Insights into Molecular Interactions between a GAPDH-Related Fish Antimicrobial Peptide, Analogs Thereof, and Bacterial Membranes.

Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.

The Tape Measure Protein Is Involved in the Heat Stability of Lactococcus lactis Phages.

Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low-quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (of the Lactococcus lactis 936 [now Sk1virus] phage group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the Sk1virus group.IMPORTANCE Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low-quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese by-products. Recently, a few Lactococcus lactis phages, members of the Sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent Sk1virus phage. Identifying such a genetic determinant for heat stability is a first step in understanding the emergence of this group of thermostable phages.

Molecular and biopharmaceutical investigation of alginate-inulin synbiotic coencapsulation of probiotic to target the colon.

Colon targeting, as a site-specific delivery for oral formulation, remains a major challenge, especially for sensitive bioactive components such as therapeutic forms of phages, live attenuated virus and prebiotics-probiotics association. Synbiotics could be used to protect encapsulated probiotics during the gastrointestinal tract and control their release in the colon. To achieve these goals, effective prebiotics, such as inulin, could be combined with alginate - the most exploited polymer used for probiotic encapsulation - in the form of beads. This work aimed to study the biopharmaceutical behaviour of alginate beads (A) and inulin-alginate beads of different inulin concentrations (5 or 20%) in 2% alginate (AI5, AI20). Beads were loaded with three probiotic strains (Pediococcus acidilactici Ul5, Lactobacillus reuteri and Lactobacillus salivarius). Dissolution of beads was studied by USP4 under conditions simulating the gastrointestinal condition. The survival rates of the bacterial strains were measured by a specific qPCR bacterial count. Mucoadhesiveness of beads was studied by an ex vivo method using intestinal mucosa. To understand the behaviour of each formulation, the ultrastructure of the polymeric network was studied using scanning electron microscopy (SEM). Molecular interactions between alginate and inulin were studied by Fourier transform infra-red spectroscopy (FTIR). Dissolution results suggested that the presence of inulin in beads provided more protection for the tested bacterial strains against the acidic pH. AI5 was the most effective formulation to deliver probiotics to the colon simulation conditions. FTIR and SEM investigations explained the differences in behaviour of each formula. The developed symbiotic form provided a promising matrix for the development of colonic controlled release systems.

Collagencin, an antibacterial peptide from fish collagen: Activity, structure and interaction dynamics with membrane.

In this study, we first report characterization of collagencin, an antimicrobial peptide identified from fish collagen hydrolysate. The peptide completely inhibited the growth of Staphylococcus aureus at 1.88 mM. Although non-toxic up to 470 µM, collagencin was hemolytic at higher concentrations. The secondary structure of collagencin was mainly composed by ß-sheet and ß-turn as determined by CD measurements and molecular dynamics. The peptide is likely to form ß-sheet structure under hydrophobic environments and interacts with both anionic (phosphatidylglycerol) and zwitterionic (phosphoethanolamine and phosphatidylcholine) lipids as shown with CD spectroscopy and molecular dynamics. The peptide formed several hydrogen bonds with both POPG and POPE lipids and remained at membrane-water interface, suggesting that collagencin antibacterial action follows a carpet mechanism. Collagenous fish wastes could be processed by enzymatic hydrolysis and transformed into products of high value having functional or biological properties. Marine collagens are a promising source of antimicrobial peptides with new implications in food safety and human health.

A prebiotic matrix for encapsulation of probiotics: physicochemical and microbiological study.

This work aims to develop an encapsulated oral-synbiotic supplement by studying the effect of adding inulin in alginate beads and observing its ability to protect three probiotic strains: Pediocucus acidilactici, Lactobacillus reuteri and Lactobacillus salivarius. Beads of different inulin concentrations 0%, 5%, 10%, 15% and 20% (w/v) in 2% (w/v) alginate solution were prepared by the extrusion/ionotropic gelation method. Polymer distribution within beads was characterised using confocal laser scanning microscopy. Interactions between alginate and inulin were monitored by Fourier transform infra-red spectroscopy (FTIR). Effect of encapsulation on viability, antimicrobial ability, acid tolerance and bile tolerance of probiotic strains were investigated. Antimicrobial and probiotic properties of bacterial strains were not affected by encapsulation. Bacterial protection against acidity was increased by adding inulin. Beads with 5% w/v inulin were the most effective in bacterial protection against bile-salts. To our knowledge, this work is the first to use such high concentrations of inulin.

Lasso-inspired peptides with distinct antibacterial mechanisms.

Microcin J25 (MccJ25) is an antibacterial peptide with a peculiar molecular structure consisting of 21 amino acids and a unique lasso topology that makes it highly stable. We synthesized various MccJ25-derived peptides that retained some of the inhibitory activity of the native molecule against Salmonella enterica and Escherichia coli. Of the tested peptides, C1, 7-21C and WK_7-21 were the most inhibitory peptides (MIC = 1-250 µM), but all three were less potent than MccJ25. While MccJ25 was not active against Gram-positive bacteria, the three derived peptides were slightly inhibitory to Gram-positive bacteria (MIC ≥ 250 µM). At 5 µM, C1, 7-21C and WK_7-21 reduced E. coli RNA polymerase activity by respectively, 23.4, 37.4 and 65.0 %. The MccJ25 and its derived peptides all appeared to affect the respiratory apparatus of S. enterica. Based on circular dichroism and FTIR spectroscopy, the peptides also interact with bacterial membrane phospholipids. These results suggest the possibility of producing potent MccJ25-derived peptides lacking the lasso structure.

Development of a novel drug delivery system: chitosan nanoparticles entrapped in alginate microparticles.

A novel carrier using chitosan nanoparticles entrapped into alginate microparticles is proposed for protecting molecules of interest from degradation in the digestive tract. The effects of polymer concentration, sonication, stirring, pH, and processing conditions on the physical characteristics of the carrier were studied. FITC and RBITC were used to localise the polymers within particles using CLSM. Diffusion of amaranth red (AR) from nanoparticles was quantified during dissolution under gastric and intestinal conditions. Under optimal preparation conditions, the size distribution of nanoparticles loaded with AR was uniform (690 nm) with an encapsulation efficacy of 21.9%. Alginate microparticles (285 µm) containing a homogenous distribution of nanoparticles and polymers were obtained. At gastric pH, the carrier released less than 5% of the loaded AR and, at intestinal pH, the release was rapid and complete. The drug carriers developed shows a promising use as a vehicle suitable to protect molecules of interest after oral administration.

Efficacy of mucoadhesive hydrogel microparticles of whey protein and alginate for oral insulin delivery.

PURPOSE: To evaluate the efficacy of mucoadhesive insulin-loaded whey protein (WP) /alginate (ALG) microparticles (MP) for oral insulin administration. METHODS: Insulin-loaded microparticles (ins-MP) made of whey protein and alginate were prepared by a cold gelation technique and an adsorption method, without adjunction of organic solvent in order to develop a biocompatible vehicle for oral administration of insulin. In vitro characterization, evaluations of ins-MP in excised intestinal tissues and hypoglycaemic effects after intestinal administration in healthy rats were performed RESULTS: The release properties and swelling behaviors, investigated in different pH buffers, demonstrated a release based on diffusion mechanism following matrix swelling. Mucoadhesion studies in rabbits and insulin transport experiments with excised intestinal rat tissues revealed that encapsulation in microparticles with mucoadhesive properties promotes insulin absorption across duodenal membranes and bioactivity protection. In vivo experiments reinforced the interest of encapsulation in whey protein/alginate combination. Confocal microscopic observations associated with blood glucose levels bring to light duodenal absorption of insulin biologically active following in vivo administration. CONCLUSIONS: Insulin-loaded WP/ALG MP with high quantities of drug entrapped, in vitro matrix swelling and protective effect as well as excellent mucohadesive properties was developped. Improvement of intestinal delivery of insulin and increased in bioavailability were recorded.

Development and characterization of coated-microparticles based on whey protein/alginate using the Encapsulator device.

The aim of this study is to prepare whey protein (WP)-based microparticles (MP) using the Encapsulator(®) device. The viscosity dependence of the extrusion device required to mix WP with a food-grade and less viscous polymer. Mixed WP/ALG MP were obtained with the optimized WP/alginate (ALG) ratio (62/38). These particles were further coated with WP or ALG using non-traumatic and solvent-free coating process developed in this study. Size and morphology of coated and uncoated MP were determined. Then, swelling and degradation (WP release) of formulations were investigated in pH 1.2 and 7.5 buffers and in simulated gastric and intestinal fluids (SGF, SIF) and compared to pure ALG and pure WP particle behaviours. At pH 1.2, pure ALG shrank and pure WP swelled, whereas the sizes of mixed WP/ALG matrix were stable. In SGF, WP/ALG MP resisted to pepsin degradation compare to pure WP particles due to ALG shrinkage which limited pepsin diffusion within particles. Coating addition with WP or ALG slowed down pepsin degradation. At pH 7.5, WP/ALG particles were rapidly degraded due to ALG sensitivity but the addition of a WP coating limited effectively the swelling and the degradation of MP. In SIF, pancreatin accelerated MP degradation but ALG-coated MP exhibited interesting robustness. These results confirmed the interest and the feasibility to produce coated WP-based MP which could be a potential orally controlled release drug delivery system.

Bacteriocin-Based Synergetic Consortia: a Promising Strategy to Enhance Antimicrobial Activity and Broaden the Spectrum of Inhibition.

Bacteria-derived natural antimicrobial compounds such as bacteriocins, reruterin, and organic acids have recently received substantial attention as food preservatives or therapeutic alternatives in human or animal sectors. This study aimed to evaluate the antimicrobial activity of different bacteria-derived antimicrobials, alone or in combination, against a large panel of Gram-negative and Gram-positive bacteria. Bacteriocins, including microcin J25, pediocin PA-1, nisin Z, and reuterin, were investigated alone or in combination with lactic acid and citric acid, using a checkerboard assay. Concentrations were selected based on predetermined MICs against Salmonella enterica subsp. enterica serovar Newport ATCC 6962 and Listeria ivanovii HPB28 as Gram-negative and Gram-positive indicator strains, respectively. The results demonstrated that the combination of microcin J25 + citric acid + lactic acid; microcin J25 + reuterin + citric acid; and microcin J25 + reuterin + lactic acid tested against S. Newport ATCC 6962 showed synergistic effects (FIC index = 0.5). Moreover, a combination of pediocin PA-1 + citric acid + lactic acid; and reuterin + citric acid + lactic acid against L. ivanovii HPB28 showed a partially synergistic interactions (FIC index = 0.75). Nisin Z exerted a partially synergistic effect in combination with acids (FIC index = 0.625 -0.75), whereas when it was combined with reuterin or pediocin PA-1, it showed additive effects (FIC index = 1) against L. ivanovii HPB28. The inhibitory activity of synergetic consortia were tested against a large panel of Gram-positive and Gram-negative bacteria. According to our results, combining different antimicrobials with different mechanisms of action led to higher potency and a broad spectrum of inhibition, including multidrug-resistance pathogens. IMPORTANCE Reuterin and bacteriocins, including microcin J25, pediocin PA-1, nisin were produced and purified with >90% purity. Using the broth-based checkerboard assay the interaction between these compounds (synergetic, additive, or antagonistic) was assessed. By combining different natural antimicrobials with different modes of action and structure (reuteirn, microcin J25, pediocin PA-1, and organic acids), we successfully developed five different synergetic consortia with improved antimicrobial activity and a broad spectrum of inhibition. These consortia were shown to be effective against a large panel of pathogenic and spoilage microorganisms as well as clinically important multidrug-resistance bacteria. Moreover, because the lower concentrations of bacteriocins and reuterin are used in the synergetic consortia, there is a limited risk of toxicity and resistance development for these compounds.

In vitro assessment of skin sensitization, irritability and toxicity of bacteriocins and reuterin for possible topical applications.

Bacteriocins and reuterin are promising antimicrobials for application in food, veterinary, and medical sectors. In the light of their high potential for application in hand sanitizer, we investigated the skin toxicity of reuterin, microcin J25, pediocin PA-1, bactofencin A, and nisin Z in vitro using neutral red and LDH release assays on NHEK cells. We determined their skin sensitization potential using the human cell line activation test (h-CLAT). Their skin irritation potential was measured on human epidermal model EpiDerm™. We showed that the viability and membrane integrity of NHEK cells remained unaltered after exposure to bacteriocins and reuterin at concentrations up to 400 µg/mL and 80 mg/mL, respectively. Furthermore, microcin J25 and reuterin showed no skin sensitization at concentrations up to 100 µg/mL and 40 mg/mL, respectively, while pediocin PA-1, bactofencin A, and nisin Z caused sensitization at concentrations higher than 100 µg/mL. Tissue viability was unaffected in presence of bacteriocins and reuterin at concentrations up to 200 µg/mL and 40 mg/mL, respectively, which was confirmed by measuring cytokine IL-1α and IL-8 levels and by histological analysis. In conclusion, the current study provides scientific evidence that some bacteriocins and reuterin, could be safely applied topically as sanitizers at recommended concentrations.

In vitro investigation of gastrointestinal stability and toxicity of 3-hyrdoxypropionaldehyde (reuterin) produced by Lactobacillus reuteri.

Reuterin (3-hyrdoxypropionaldehyde (3-HPA)) is a highly potent metabolite of L. reuteri, which has applications in food, health, and veterinary sectors. Similar to other natural antimicrobial compounds, the approval of reuterin as a bio-preservative or therapeutic agent by regulatory agencies relies on sufficient data on its cytotoxicity and behavior in the gastrointestinal environment. Although the antimicrobial activity of reuterin has been broadly studied, its safety and toxicity are yet to be explored in detail. In this study, the stability and activity of reuterin were investigated in the gastrointestinal tract using in vitro models simulating gastrointestinal conditions. In addition, hemolytic activity and in vitro cytotoxicity of reuterin were evaluated by neutral red assay and lactate dehydrogenase (LDH) colorimetric assay using the same cell line. Activity of reuterin was observed to be stable during gastrointestinal transit. Viability and membrane integrity of cells remained unaltered by reuterin up to 1080 mM concentration. Furthermore, no hemolysis was observed in blood cells exposed to 270 mM reuterin. This study provides unique and highly relevant in vitro data regarding gastrointestinal behavior and toxicity of reuterin. In conclusion, the current study indicates that within a certain concentration range, reuterin can be safely used in bio-preservation and therapeutics applications. However, further in vivo studies are required to confirm these findings.

Gastrointestinal Stability and Cytotoxicity of Bacteriocins From Gram-Positive and Gram-Negative Bacteria: A Comparative in vitro Study.

Bacteriocins are receiving increased attention as potent candidates in food preservation and medicine. Although the inhibitory activity of bacteriocins has been studied widely, little is known about their gastrointestinal stability and toxicity toward normal human cell lines. The aim of this study was to evaluate the gastrointestinal stability and activity of microcin J25, pediocin PA-1, bactofencin A and nisin using in vitro models. In addition cytotoxicity and hemolytic activity of these bacteriocins were investigated on human epithelial colorectal adenocarcinoma cells (Caco-2) and rat erythrocytes, respectively. Pediocin PA-1, bactofencin A, and nisin were observed to lose their stability while passing through the gastrointestinal tract, while microcin J25 is only partially degraded. Besides, selected bacteriocins were not toxic to Caco-2 cells, and integrity of cell membrane was observed to remain unaffected in presence of these bacteriocins at concentrations up to 400 µg/mL. In hemolysis study, pediocin PA-1, bactofencin A, and nisin were observed to lyse rat erythrocytes at concentrations higher than 50 µg/mL, while microcin J25 showed no effect on these cells. According to data indicating gastrointestinal degradation and the absence of toxicity of pediocin PA-1, bactofencin A, and microcin J25 they could potentially be used in food or clinical applications.

Coated whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast.

Viable Saccharomyces boulardii, used as a biotherapeutic agent, was encapsulated in food-grade whey protein isolate (WP) and alginate (ALG) microparticles, in order to protect and vehicle them in gastrointestinal environment. Yeast-loaded microparticles with a WP/ALG ratio of 62/38 were produced with high encapsulation efficiency (95%) using an extrusion/cold gelation method and coated with ALG or WP by a simple immersion method. Swelling, yeast survival, WP loss and yeast release in simulated gastric and intestinal fluids (SGF and SIF, pH 1.2 and 7.5) with and without their respective digestive enzymes (pepsin and pancreatin) were investigated. In SGF, ALG network shrinkage limited enzyme diffusion into the WP/ALG matrix. Coated and uncoated WP/ALG microparticles were resistant in SGF even with pepsin. Survival of yeast cells in microparticles was 40% compared to 10% for free yeast cells and was improved to 60% by coating. In SIF, yeast cell release followed coated microparticle swelling with a desirable delay. Coated WP/ALG microparticles appear to have potential as oral delivery systems for Saccharomyces boulardii or as encapsulation means for probiotic cells in pharmaceutical or food processing applications.

Divergicin M35-Chitosan Film: Development and Characterization.

Chitosan films loaded with bacteriocin were examined by FTIR spectroscopy, tested for color, puncture strength, water vapor permeability, and as antimicrobials of Listeria innocua HPB13. Divergicin M35, a bacteriocin produced by Carnobacterium divergens, was incorporated into films made with chitosan of molecular mass 2 kDa, 20 kDa, or 100 kDa and de-acetylated either 87% or 95%. Only 100 kDa chitosan yielded films that could be peeled and handled easily. The higher degree of de-acetylation increased the total color factor (ΔE) of bacteriocin-loaded films, their permeability, and puncture strength. Incorporation of divergicin M35 into the films increased amide I peak intensity but otherwise did not induce significant structural change. The FTIR spectra of divergicin M35 shed from the films did not differ from those of the original free bacteriocin, except in overall peak intensity. The release of active divergicin M35 from the film was faster into the buffer than into tryptic soy broth and peaked at 10-12 h in both cases. Chitosan 95% de-acetylated and loaded with divergicin M35 was the most active, producing a six-log drop in Listeria innocua HPB13 viable count within 24 h. These results suggest that the biocompatible and biodegradable films developed here have the potential for application as antimicrobials of Listeria spp. in foods, especially ready-to-eat, minimally processed products.

Novel design for alginate/resistant starch microcapsules controlling nisin release.

This study aimed to develop a novel nontoxic, biocompatible, biodegradable, and cost-efficient matrix for the encapsulation of antimicrobial component (nisin) to be used as bio-preservative agent in cheddar cheese. Nisin A loaded beads were prepared from alginate at 0.5%, 1% and 2%; and hi-maize resistant starch at 0.5 or 1%. Beads were characterized by microscopic examination and transmission electron microscopy. Molecular structures were investigated by FTIR, and particle size distributions were measured. The Entrapment efficiency (EE) was measured microbiologically by agar diffusion. The encapsulated nisin showed similar inhibition activities in all developed formulas with an inhibition zone of 15 ± 2 mm. The FTIR analysis confirmed the compatibility of the nisin with sodium alginate and starch. The formulas composed of 1% Alginate and 0.5% Non-Gelatinized Starch had the highest encapsulation efficiency among other formulas (33%). Moreover, that formula allowed the protection and gradual release of the encapsulated nisin during a long-term storage for up to two months. Application in cheddar cheese proved the inhibition of encapsulated nisin on the growth of C. tyrobutyricum at the large-scale production. In conclusion, the alginate (Alg)/non-gelatinized resistant starch formula is suitable for the protection and controlled release of nisin in food applications.

Elaboration and characterization of soy/zein protein microspheres for controlled nutraceutical delivery.

Microspheres (15-25 microm) of soy protein isolate (SPI), zein, and SPI/zein blends were prepared using a cold gelation method as possible delivery systems for nutraceutical products. Microsphere matrix crystalline structure, swelling behavior, and nutrient load release kinetics in simulated gastrointestinal fluids were investigated. SPI microspheres showed early burst release of the model nutrient, whereas zein microspheres showed very slow release in both simulated gastric and intestinal fluids. Blending of SPI and zein provides a convenient method of adjusting the hydrophobicity and crystallinity of the protein matrix and hence its swelling behavior and in vivo nutrient release kinetics. Diffusion plays a major role in regulating nutrient release. SPI/zein microspheres blended at ratios of 5:5 and 3:7 showed near zero-order release kinetics over the test period in simulated intestinal buffer and thus have potential as delivery vehicles for nutraceutical products in functional foods.

Investigation of coated whey protein/alginate beads as sustained release dosage form in simulated gastrointestinal environment.

AIM: The biopharmaceutical behavior of new formulations based on both food-grade polymers, whey protein (WP) and alginate (ALG) was studied using different in vitro methods. The Biopharmaceutical Classification System (BCS) class I drug Theophylline was chosen as drug model. METHOD: Drug release was studied (i) at pH 1.2 (2 hours) followed by pH 7.5, and in simulated gastric fluid (SGF; 2 hours) followed by simulated intestinal fluid (SIF) using the paddle method and (ii) in an artificial digestive system. RESULTS: Freeze-dried mixed WP/ALG (62/38) beads were coated with WP or ALG with encapsulation efficiency 34.9% and 18.3%, respectively. At pH 1.2, coated beads exhibited gastroresistant properties (< 10% of drug released after 2 hours) followed at pH 7.5 by a sustained release behavior (< 60% of drug released at 24 hours) controlled by an erosion mechanism. In SGF, despite enzyme hydrolysis, drug release was still controlled due to ALG shrinkage. After transfer in SIF, formulations were completely degraded in less than 2 h with total drug release. In an artificial digestive system, coated beads appeared gastroresistant, intestinal part sustained drug release was controlled by erosion. CONCLUSION: Combination of in vitro methods allowed prediction of the in vivo potentialities of WP- and ALG- coated WP/ALG beads as oral sustained release systems.

Interaction of oil bodies proteins with phospholipid bilayers: A molecular level elucidation as revealed by infrared spectroscopy.

It is crucial to develop new natural sources of emulsifiers to substitute the synthetic molecules. An ideal emulsifying system exists in plants that is consisting of oil bodies proteins and phospholipids. In this study, Fourier transformed infrared (FTIR) spectroscopy was used to investigate the interactions between oil bodies proteins (OBP) and model phospholipid (PL) membranes. The secondary structure and PL thermotropism were investigated. Different PL varying in chain length and polar head were used including two zwitterionic phospholipids, dimyristoylphosphatidylcholine and dioleoylphosphatidylcholine, and two anionic phospholipids, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol. The changes in lipid physical state and protein denaturation were investigated as a function of temperature from 20 to 80 °C. OBP in solution is composed of unordered structures and ß-sheets with signs of aggregation. Anionic PL interacts with OBP whereas zwitterionic PL does not or only slightly interacts with the protein. Unsaturated PL promoted the α-helix structure in OBP. The interactions between OBP and PL depended on the protein charge inducing different protein conformations. Overall, the study showed that OBP and commercial anionic phospholipids have a potential in developing stable emulsifier for food industry.

Kinetics of the breakdown of cross-linked soy protein films for drug delivery.

The aim of the present work was to investigate the potential of soy protein isolate (SPI) films as controlled release systems for active compounds. Mechanical properties, dissolution and compound release kinetics of SPI films prepared with different concentrations of formaldehyde were measured over time in the absence or presence of digestive enzymes at gastric or intestinal pH. The effect of formaldehyde on tensile strength, elastic modulus, % elongation and swelling suggested that increasing its concentration increased film cross-linking density. Film bulk erosion in the presence of digestive enzymes followed first-order kinetics. Methylene blue or rifampicin release followed variable kinetics depending on compound solubility during a 1-2h initial phase, followed by zero-order release. Cross-linking density appears to provide effective means of regulating the erosion and release rate of SPI films. SPI film networks displayed excellent compound binding capacity, especially for hydrophobic molecules, and hence potential for use in controlled release systems based on matrix erosion.