In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells.

AIM: To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form. METHODS AND RESULTS: Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD(50) showed that the fibroblasts were able to tolerate up to 80 microg ml(-1) for 24 h, dropping thereafter to 62 mug ml(-1) after 72 h of contact, compared to 160 microg ml(-1) after 24 h, and 80 microg ml(-1) after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25 microg ml(-1)) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition. CONCLUSION: These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.

Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form.

AIMS: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. METHODS AND RESULTS: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 microg ml(-1)) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast's transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 microg ml(-1). This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. CONCLUSION: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.

Resveratrol binding to human serum albumin.

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419-426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclooxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents (microM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure. Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K(Res) = 2.56 x 10(5) M(-1). The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of alpha-helix from 57% (free HSA) to 62% and a decrease of beta-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane.

Interactions between beta-lactoglobulin (beta-lg) and dimyristoylphosphatidylglycerol (DMPG) bilayers were studied using one- and two-dimensional infrared spectroscopy above (pD 7.4) and below (pD 4.4) the protein's (beta-lg's) isoelectric point (pI=5.2). The aim of the study was threefold: (1) gain a better understanding of beta-lg-phospholipid interaction; (2) provide information relative to the structure of beta-lg as it interacts with membranes; (3) determine whether the conformational modifications of the protein in the presence of lipids are strictly caused by thermal effects or whether they are modulated by the chain-melting phase transition. At pD 7.4, the lipid thermotropism, the acyl-chain order, and the membrane interfacial region were essentially unaffected by the presence of beta-lg, whereas the protein amide I region showed dramatic alterations. The results suggested the predominance of beta-sheets and alpha-helix elements, with a lost of structural integrity. At pD 4.4, beta-lg induced an approximately 2 degrees C downshift of the transition temperature, whereas the conformational order of the lipid chain decreased in the gel phase and increased in the liquid-crystalline phase. The hydration state of the DMPG C==O groups increased in the liquid-crystalline phase. The conformation of beta-lg at pD 4.4 in the presence of DMPG showed similarities with that observed at pD 7.4, but an increase in the alpha-helix content and a reduced thermal stability were noticed. In contrast to the protein alone, beta-lg aggregates in the presence of DMPG at pD 4.4 above 50 degrees C. At both pD values, the charged surface of the membrane seemed to be the main factor for inducing protein conformational changes by altering the intramolecular interactions that stabilize the native structure. However, protein incorporation within the membrane seemed to be involved at pD 4.4. The two-dimensional analysis performed with spectra recorded upon heating showed that spectral intensity changes at pD 4.4 and 7.4 occurred at the same frequencies in the amide I' region. The heat-induced structural changes of beta-lg were not correlated with the conformational modifications of the phospholipids along the phase transition, indicating that the thermal behavior of the protein was not modulated by the lipid chain melting, but rather represented the heat-induced protein rearrangement in the presence of DMPG.

Structural characteristics of a globular protein investigated by X-ray photoelectron spectroscopy: comparison between a legumin film and a powdered legumin.

Films of legumin, a pea protein, were deposited onto a glass support using the Langmuir-Blodgett method, at various surface pressures. XPS study of these films show that their thickness increases with the deposition pressure. At the pressure limits of films stability, the thickness values (respectively 73 and 110 A) are close to the protein dimensions. Layered at low pressure, the oblate protein stands up when pressure increases. Furthermore, XPS study shows that the orientation of the external flexible loops depends on the obtention conditions. Thus, in the case of Langmuir-Blodgett films, hydrophobic residues are turned towards the external surface, and the hydrophilic ones towards the glass substrate. But, in the opposite, when protein is obtained by lyophilization, the hydrophilic residues are orientated outsides. It seems possible to determine by XPS the nature of the residues which give to the protein its reactivity, since they are located at its external surface.

Conformational changes upon dissociation of a globular protein from pea: a Fourier transform infrared spectroscopy study.

Fourier transform infrared spectroscopy shows that the secondary structure of legumin, a globular protein from pea seeds, is composed of 41% beta-sheets and 16% alpha-helices and furthermore reveals the presence of beta-turns. The conformation prediction from the analysis of the amino-acid sequence of legumin using hydrophobic cluster analysis reveals that the C-terminal part of the alpha-polypeptide is devoid of defined secondary structures, whereas the beta-polypeptide is highly ordered. Comparison with analogous 11S globulins from other plant families indicates that ordered domains are highly preserved, phenomenon that may be associated with the similarity of the quaternary structure of these proteins. The results also reveal the presence of a large hypervariable region, located at the surface of the protein, that could be at the origin of the different functional properties of the 11S type globulins. The step-by-step destruction of the quaternary oligomeric structure of the native protein is accompanied by conformational changes that depend on the dissociation conditions. Whereas acylation leads to a decrease of the alpha-helix content by 10% at the expense of the beta-sheet content, addition of sodium perchlorate results in the conversion of 10% of the protein secondary structure from beta-sheet to unordered. These observations provide further evidence of the existence of different monomeric states that differ from their secondary structure and, therefore, exhibit different surface-active properties.

Interaction of beta-lactoglobulin with phospholipid bilayers: a molecular level elucidation as revealed by infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy has been used to study, at a molecular level, the interactions between beta-lactoglobulin (BLG), the most abundant globular protein in milk, and some lipids (sphingomyelin, SM; dimyristoylphosphatidylcholine, DMPC; dipalmytoylphosphatidylcholine, DPPC; dimyristoylphosphatidylserine-sodium salt, DMPS; dipalmitoylphosphatidylserine-sodium salt, DPPS) constituting the milk fat globule membrane (MFGM). The interactions were monitored with respect to alteration in the secondary structure of BLG, as registered by the amide I' band, and phospholipid conformation, as revealed by the acyl chain and carbonyl bands. The results show that neither the conformation nor the thermotropism of neutral bilayers containing DMPC or DPPC is affected by BLG. Reciprocally, the secondary structure and thermal behaviour of pure BLG remain the same in the presence of PC. These results suggest that no interaction occurs between PC and BLG, in agreement with previous studies. However, it is found that BLG interacts with neutral bilayers constituted by milk SM lipids, increasing gauche conformers and thus conformational disorder of the lipid acyl chains. This perturbing effect has been attributed to a partial penetration of BLG into the hydrophobic core of the bilayer, which allows hydrophobic interactions between BLG and SM. Moreover, the fact that SM possesses the same headgroup of PC implies that the head group does not prevent the occurrence of BLG-lipid interactions and other lipid regions can control the binding of BLG to lipids. Furthermore, BLG was found to interact electrostatically with charged bilayers containing PS, leading to a rigidification of the lipid hydrocarbon chains and a dehydration of the interfacial region. This last effect suggests that the protein limits the accessibility of water molecules to the interfacial region of the phospholipids by its presence at the membrane surface.

Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films.

Fourier transform infrared spectroscopy has been used to investigate the conformational changes of glycinin. a major storage protein of soybean seeds, upon film-forming. The results show that the secondary structure of glycinin is mainly composed of a beta-sheet (48%) and unordered (49%) structures. The amide I band of glycinin in film-forming conditions, i.e. in alkaline media and in the presence of plasticizing agent, reveals the conversion of 18% of the secondary structure of the protein from the beta-sheet (6%) and random coil (12%) to the alpha-helical conformation due to the helicogenic effect of the ethylene glycol used as the plasticizing agent. Conformational changes also occur upon the film-forming process leading to the formation of intermolecular hydrogen-bonded beta-sheet structures. Results obtained from other plant families indicate that, whatever the origin and conformation of protein, formation of films leads to the appearance of intermolecular hydrogen-bonded beta-sheet structures, suggesting that this type of structure might be essential for the network formation in films. Thus, it is hypothesized that, in the film state, intermolecular hydrogen bonding between segments of beta-sheet may act as junction zones in the film network. This study reveals for the first time that there is a close relationship between the conformation of proteins and the mechanical properties of films.

Relationships between conformation of beta-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy.

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of beta-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of beta-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon beta-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding beta-sheet structure as evidenced by the appearance of a strong band at 1614 cm(-1) at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of beta-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular beta-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of beta-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular beta-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.

Preparation and in vitro evaluation of calcium-induced soy protein isolate nanoparticles and their formation mechanism study.

Soy protein isolate (SPI) nanoparticles (28-179 nm) were prepared by employing a cold gelation method with a slight modification. The obtained nanoparticles exhibited uniform size distribution and spherical shape with a unique honeycomb-like core structure. Nanoparticle characteristics including size, surface charge and hydrophobicity could be adjustable by changing calcium concentration and environmental pH. Generally, higher calcium concentration and lower pH led to formation of nanoparticles with larger size, lower surface charge and hydrophobicity. Both protein conformation and nanoparticle dissociation studies indicated that calcium likely shielded negative charges on the SPI polypeptide chains, and functioned as a salt-bridge to permit polypeptide chains to approach one another. In this process, calcium favoured the development of ß-sheet structures to form SPI aggregates stabilised by hydrogen bonding. These aggregates were then associated to build SPI nano-networks through hydrophobic interactions. In vitro study indicated that the SPI nanoparticles were non-toxic and mainly located in the cytoplasm when uptaken into Caco-2 cells.

Design and evaluation of succinylated soy protein tablets as delayed drug delivery systems.

The impact of succinylation on soy proteins as excipients for delayed delivery of drugs in the gastrointestinal tract was studied. Succinylation decreased protein solubility and protein charge density at pH 1.2 and increased solubility and zeta potential at pH above 4.5. Tablet erosion and swelling were decreased at pH 1.2 and increased at pH 7.5. FTIR analysis indicated polypeptide chain unfolding as a result of succinylation. Tablets of protein succinylated 50% or 100% released less than 10% of loaded riboflavin or rifampicin in 2h at gastric pH in the presence of pepsin but released these compounds rapidly at intestinal pH. Succinylated soy protein tablets were thus gastroresistant, suggesting their use as excipients for controlled release of medicinal or nutraceutical agents.

Inhibition of Listeria monocytogenes by a combination of chitosan and divergicin M35.

The antimicrobial activities of the class IIa bacteriocin divergicin M35 and several types of chitosan against Listeria monocytogenes were quantified by agar diffusion, critical micro-dilution, and viable count and observed by electron microscopy. Antimicrobial activity of chitosan depended on its molecular mass (MM) and the pH. Three chitosans with MM values of 2, 20, and 100 kDa and 87.4% degree of deacetylation (DDA) were chosen for further study, based on high anti-listerial activity at pH 4.5. Electron microscopy suggested that the mechanism of anti-listerial activity also varied with the MM. Low-MM chitosan appeared to inhibit L. monocytogenes by affecting cell permeability and growth, whereas medium- and high-MM chitosan may form a barrier on the cell surface that prevents entry of nutrients. The minimum inhibitory concentrations (MICs) of 2, 20, and 100 kDa chitosan and divergicin M35 against a divergicin-resistant strain of L. monocytogenes (LSD 535) were 2.5, 2.5, 0.625, and 0.25 mg/mL, respectively. The combination of any of these 3 chitosans and divergicin M35 appeared to have an additive effect against L. monocytogenes, as determined by fractional inhibitory concentration (FIC) index. This study provides useful data for the development of chitosan films incorporating divergicin M35 for inhibiting L. monocytogenes in foods.

Characterization of amino cross-linked soy protein hydrogels.

This study focused on amino cross-linking as means of forming soy protein hydrogels with modifiable properties. The efficiency of glyceraldehyde, a potential food-grade cross-linking agent, was compared to glutaraldehyde, a well-known dialdehyde. The influence of the concentration of these agents on the degree of cross-linking as well as macroscopic and supramolecular properties was studied. The effect of increasing the cross-linker concentration was mainly an increase in degree of cross-linking and gel storage modulus (G') and a decrease in gel swelling ratio and increase in gel deswelling ratio. However, the cross-linking influence was less pronounced in the case of glyceraldehyde. Glutaraldehyde displayed greater ability to form hydrogels with modifiable properties. Finally, electron micrographs suggested that cross-linking agent type had no impact on gel microstructure.

Molecular differences in the formation and structure of fine-stranded and particulate beta-lactoglobulin gels.

In order to reveal at a molecular level differences between fine-stranded and particulate gels, we present an Fourier transform infrared spectroscopic study of the thermal behavior of beta-lactoglobulin (beta-lg) in salt-free D(2)O solutions and low ionic strength at different pDs. Differences are found in the denaturation mechanism, in the unfolded state of the protein, in the aggregate formation, and in the strength of the intermolecular interactions. For fine-stranded gels (pD 2.8 and 7.8), heating induces the dissociation of the dimers into monomers. The protein undergoes extensive structural modifications before aggregation begins. Aggregation is characterized by the appearance of a new band attributed to intermolecular beta-sheets which is located in the 1613-1619 cm(-1) range. For particulate gels (pD 4.4 and 5.4), the protein structure is almost preserved up to 75-80 degrees C with no splitting of the dimers. The band characteristic of aggregation originates from the component initially located at 1623 cm(-1), suggesting that at the beginning of aggregation, globular beta-lg in the dimeric form associate to constitute oligomers with higher molecular mass. Aggregation may result in the association of globular slightly denatured dimers, leading to the formation of spherical particles rather than linear strands. The aggregation band is always located in the 1620-1623 cm(-1) range for particulate gels showing that hydrogen bonds are weaker for these aggregates than for fine-stranded ones. This has been related to a more extensive protein unfolding for fine-stranded gels that allows a closer alignment of the polypeptide chains, and then to the formation of much stronger hydrogen bonds. Small differences are also found in protein organization and in intermolecular hydrogen bond strength vs pD within the same type of gel. Protein conformation and protein-protein interactions in the gel state may be responsible of the specific macroscopic properties of each gel network. A coarse representation of the different modes of gelation is described.

Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy.

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.