Protein-based emulsion electrosprayed micro- and submicroparticles for the encapsulation and stabilization of thermosensitive hydrophobic bioactives

This work shows the potential of emulsion electrospraying of proteins using food-grade emulsions for the microencapsulation and enhanced protection of a model thermosensitive hydrophobic bioactive. Specifically, gelatin, a whey protein concentrate (WPC) and a soy protein isolate (SPI) were compared as emulsion stabilizers and wall matrices for encapsulation of α-linolenic acid. In a preliminary stage, soy bean oil was used as the hydrophobic component for the implementation of the emulsion electrospraying process, investigating the effect of protein type and emulsion protocol used (i.e. with or without ultrasound treatment) on colloidal stability. This oil was then substituted by the ω-3 fatty acid and the emulsions were processed by electrospraying and spray-drying, comparing both techniques. While the latter resulted in massive bioactive degradation, electrospraying proved to be a suitable alternative, achieving microencapsulation efficiencies (MEE) of up to ∼70%. Although gelatin yielded low MEEs due to the need of employing acetic acid for its processing by electrospraying, SPI and WPC achieved MEEs over 60% for the non-sonicated emulsions. Moreover, the degradation of α-linolenic acid at 80 °C was significantly delayed when encapsulated within both matrices. Whilst less than an 8% of its alkene groups were detected after 27 h of thermal treatment for free α-linolenic acid, up to 43% and 67% still remained intact within the electrosprayed SPI and WPC capsules, respectively.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO project AGL2012-30647 for financial support. Sara Díaz Cuesta is also acknowledged for experimental support.Peer reviewe

Stability and bioaccessibility of EGCG within edible micro-hydrogels. Chitosan vs. gelatin, a comparative study

Micro-hydrogels are very promising systems for the protection and controlled delivery of sensitive bioactives, but limited knowledge exists regarding the impact of this encapsulation on their bioaccessibility. In this work, two different hydrogel-forming biopolymers (gelatin and chitosan) were compared as wall materials for the microencapsulation of a model flavonoid, (−)-epigallocatechin gallate (EGCG). Results showed that gelatin was more adequate as wall material for the encapsulation of EGCG than chitosan, achieving higher encapsulation efficiencies (95% ± 6%), being more effective in delaying EGCG release and degradation in aqueous solution and exhibiting a 7 times higher bioaccessibility of the bioactive compound (in terms of antioxidant activity) after in-vitro gastrointestinal digestion. A very low bioaccessibility of EGCG in chitosan was observed, due to the neutralization of the carbohydrate in the basic simulating salivary conditions, thus precluding subsequent flavonoid release. Moreover, gelatin micro-hydrogels also hindered dimer formation during in-vitro digestion, thus suggesting greater bioavailability when compared with free EGCG.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO projectAGL2015-63855-C2-1 for financial support.Peer reviewe

Impact of molecular weight on the formation of electrosprayed chitosan microcapsules as delivery vehicles for bioactive compounds

The molecular weight of chitosan is one of its most determinant characteristics, which affects its processability and its performance as a biomaterial. However, information about the effect of this parameter on the formation of electrosprayed chitosan microcapsules is scarce. In this work, the impact of chitosan molecular weight on its electrosprayability was studied and correlated with its effect on the viscosity, surface tension and electrical conductivity of solutions. A Discriminant Function Analysis revealed that the morphology of the electrosprayed chitosan materials could be correctly predicted using these three parameters for almost 85% of the samples. The suitability of using electrosprayed chitosan capsules as carriers for bioactive agents was also assessed by loading them with a model active compound, (−)-epigallocatechin gallate (EGCG). This encapsulation, with an estimated efficiency of around 80% in terms of preserved antioxidant activity, showed the potential to prolong the antiviral activity of EGCG against murine norovirus via gradual bioactive release combined with its protection against degradation in simulated physiological conditions.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Gloria Sanchez was supported by the “Ramón y Cajal” Young Investigator Program. The authors would like to thank the Spanish MINECO project AGL2015-63855-C2-1 and INIA grant RTA2014-00024-C04-03 for financial support.Peer reviewe

Electrosprayed gelatin submicroparticles as edible carriers for the encapsulation of polyphenols of interest in functional foods

In this work, the potential of the electrospraying technique to obtain food-grade gelatin capsules in the submicron range for sensitive bioactive protection was explored, studying the influence of the protein concentration on the size and morphology of the obtained particles. Gelatin was selected as encapsulating material because, being commonly used as a food ingredient, it possesses unique gelation properties and is commercially available at a low cost. The electrosprayed matrices were used to encapsulate a model antioxidant molecule, (−)-epigallocatechin gallate (EGCG). Very high encapsulation efficiencies, close to 100%, were achieved, and the antioxidant activity of the bioactive was fully retained upon encapsulation. The EGCG release profiles showed a delayed release of the encapsulated antioxidant in aqueous solutions. Furthermore, while free EGCG in PBS lost a 30% of their antioxidant activity being completely degraded in 100 h, encapsulated EGCG retained its whole antioxidant activity within this time period.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO project AGL2012-30647 for financial support.Peer reviewe

Microencapsulation of a whey protein hydrolysate within micro-hydrogels: Impact on gastrointestinal stability and potential for functional yoghurt development

Gelatin and chitosan micro-hydrogels containing a potentially bioactive whey protein hydrolysate were developed through spray drying and the impact of microencapsulation on protection during digestion and peptide stability against lactic acid fermentation during yoghurt manufacturing was assessed. The results showed that the protection exerted by the encapsulation structures during milk fermentation was sequence- and matrix-dependent, being chitosan more effective than gelatin in stabilising the peptides. However, only 5 out of the 21 fermentation-susceptible peptides identified could be protected through encapsulation within chitosan (1 of which was also protected by gelatin). Moreover, the encapsulation within chitosan microparticles did not substantially affect the peptide profile of the digested hydrolysate, and therefore, the peptide bioaccessibility was not expected to be compromised.Laura G. Gómez-Mascaraque is the recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO projects AGL2015-63855-C2-1 and AGL2015-66886-R for financial support.Peer reviewe

Nano- and microstructural evolution of alginate beads in simulated gastrointestinal fluids. Impact of M/G ratio, molecular weight and pH

peer-reviewedAlginate microcapsules were prepared using three different alginate grades and incubated under simulated digestion conditions. Their micro- and nanostructural changes were studied using microscopy, laser diffraction and small angle X-ray scattering. Both the molecular weight and M/G ratio affected the size and nanostructural features of the capsules, but the changes in gastrointestinal conditions were mainly determined by the latter. All microcapsules swelled slightly in simulated gastric fluid (pH = 3) and swelled further in simulated intestinal fluid (pH = 7), particularly those with high mannuronic acid (M) contents. While high guluronic acid (G) beads maintained the nanostructural features characteristic of alginate gels (junction zones) in both media, these were rapidly disrupted in the M-rich capsules. Decreasing the pH of the gastric phase from 3 to 2 had dramatic structural impacts, resulting in a greater integrity of the microcapsules, thus highlighting the importance of the selected digestion protocol for rational microcapsule design

Optimization of electrospraying conditions for the microencapsulation of probiotics and evaluation of their resistance during storage and in-vitro digestion

Electrospraying has recently emerged as a novel microencapsulation technique with potential for the protection of probiotics. However, research efforts are still needed to minimize the viability loss observed during the processing of sensitive strains, and to maximize productivity. The aim of the present work was the optimization of the electrospraying conditions for the microencapsulation of a model probiotic microorganism, Lactobacillus plantarum, within a whey protein concentrate matrix. In a pre-optimization step, the convenience of encapsulating fresh culture instead of freeze-dried bacteria was established. Additionally, a surface response methodology was used to study the effect of the applied voltage, surfactant concentration, and addition of a prebiotic to the formulation on cell viability and productivity. Viability losses lower than 1 log10 CFU were achieved and the bacterial counts of the final products exceeded 8.5 log10 CFU/g. The protection ability of the developed structures during storage and in-vitro digestion was also evaluated.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Russell Cruz Morfin received a scholarship from the Mexican National Council for Science and Technology (CONACYT), Call 2014. Gloria Sánchez is supported by the “Ramón y Cajal” Young Investigator program of the MINECO. This work was financially supported by the Spanish MINECO project AGL2012-30647 and by the CSIC project 201470I002.Peer reviewe

X-ray computerized microtomography and confocal Raman microscopy as complementary techniques to conventional imaging tools for the microstructural characterization of Cheddar cheese

peer-reviewedThis study explored the use of X-ray computerized microtomography (micro-CT) and confocal Raman microscopy to provide complementary information to well-established techniques, such as confocal laser scanning microscopy (CLSM), for the microstructural characterization of cheese. To evaluate the potential of these techniques, 5 commercial Cheddar cheese samples, 3 with different ripening times and 2 with different fat contents, were analyzed. Confocal laser scanning microscopy was particularly useful to describe differences in fat and protein distribution, especially between the 2 samples with different fat contents. The quantitative data obtained through image analysis correlated well with the nutritional information provided in the product labels. Conversely, micro-CT was more advantageous for studying the size and spatial distribution of microcrystals present within the cheese matrix. Two types of microcrystals were identified that differed in size, shape, and X-ray attenuation. The smallest, with a diameter of approximately 10 to 20 μm, were more abundant in the samples and presented a more uniform roundish shape and higher X-ray attenuation. Larger and more heterogeneous crystals with diameters reaching 50 μm were also observed in scarcer numbers and showed lower X-ray attenuation. Confocal Raman microscopy was useful not only for identifying the distribution of all these components but also allowed comparing the presence of micronutrients such as carotenoids in the cheeses and provided compositional information on the crystals detected. Small and large crystals were identified as calcium phosphate and calcium lactate, respectively. Overall, using micro-CT, confocal Raman microscopy, and CLSM in combination generated novel and complementary information for the microstructural and nutritional characterization of Cheddar cheese. These techniques can be used to provide valuable knowledge when studying the effect of milk composition, processing, and maturation on the cheese quality attributes

Characterization and gelling properties of a bioactive extract from Ascophyllum nodosum obtained using a chemical-free approach

peer-reviewedThe bioactivity and gelling properties of a carbohydrate-rich algal extract obtained from locally harvested Ascophyllum nodosum seaweed using a chemical-free approach were investigated for its potential interest in food applications. Physicochemical characterisation and compositional analysis of the extract, using FTIR, biochemical methods and monosaccharide analysis, confirmed the presence of alginates and fucoidans, although the main polysaccharide present in it was laminarin. Significant amounts of phenolic compounds (~9 ​mg phloroglucinol/100 ​mg sample) were also detected. As a result, the extract exhibited good antioxidant activity. It also showed promising prebiotic potential, promoting the growth of beneficial Lactobacillus sp. and Bifidobacteria sp. when compared with commercial prebiotics, but not that of pathogenic bacteria such as E. coli or P. aeruginosa. The gelling properties of the raw extract were explored to optimize hydrogel bead formation by external gelation in CaCl2 solutions. This was enhanced at neutral to alkaline pHs and high extract and CaCl2 concentrations. The mechanical strength, nano- and microstructure of the hydrogel beads prepared under optimised conditions were determined using compression tests, synchrotron small- and wide-angle X-ray scattering (SAXS/WAXS) and scanning electron microscopy (SEM). It was concluded that the raw algal extract at neutral pH had potential for use as a gelling agent, although further enrichment with alginate improved the mechanical properties of the obtained gels. The advantages and disadvantages of applying the non-purified algal extract in comparison with purified carbohydrates are discussed

Microstructural Analysis of Whey/Soy Protein Isolate Mixed Gels Using Confocal Raman Microscopy

This work explores the potential of confocal Raman microscopy to investigate the microstructure of mixed protein gel systems. Heat-set protein gels were prepared using whey protein isolate (WPI), soy protein isolate (SPI), and mixtures thereof, with a total of five different whey-to-soy protein ratios (100, 75, 50, 25, and 0%). These were analysed using confocal Raman microscopy, and different data analysis approaches were used to maximize the amount of structural and compositional information extracted from the spectral datasets generated, including both univariate and multivariate analysis methods. Small spectral differences were found between pure WPI and SPI gels, mainly attributed to conformational differences (amide bands), but SPI exhibited considerably greater auto-fluorescence than WPI. The univariate analysis method allowed for a rapid microstructural analysis, successfully mapping the distribution of protein and water in the gels. The greater fluorescence of the capsule-like structures found in the mixed gels, compared to other regions rich in proteins, suggested that these may be enriched in soy proteins. Further analysis, using a multivariate approach, allowed us to distinguish proteins with different levels of hydration within the gels and to detect non-proteinaceous compounds. Raman microscopy proved to be particularly useful to detect the presence of residual lipids in protein gels