARISH, LOUISIANA.

Information of species presence and abundance is useful for taking a targeted approach to controlling populations of medically important and nuisance mosquito species. In April 2023, staff at St. Tammany Parish Mosquito Abatement District (STPMAD), in Slidell, Louisiana, identified Aedes japonicus (Theobald, 1901) mosquitoes from a larval sample brought in from the field for identification. Although invasive Aedes species like Aedes albopictus are commonly found in Louisiana, this is the first record of Ae. japonicus in St. Tammany Parish.

Saturated fat replacement in short dough biscuits with HPMC and lecithin stabilised nanoemulsions.

Biscuits contain high proportions of saturated fats, which could lead to an adverse health effect. The objective of this study was to study the functionality of a complex nanoemulsion (CNE), stabilised with hydroxypropyl methylcellulose and lecithin, when used as a saturated fat replacer in short dough biscuits. Four biscuit formulations were studied including a control (butter) and three formulations where 33% of the butter was replaced with either extra virgin olive oil (EVOO), with CNE, or with the individual ingredients of the nanoemulsion added separately (INE). The biscuits were evaluated by texture analysis, microstructural characterisation, and quantitative descriptive analysis by a trained sensory panel. The results showed that incorporation of CNE and INE yielded doughs and biscuits with significantly higher (p < 0.05) hardness and fracture strength values than the control. The doughs made of CNE and INE showed significantly less oil migration during the storage than EVOO formulations, which was confirmed by the confocal images. The trained panel did not find significant differences in crumb density and hardness on the first bite among CNE, INE and the control. In conclusion, nanoemulsions stabilised with hydroxypropyl methylcellulose (HPMC) and lecithin can work as saturated fat replacers in short dough biscuits, providing satisfactory physical characteristics and sensory attributes.

Development of Saturated Fat Replacers: Conventional and Nano-Emulsions Stabilised by Lecithin and Hydroxylpropyl Methylcellulose.

The combination of two emulsifiers, lecithin and hydroxypropyl methylcellulose (HPMC), into emulsions is an interesting strategy to design fat replacers in food matrices. The objective of this study was to investigate the effect of HPMC type and concentration on the formation, stability, and microstructure of conventional emulsions and nanoemulsions. Two different types of HPMC with low and high content of methyl and hydroxypropyl groups (HPMC-L and HPMC-H) were evaluated. The results showed that the molecular structure and concentration of HPMC play a major role in the viscoelastic behaviour, the gelation temperature, and the strength of gel formed. The firmness and work of shear of HPMC solutions increased significantly (p < 0.05) with increasing concentration. HPMC-L illustrated a more stable gel structure than the HPMC-H solution. Nanoemulsions showed lower moduli values, firmness, and work of shear than conventional emulsions due to the influence of high-pressure homogenization. A combination of lecithin and HPMC improved the physical and lipid oxidative stability of the emulsions, presenting a lower creaming index and thiobarbituric acid reactive substances (TBARS). In conclusion, HPMC-L at 2% w/w could be a suitable type and concentration combined with lecithin to formulate a saturated fat replacer that could mimic butter technological performance during food manufacturing operations.

Physical and Chemical Characterisation of Conventional and Nano/Emulsions: Influence of Vegetable Oils from Different Origins.

The processes of oil production play an important role in defining the final physical and chemical properties of vegetable oils, which have an influence on the formation and characteristics of emulsions. The objective of this work was to investigate the correlations between oils' physical and chemical properties with the stability of conventional emulsions (d > 200 nm) and nanoemulsions (d < 200 nm). Five vegetable oils obtained from different production processes and with high proportion of unsaturated fatty acids were studied. Extra virgin olive oil (EVOO), cold-pressed rapeseed oil (CPRO), refined olive oil (OO), refined rapeseed oil (RO) and refined sunflower oil (SO) were used in this study. The results showed that the physicochemical stability of emulsion was affected by fatty acid composition, the presence of antioxidants, free fatty acids and droplet size. There was a significant positive correlation (p < 0.05) between the fraction of unsaturated fatty acids and emulsion oxidative stability, where SO, OO and EVOO showed a significantly higher lipid oxidative stability compared to RO and CPRO emulsions. Nanoemulsions with a smaller droplet size showed better physical stability than conventional emulsions. However, there was not a significant correlation between the oxidative stability of emulsions, droplet size and antioxidant capacity of oils.

Lipid composition in fungal membrane models: effect of lipid fluidity.

The creation of effective fungal membrane models for neutron and X-ray reflectometry experiments is a key step in the development of new antifungal pharmaceuticals and agrochemicals to allow in vitro investigation of their mode of interaction with target cells. The structure of the obtained models depends on the properties of the lipids used and the final composition of the leaflets, and can be subject to the spontaneous translocation of phospholipids across the bilayer. The effect of phospholipid acyl-chain unsaturation and the presence of steroids in the membrane on the bilayer asymmetry were examined by means of neutron reflectometry. The measurements showed that membrane stability was higher if a zwitterionic, saturated acyl-chain phospholipid is present as the inner leaflet. Furthermore, membrane asymmetry was higher in the case of fully saturated lipid systems. As a result, membrane models consisting of fully saturated acyl chains within the inner leaflet are recommended as the starting point for subsequent studies of antifungal interactions owing to the simplicity of the models and their relative stability, thus allowing better control over the exact lipid composition facing the tested antifungal.

Tryptophan to Arginine Substitution in Puroindoline-b Alters Binding to Model Eukaryotic Membrane.

We have studied how puroindoline-b (PINB) mutants bind to model eukaryotic membranes dependent on binary composition of anionic:zwitterionic phospholipids and the presence of cholesterol and sphingomyelin in the model membrane. We have found that the trends in lipid binding behavior are different for wild-type PINB compared to its naturally occurring PINB(Trp44Arg) mutant form and have seen evidence of protein-induced domain formation within the lipid layer structure. Results show that selective binding of antimicrobial peptides to different membrane types is as a result of differences in lipid composition and the arrangement of lipids within the membrane surface. However, membrane-binding behavior is not easily predicted; it is determined by net charge, hydrophobicity, and the amphiphilicity of the protein/peptide lipid-binding domain.

Enzymatic hydrolysis of phytate and effects on soluble oxalate concentration in foods.

Soluble oxalate in foods is major concern for kidney stone formers due to its tendency to increase urinary oxalate concentration. Phytate forms complexes with cations, which increases soluble oxalate by making cations unavailable to precipitate oxalate. Thus, in order to reduce soluble oxalate, bran samples (wheat, oat and barley) and bean samples (red kidney bean and white bean) were treated with phytase. Release of phosphate after phytate degradation and its association with calcium was determined. Phosphate concentration increased after application of phytase in all samples, but effect on soluble oxalate concentration varied. Wheat and oat bran showed significant reduction (P<0.05) in soluble oxalate compared to bean samples. Wheat bran, oat bran and white bean had a lower calcium:phosphate ratio than barley bran and red kidney beans. Correlation of the calcium:phosphate molar ratio with release of phosphate depends on concentration of calcium ions and this influences soluble oxalate concentration.

Astringency reduction in red wine by whey proteins.

Whey is a by-product of cheese manufacturing and therefore investigating new applications of whey proteins will contribute towards the valorisation of whey and hence waste reduction. This study shows for the first time a detailed comparison of the effectiveness of gelatin and ß-lactoglobulin (ß-LG) as fining agents. Gelatin was more reactive than whey proteins to tannic acid as shown by both the astringency method (with ovalbumin as a precipitant) and the tannins determination method (with methylcellulose as a precipitant). The two proteins showed similar selectivity for polyphenols but ß-LG did not remove as much catechin. The fining agent was removed completely or to a trace level after centrifugation followed by filtration which minimises its potential allergenicity. In addition, improved understanding of protein-tannin interactions was obtained by fluorescence, size measurement and isothermal titration calorimetry (ITC). Overall this study demonstrates that whey proteins have the potential of reducing astringency in red wine and can find a place in enology.

Role of Lipid Composition on the Interaction between a Tryptophan-Rich Protein and Model Bacterial Membranes.

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements, and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition, and fluidity on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

High pressure intensification of cassava resistant starch (RS3) yields.

Cassava starch, typically, has resistant starch type 3 (RS3) content of 2.4%. This paper shows that the RS3 yields can be substantially enhanced by debranching cassava starch using pullulanase followed by high pressure or cyclic high-pressure annealing. RS3 yield of 41.3% was obtained when annealing was carried out at 400MPa/60°C for 15 min, whereas it took nearly 8h to obtain the same yield under conventional atmospheric annealing at 60°C. The yield of RS3 could be further significantly increased by annealing under 400 MPa/60°C pressure for 15 min followed by resting at atmospheric pressure for 3h 45 min, and repeating this cycle for up to six times. Microstructural surface analysis of the product under a scanning electron microscope showed an increasingly rigid density of the crystalline structure formed, confirming higher RS3 content.

Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin.

Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 10(4)-10(6) M(-1) and stoichiometries, n1, of 2-13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 10(3)-10(5) M(-1) and stoichiometries, n2, of 16-30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern-Volmer quenching constant, K(SV).

Effects of phytate and minerals on the bioavailability of oxalate from food.

Phytate and mineral cations are both considered as important dietary factors for inhibiting the crystallisation of calcium oxalate kidney stones in susceptible individuals. In this paper, the phytate and mineral composition of whole bran cereals (wheat, barley and oat) and legumes were determined together with their soluble and insoluble oxalate concentrations in order to investigate the effects on oxalate solubility. The oat bran sample had the highest soluble oxalate concentration at 79±1.3 mg/100 g, while total and soluble oxalate concentrations in the food samples studied range from 33 to 199 mg/100 g and 14 to 79 mg/100 g, respectively. The phytate concentration was in the range from 227 to 4393 mg/100 g and the concentrations of cations were in the range 54-70 mg/100 g for calcium, 75-398 mg/100 g for magnesium, 244-1529 mg/100 g for potassium and 4-11 mg/100 g for iron. Soluble oxalate concentration did not increase in proportion to total oxalate, and the phytate concentration in all foods was sufficient to contribute to an increase in soluble oxalate concentration by binding calcium.

Selected wheat seed defense proteins exhibit competitive binding to model microbial lipid interfaces.

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. This study examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b, and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and ß-purothionin (ß-Pth) model anionic lipid layers using a combination of surface pressure measurements, external reflection FTIR spectroscopy, and neutron reflectometry. Results highlighted differences in the protein binding mechanisms and in the competitive binding and penetration of lipid layers between respective Pins and ß-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of ß-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with ß-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of ß-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity.

The role of protein hydrophobicity in thionin-phospholipid interactions: a comparison of α1 and α2-purothionin adsorbed anionic phospholipid monolayers.

The plant defence proteins α1- and α2-purothionin (Pth) are type 1 thionins from common wheat (Triticum aestivum). These highly homologous proteins possess characteristics common amongst antimicrobial peptides and proteins, that is, cationic charge, amphiphilicity and hydrophobicity. Both α1- and α2-Pth possess the same net charge, but differ in relative hydrophobicity as determined by C18 reversed phase HPLC. Brewster angle microscopy, X-ray and neutron reflectometry, external reflection FTIR and associated surface pressure measurements demonstrated that α1 and α2-Pth interact strongly with condensed phase 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) monolayers at the air/liquid interface. Both thionins disrupted the in-plane structure of the anionic phospholipid monolayers, removing lipid during this process and both penetrated the lipid monolayer in addition to adsorbing as a single protein layer to the lipid head-group. However, analysis of the interfacial structures revealed that the α2-Pth showed faster disruption of the lipid film and removed more phospholipid (12%) from the interface than α1-Pth. Correlating the protein properties and lipid binding activity suggests that hydrophobicity plays a key role in the membrane lipid removal activity of thionins.

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and ß-purothionin.

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and ß-purothionin (ß-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and ß-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and ß-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. ß-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and ß-Pth hydrophobic regions.

Puroindoline-a, a lipid binding protein from common wheat, spontaneously forms prolate protein micelles in solution.

The self-assembly in solution of puroindoline-a (Pin-a), an amphiphilic lipid binding protein from common wheat, was investigated by small angle neutron scattering, dynamic light scattering and size exclusion chromatography. Pin-a was found to form monodisperse prolate ellipsoidal micelles with a major axial radius of 112 ± 4.5 Å and minor axial radius of 40.4 ± 0.18 Å. These protein micelles were formed by the spontaneous self-assembly of 38 Pin-a molecules in solution and were stable over a wide pH range (3.5-11) and at elevated temperatures (20-65 °C). Pin-a micelles could be disrupted upon addition of the non-ionic surfactant dodecyl-ß-maltoside, suggesting that the protein self-assembly is driven by hydrophobic forces, consisting of intermolecular interactions between Trp residues located within a well-defined Trp-rich domain of Pin-a.

Binding of pentagalloyl glucose to two globular proteins occurs via multiple surface sites.

The interaction between pentagalloyl glucose (PGG) and two globular proteins, bovine serum albumin (BSA) and ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco), was investigated by isothermal titration calorimetry (ITC). ITC data fit to a binding model consisting of two sets of multiple binding sites, which reveal similarities in the mode of binding of PGG to BSA and rubisco. In both cases, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface, which promotes aggregation and precipitation of the PGG-protein complex. This model was confirmed by turbidimetry analysis of the PGG-BSA interaction. Analysis of tryptophan fluorescence quenching during the interaction of PGG with BSA suggests that binding of PGG leads to some conformational changes that are energetically closer to the unfolded state of the BSA structure, because small red shifts in the resulting emission spectra were observed.

Efeito do tratamento sob alta pressão isostática sobre os teores de fitato e inibidor de tripsina de soja / Efect of tratment isostatic high pressure on phytate and trypsin inhibitors soybean

A soja constitui excelente fonte de proteína para a alimentação humana e animal, porém contém alguns componentes de ação antinutricional, como os inibidores de proteases, lectinas, fitatos e saponinas. Neste trabalho foram avaliados os efeitos do tratamento sob alta pressão isostática,considerado brando em relação ao tratamento térmico, sobre os fatores antinutricionais como teor de fitato e inibidor de tripsina de solução com 5% de isolado proteico de soja, processada na faixa de 200 a 700 MPa. Foram realizadas duas extrações de fitato e inibidor de tripsina de bateladas diferentes, sendo as amostras analisadas em triplicata. Há indícios que o tratamento sob alta pressão isostática seja eficiente para eliminar o fitato presente nas amostras de isolado proteico de soja, mas não se mostrou efetivo para alterar os teores de inibidor de tripsina.