Hydroxypropyl methylcellulose-based micro- and nanostructures for encapsulation of melanoidins: Effect of electrohydrodynamic processing variables on morphological and physicochemical properties.

Electrohydrodynamic processing (EHDP) allows the use of a wide range of biopolymers and solvents, including food-grade biopolymers and green solvents, for the development of micro- and nanostructures. These structures present a high surface-area-to-volume ratio and different shapes and morphologies. The aim of this work was to design and produce hydroxypropyl methylcellulose (HPMC)-based micro- and nanostructures through EHD processing using green solvents, while exploring the influence of process and solution parameters, and incorporating a bioactive extracted from a food by-product. Low (LMW) and high (HMW) molecular weight HPMC have been used as polymers. The design-of-experiments methodology was used to determine the effects of process parameters (polymer concentration, flow rate, tip-to-collector distance, and voltage) of EHDP on the particle and fibre diameter, aspect ratio, diameter distribution, aspect ratio distribution, and percentage of fibre breakage. Additionally, melanoidins extracted from spent coffee grounds were encapsulated into the HPCM-based structures at a concentration of 2.5 mg melanoidins/mL of the polymer solution. Polymer solutions were characterised regarding their viscosity, surface tension and conductivity, and showed that the incorporation of melanoidins increased the viscosity and conductivity values of the polymer solutions. The developed structures were characterised regarding their thermal properties, crystallinity and morphology before and after melanoidin incorporation and it was observed that melanoidin incorporation did not significantly influence the characteristics of the produced micro- and nanostructures. Based on the results, it is possible to envision the use of the produced micro- and nanostructures in a wide range of applications, both in food and biomedical fields.

Electrosprayed cashew gum microparticles for the encapsulation of highly sensitive bioactive materials.

This study focuses on the production and characterization of electrosprayed cashew gum (CG) microparticles that encapsulate ß-carotene. CG is an inexpensive, non-toxic polysaccharide obtained from Anacardium occidentale trees. Encapsulation of ß-carotene in CG was performed by electrospraying from two emulsion formulations (water : oil ratios 80:20 and 90:10 (v/v)) in which the dispersed phase consisted of ß-carotene dissolved in castor oil, and the continuous phase was a CG aqueous solution. Spherical particles with smooth surface and medium size between 3 and 6 µm were obtained. The particles produced from the 90:10 (v/v) emulsion showed a loading capacity of 0.075 ± 0.006 % and a minor amount of extractable ß-carotene, 10.75 ± 2.42 %. ATR-FTIR confirmed the absence of interaction between the particles' components. CG demonstrated to offer thermoprotection, and photoprotection for short periods of time. These results make CG a viable candidate to encapsulate bioactive compounds via electrospraying for agricultural, food and pharmaceutical applications.

Biosynthesis of silver nanoparticles and polyhydroxybutyrate nanocomposites of interest in antimicrobial applications.

This study deals with the optimization and scaling up of the production of poly(3-hydroxybutyrate), PHB, nanocomposites containing biosynthesized silver nanoparticles (AgNPs) to generate materials with antimicrobial performance. First, a comparative study of the chemical and biological synthesis of AgNPs during the fermentation process of Cupriavidus necator at shake flask-scale was carried out. These experiments demonstrated the inherent capacity of C. necator to reduce the silver salt and produce AgNPs without the need for adding a reducing agent and, that the method of synthesis (with or without reducing agent) affects the dispersion of the AgNPs and their antimicrobial performance. Finally, the process was scaled-up to a 10Liters bioreactor and the relevant physical properties of the PHB-AgNPs nanocomposites pressed into films were determined. From the characterization work, the AgNPs were found to be well dispersed and distributed into the polymer matrix, having a maximum frequency of particles with average diameter of 76-95nm. Moreover, the presence of AgNPs did not cause any effect on the thermal properties of the biopolymer, although a slight reduction in crystallinity was seen. The developed materials presented a strong antimicrobial activity against the food-borne pathogens Salmonella enterica and Listeria monocytogenes, which makes them potentially suitable for active coatings and packaging applications. Complete biodisintegration of the samples occurred during composting conditions within the first 40days. Interestingly, the presence of the AgNPs did not impair the profile of biodegradation of the microbial polymer.

Novel poly(ε-caprolactone)/gelatin wound dressings prepared by emulsion electrospinning with controlled release capacity of Ketoprofen anti-inflammatory drug.

In the present study, a single and binary Ketoprofen-loaded mats of ultrathin fibers were developed by electrospinning and their physical properties and drug release capacity was analyzed. The single mat was prepared by solution electrospinning of poly(ε-caprolactone) (PCL) with Ketoprofen at a weight ratio of 5wt%. This Ketoprofen-containing PCL solution was also used as the oil phase in a 7:3 (wt/wt) emulsion with gelatin dissolved in acidified water. The resultant stable oil-in-water (O/W) emulsion of PCL-in-gelatin, also containing Ketoprofen at 5wt%, was electrospun to produce the binary mat. Cross-linking process was performed by means of glutaraldehyde vapor on the electrospun binary mat to prevent dissolution of the hydrophilic gelatin phase. The performed characterization indicated that Ketoprofen was successfully embedded in the single and binary electrospun mats, i.e. PCL and PCL/gelatin, and both mats showed high hydrophobicity but poor thermal resistance. In vitro release studies interestingly revealed that, in comparison to the single PCL electrospun mat, the binary PCL/gelatin mat significantly hindered Ketoprofen burst release and exhibited a sustained release capacity of the drug for up to 4days. In addition, the electrospun Ketoprofen-loaded mats showed enhanced attachment and proliferation of L929 mouse fibroblast cells, presenting the binary mat the highest cell growth yield due to its improved porosity. The here-developed electrospun materials clearly show a great deal of potential as novel wound dressings with an outstanding controlled capacity to release drugs.

Efficacy of Cinnamaldehyde Against Enteric Viruses and Its Activity After Incorporation Into Biodegradable Multilayer Systems of Interest in Food Packaging.

Cinnamaldehyde (CNMA), an organic compound that gives cinnamon its flavor and odor, was investigated for its virucidal activity on norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), and hepatitis A virus (HAV). Initially, different concentrations of CNMA (0.1, 0.5 and 1 %) were individually mixed with each virus at titers of ca. 6-7 log10 TCID50/ml and incubated 2 h at 4 and 37 °C. CNMA was effective in reducing the titers of norovirus surrogates in a dose-dependent manner after 2 h at 37 °C, while HAV titers were reduced by 1 log10 after treatment with 1 % of CNMA. When incubation time was extended, HAV titers were reduced by 3.4 and 2.7 log10 after overnight incubation at 37 °C with 1 and 0.5 % of CNMA, respectively. Moreover, this paper analyzed, for the first time, the antiviral activity of adding an active electrospun interlayer based on zein and CNMA to a polyhydroxybutyrate packaging material (PHB) in a multilayer form. Biodegradable multilayer systems prepared with 2.60 mg/cm(2) (~9.7 %) of CNMA completely inactivated FCV according to ISO 22196:2011, while MNV titers were reduced by 2.75 log10. When the developed multilayer films were evaluated after one month of preparation or at 25 °C, the antiviral activity was reduced as compared to freshly prepared multilayer films evaluated at 37 °C. The results show the excellent potential of this system for food contact applications as well as for active packaging technologies in order to maintain or extend food quality and safety.

Stabilization of antimicrobial silver nanoparticles by a polyhydroxyalkanoate obtained from mixed bacterial culture.

The incorporation of antimicrobials into polymer matrices is a promising technology in the food packaging and biomedical areas. Among the most widely used antimicrobials, silver nanoparticles (AgNPs) have emerged as one of the most researched technologies to prevent microbial outbreaks. However, it is known that AgNPs are rather unstable and present patterns of agglomeration that might limit their application. In this work, AgNPs were produced by chemical reduction in suspensions of an unpurified poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) which was previously obtained from a mixed culture fermentation using a synthetic medium mimicking fermented cheese whey. The synthesis of AgNPs was carried out within the unpurified PHBV suspension (in situ) and by physical mixing (mix). The stability of crystalline and spherical nanoparticles (7±3nm) obtained in situ was found to be stable during at least 40 days. The results suggest that the unpurified PHBV appears to be a very efficient capping agent, preventing agglomeration and, thereby, stabilizing successfully the silver nanoparticles. The in situ obtained AgNP-PHBV materials were also found to exhibit a strong antibacterial activity against Salmonella enterica at low concentration (0.1-1ppm).

Evaluation of silver-infused polylactide films for inactivation of Salmonella and feline calicivirus in vitro and on fresh-cut vegetables.

There is a growing trend to develop packaging materials with an active role in guarantying that the quality and safety characteristics of packaged products will remain or improve from preparation throughout shelf-life. In the present study, 0.001-1.0 wt.% silver ions were satisfactorily incorporated into polylactide (PLA) films by a solvent casting technique. Silver migration from the films was measured by voltamperometry and then correlated with its antimicrobial efficacy against Salmonella enterica and feline calicivirus (FCV), a human norovirus surrogate, by using the Japanese industrial standard (JIS Z 2801). The PLA-silver films showed strong antibacterial and antiviral activity in vitro, with increasing effects at higher silver concentrations. Moreover, results show that FCV was less susceptible to silver than Salmonella. When films were applied on food samples, antibacterial and antiviral activity was reduced as compared to in vitro. Antimicrobial activity was very much dependent on the food type and temperature. In lettuce samples incubated at 4 °C during 6 days, 4 log CFU of Salmonella was inactivated for films with 1.0 wt.% and no infectious FCV was reported under the same conditions. On paprika samples, no antiviral effect was seen on FCV infectivity whereas films showed less antibacterial activity on Salmonella.

On the different growth conditions affecting silver antimicrobial efficacy on Listeria monocytogenes and Salmonella enterica.

Silver is known to inhibit microorganisms and therefore it is an ideal candidate for its incorporation in a wide variety of materials for food applications. However, there is still a need for understanding how silver prolonged exposure to bacterial contamination affects the bioavailability of the active silver species. In the present study, growth curves of Listeria monocytogenes and Salmonella enterica were performed for 3-5 days in Tryptic Soy Broth (TSB) and M9 minimal medium (M9) in the presence of silver ions and silver solutions previously in contact with the growth media. The cultivability of the bacteria under these conditions was correlated with the viability of the bacterial populations as measured by flow cytometry analysis (FC) using a LIVE/DEAD BacLight kit. It was found that, after a period where viable counts were not detected, bacterial populations recovered and were able to proliferate in most cases. The resuscitation of the cultures was explained by both the existence of a resilient fraction of bacteria in a compromised state and the parallel inactivation of the silver species. This inactivation was found to be highly influenced by time dependant chemical reactions taking place in the environment of exposure, producing differences of at least 3 fold between results for nutrient rich environments and results for limiting environments. This study points out the need for understanding these chemical interactions and bacterial mechanisms of adaptation and may have relevance in the design of silver-based antimicrobial systems for food-related applications.

Dietary glycosaminoglycans interfere in bacterial adhesion and gliadin-induced pro-inflammatory response in intestinal epithelial (Caco-2) cells.

Dietary components may have an important role in maintaining a balanced gut microbiota composition. Celiac disease is an autoimmune enteropathy caused by gliadins, and has been associated with a reduced proportion of Bifidobacterium in gut microbiota. This study evaluates the influence of glycosaminoglycans (GAGs) on bacterial adhesion and their contribution in the gliadins-induced inflammatory response. The adhesion of potential probiotic (Bifidobacterium longum CECT 7347 and Bifidobacterium bifidum CECT 7365), commensal (Escherichia coli and Bacteroides fragilis) and pathogenic (Salmonella enterica CECT 443 and Listeria monocytogenes CECT 935) bacteria to mucin and Caco-2 cell cultures was determined. Gliadins were subjected to in vitro digestion (pepsin/pancreatin-bile), with/out GAGs, and the presence or not of cell suspensions of B. longum (10(8) CFU/ml). B. longum, E. coli, and L. monocytogenes, markedly interact with the high-sulphur-containing fraction of GAGs. The GAGs reduced the gliadins-mediated production of interleukin-1ß, but not tumour necrosis factor-α. The results suggest that GAGs may ameliorate gliadin-induced inflammatory response, though they also slightly interfere with the action of B. longum.

Effects of chitosan films on the growth of Listeria monocytogenes, Staphylococcus aureus and Salmonella spp. in laboratory media and in fish soup.

The objective of this study was to assess the antimicrobial effectiveness of chitosonium acetate films on the growth of Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus. The samples were tested in both laboratory conditions using Tryptone Soy Broth (TSB) and in a real food system using fish soup. The study was carried out at different temperatures (4, 12, and 37 degrees C) in order to discern the influence of such variables. Moreover, a sensory evaluation of the final product was performed as a parameter of consumer acceptance. The results showed a significant reduction of the bacterial growth, which greatly depended on the bacteria type, the temperature of incubation and the food substrate. Although the effectiveness of chitosan films decreased in the fish soup, neither the sensory properties nor the pH of the soup was affected upon their addition. The application of chitosonium acetate as an internal coating of the packaging material could be a very suitable means to assure safety of liquid food products such as fish soup at the range of temperatures studied.

Optimization of the film-forming and storage conditions of chitosan as an antimicrobial agent.

The aim of this work was to assess the antimicrobial capacity of chitosan-based films obtained by a dissolution and solvent evaporation (solvent casting) method at various temperatures (i.e., 37, 80, and 120 degrees C) on the growth of Staphylococcus aureus and Salmonella spp. bacteria. The effect of temperature (4, 23, 37 degrees C) and relative humidity (RH; 0, 75%) during storage on the biocide performance was also investigated. Color parameters and ATR-FTIR spectra were analyzed for each sample to investigate the relationship between structural and/or chemical alterations in the films during storage and biocide performance. The results indicated that films formed at 37 and 80 degrees C presented a significant inhibitory effect for both types of bacteria; however, when cast at 120 degrees C, the films ceased to exhibit antimicrobial properties. Curiously, chitosonium acetate films were seen to diminish to a large extent their biocide properties when stored at 23 degrees C and 75% RH for 2 months or alternatively when stored and 37 degrees C and 0% RH over the same period of time. In good agreement with this behavior the FTIR results indicated that under the previous conditions a significant fraction of the biocide carboxylate chemistry remained in the polymer after contact with the bacterial solution due to a strong reduction in cast film solubility. Because biopolymer active species migration from the film to the culture media is needed for the biomaterial to exhibit measurable antimicrobial effect, proper control of temperature and humidity during film formation and storage is necessary to design the optimum performance of chitosan as a biocide.

Characterization of antimicrobial properties on the growth of S. aureus of novel renewable blends of gliadins and chitosan of interest in food packaging and coating applications.

The biocide properties of chitosan-based materials have been known for many years. However, typical antimicrobial formulations of chitosan, mostly chitosonium salts, are known to be very water sensitive materials which may impair their use in many application fields such as food packaging or food coating applications. This first work reports on the development and characterization of the antimicrobial properties of novel fully renewable blends of chitosan with more water-resistant gliadin proteins isolated from wheat gluten. Chitosan release to the nutrient broth from a wide range of blends was studied making use of the ninhydrin method. The results indicated that both pure chitosan and its blends with gliadins presented significant antimicrobial activity, which increased with increasing the amount of chitosan in the composite formulation as expected. The gliadins-chitosan blends showed good transparency and film-forming properties and better water resistance than pure chitosan. The release tests revealed that dissolution of the biocide glucosamine groups, i.e. the chitosan water soluble fractions, also increased with the amount of chitosan present in the formulation. The release of these groups was for the first time directly correlated with the antimicrobial properties exhibited by the blends. Thus, incorporation of chitosan into an insoluble biopolymer matrix was revealed as a very feasible strategy to generate novel chitosan-based antimicrobial materials with potential advantages, for instance active food packaging applications.

Surface characterization of poly(lactic acid) and polycaprolactone by inverse gas chromatography.

Inverse gas chromatography (IGC) has been used to characterise the surface properties of polycaprolactone (PCL) and poly(lactic acid) (PLA). The dispersive component of the surface free energy (gamma(S)(D)) was found to be very small for both of them--values close to 30 mJ/m(2) in the case of the PLA and ca. 40 mJ/m(2) for the PCL. The retention times of the n-alkanes, necessary to calculate the dispersive component of the surface energy, were obtained from the maximum, the centre at half height and the centre of mass of the chromatographic peak. While the values obtained using the first two parameters appear not to be affected by the peak asymmetry, in spite of having been obtained above the glass transition temperature of the polymer, the values obtained using the latter have been found to be not reliable. The drawbacks of using n-alkanes with a very small retention time have also been discussed, estimating the error it can introduce in the final results. Finally, the acid-base properties of the two biopolymers were determined using the approaches suggested by Schultz et al. and by St. Flour and Papirer. Although both methods describe the surfaces of PLA and PCL as neutral ones, differences between the values of the parameters K(A), K(D) and S(C) were obtained.

Film-forming process and biocide assessment of high-molecular-weight chitosan as determined by combined ATR-FTIR spectroscopy and antimicrobial assays.

This pioneering study reported about the film-forming properties of high-molecular-weight chitosan as followed in situ by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and has implications in fields such as biomedical, pharmaceutical, packaging, and coating applications. From the results, it was observed that immediately after dissolution in an acetic acid aqueous solution and subsequent casting over the ATR crystal, the formed carboxylate antimicrobial (-NH3+ -OOCH) species are not stable in the film formulation and become reduced over time; further assays confirmed previous research, which suggested that the presence and stability of these groups is strongly dependent, among other factors, on storage conditions. As-received chitosan and chitosan neutralized in NaOH films did not exhibit biocide performance towards Staphylococcus aureus. The antimicrobial tests were also found to strongly relate the presence of a sufficient quantity of these carboxylate groups to the chitosan activity as a biocide agent. Moreover, a novel methodology based on the use of a normalized infrared band centered at 1405 cm(-1) is proposed which can be correlated with the antimicrobial character of the biopolymer.

Water diffusion and sorption-induced swelling as a function of temperature and ethylene content in ethylene-vinyl alcohol copolymers as determined by attenuated total reflection fourier transform infrared spectroscopy.

Attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy has been advantageously used to carry out a simultaneous study of the effect of temperature on sorption, diffusion, swelling rate, and swelling rate factor of ethylene-vinyl alcohol (EVOH) cast films with three different ethylene contents (29, 38, and 44 mol % of ethylene). While the sorption and swelling levels at equilibrium did not appear to be affected by temperature in the temperature range studied, the effect of increasing ethylene content was seen to largely decrease the sorption-induced swelling. It should be noted that all samples showed significant levels of swelling ( approximately 60% in the copolymer with lowest ethylene content), suggesting that films obtained by solution-casting generate polymer morphologies that are far more prone to uptake water than typical melt-extruded ones. It was also observed that increasing the ethylene content led to a reduction of the "effective" D value, while raising the temperature increased diffusion and swelling rate factor. The activation energies obtained for the diffusion of water were relatively low and similar to the typical energy barrier required to break hydrogen bonding interactions, suggesting that water molecules diffuse very easily across the film due to its high chemical affinity with the polymer matrix.

Improving packaged food quality and safety. Part 1: synchrotron X-ray analysis.

The objective was to demonstrate, as an example of an application, the potential of synchrotron X-ray analysis to detect morphological alterations that can occur in barrier packaging materials and structures. These changes can affect the packaging barrier characteristics when conventional food preservation treatments are applied to packaged food. The paper presents the results of a number of experiments where time-resolved combined wide-angle X-ray scattering and small-angle X-ray scattering analysis as a function of temperature and humidity were applied to ethylene-vinyl alcohol co-polymers (EVOH), polypropylene (PP)/EVOH/PP structures, aliphatic polyketone terpolymer (PK) and amorphous polyamide (aPA) materials. A comparison between conventional retorting and high-pressure processing treatments in terms of morphologic alterations are also presented for EVOH. The impact of retorting on the EVOH structure contrasts with the good behaviour of the PK during this treatment and with that of aPA. However, no significant structural changes were observed by wide-angle X-ray scattering in the EVOH structures after high-pressure processing treatment. These structural observations have also been correlated with oxygen permeability measurements that are of importance when guaranteeing the intended levels of safety and quality of packaged food.

Improving packaged food quality and safety. Part 2: nanocomposites.

This paper gathers a number of significant results where nanotechnology was satisfactorily applied to improve packaged food quality and safety by increasing the barrier properties to oxygen of an ethylene-vinyl alcohol copolymer (EVOH) in dry and under humid conditions and of a poly(lactic acid) (PLA) biopolymer. The nanodispersion in the polymer matrix of modified monolayers of clays included in positive lists for food-contact applications is an adequate methodology to increase packaged food shelf-life. In spite of the fact that, in principle, there is no reason to believe that 'adequately' modified nanocomposites making use of substances in positive lists can impose any immediate risk threat for food-contact applications, further studies concerning potential migration issues and life-cycle analysis have to still emerge within the overall field of nanotechnology to corroborate the fact.

Modelling permeation through porous polymeric films for modified atmosphere packaging.

The use of perforated packaging films is increasing with the application of modified atmosphere packaging for fresh produce. These films provide high to very high mass exchange rates. However, irrespective of the chemistry of the material, mass transport through such films cannot be described using conventional permeability equations (Henry's plus Fick's laws). Other expressions such as Knudsen's law, gas diffusivities or Poiseuille's hydrodynamic flow can be applicable. The application of these laws is discussed and their corresponding range of validity is provided. These laws were also applied to model experimental permeation rates of oxygen and water and were further used to describe the headspace evolution of two fruit products in modified atmosphere packaging. In the light of these results, the contribution of different factors to the headspace evolution is discussed.