Encapsulation and dispersion of Lactobacillus acidophilus in a chocolate coating as a strategy for maintaining cell viability in cereal bars.

Flour from Pereskia aculeata leaf and green banana were used as ingredients in the formulation of a cereal bar with added Lactobacillus acidophilus LA02-ID-1688. Encapsulation in a calcium-alginate hydrogel reinforced with magnesium hydroxide was used as a strategy to protect the probiotic cells under gastrointestinal conditions and to prolong shelf-life. The results are relevant especially for maintaining cell viability during shelf-life; a challenge for the food industry in relation to dry probiotic products. Encapsulation promoted the protection of probiotic cells in simulated gastric and intestinal conditions, allowing the maintenance of high viable cell counts (> 10 log CFU, colony forming unit). Encapsulation also contributed to cellular protection under extreme temperature conditions, with reductions of cell viability of < 1 logarithmic cycle when the capsules were subjected to 55ºC/10 min. Even at 75ºC/10 min, encapsulation protected the probiotic cells 3-times greater than the free-cells. The food bar proved to be rich in dietary fiber (19 g 100 g-1), lipids (12.63 g 100 g-1) and showed an appreciable protein content (5.44 g 100 g-1). A high viable probiotic cell count on storage over 120 days (12.54 log CFU) was observed, maintaining a probiotic survival rate > 90% and viability levels sufficient to promote health benefits.

Efficacy of Salmonella Bacteriophage S1 Delivered and Released by Alginate Beads in a Chicken Model of Infection.

Modern bacteriophage encapsulation methods based on polymers such as alginate have been developed recently for their use in phage therapy for veterinary purposes. In birds, it has been proven that using this delivery system allows the release of the bacteriophage in the small intestine, the site of infection by Salmonella spp. This work designed an approach for phage therapy using encapsulation by ionotropic gelation of the lytic bacteriophage S1 for Salmonella enterica in 2% w/v alginate beads using 2% w/v calcium chloride as crosslinking agent. This formulation resulted in beads with an average size of 3.73 ± 0.04 mm and an encapsulation efficiency of 70%. In vitro, the beads protected the bacteriophages from pH 3 and released them at higher pH. To confirm that this would protect the bacteriophages from gastrointestinal pH changes, we tested the phage infectivity in vivo assay. Using a model chicken (Gallus gallus domesticus) infected with Salmonella Enteritidis, we confirmed that after 3 h of the beads delivery, infective phages were present in the chicken's duodenal and caecal sections. This study demonstrates that our phage formulation is an effective system for release and delivery of bacteriophage S1 against Salmonella Enteritidis with potential use in the poultry sector.

Neovascularized implantable cell homing encapsulation platform with tunable local immunosuppressant delivery for allogeneic cell transplantation.

Cell encapsulation is an attractive transplantation strategy to treat endocrine disorders. Transplanted cells offer a dynamic and stimulus-responsive system that secretes therapeutics based on patient need. Despite significant advancements, a challenge in allogeneic cell encapsulation is maintaining sufficient oxygen and nutrient exchange, while providing protection from the host immune system. To this end, we developed a subcutaneously implantable dual-reservoir encapsulation system integrating in situ prevascularization and local immunosuppressant delivery, termed NICHE. NICHE structure is 3D-printed in biocompatible polyamide 2200 and comprises of independent cell and drug reservoirs separated by a nanoporous membrane for sustained local release of immunosuppressant. Here we present the development and characterization of NICHE, as well as efficacy validation for allogeneic cell transplantation in an immunocompetent rat model. We established biocompatibility and mechanical stability of NICHE. Further, NICHE vascularization was achieved with the aid of mesenchymal stem cells. Our study demonstrated sustained local elution of immunosuppressant (CTLA4Ig) into the cell reservoir protected transcutaneously-transplanted allogeneic Leydig cells from host immune destruction during a 31-day study, and reduced systemic drug exposure by 12-fold. In summary, NICHE is the first encapsulation platform achieving both in situ vascularization and immunosuppressant delivery, presenting a viable strategy for allogeneic cell transplantation.

ENCAPSULACIÓN DEL PÉPTIDO ANTIMICROBIANO AP7121 PARA SU ADMINISTRACIÓN POR VÍA ORAL / Encapsulation of antimicrobial peptide AP7121 for oral administration

El péptido antimicrobiano AP7121, producido por Enterococcus faecalis CECT7121, presenta actividad bactericida sobre patógenos Gram positivos. Sin embargo, su vía de administración oral está inhibida por la acidez gástrica y enzimas proteolíticas digestivas. El objetivo del presente trabajo fue analizar la eficacia de la encapsulación de AP7121 para su administración por vía oral. Para ello, se realizó la encapsulación del péptido antimicrobiano mediante la formación de gotas de alginato de sodio estéril 2,2% conteniendo AP7121 (30,0 mg/L) y circulando en dispositivo extrusor. Se evaluó la actividad inhibitoria de cápsulas obtenidas mediante la determinación de la Concentración Inhibitoria Mínima de AP7121 (CIMAP7121), con Listeria monocytogenes ATCC 19111 (LM, CIMAP7121: 0,8 mg/L). Asimismo, se investigó la estabilidad del péptido antimicrobiano frente a las enzimas proteolíticas tripsina, α-quimotripsina, proteinasa K y pronasa E (1 mg/mL) y se evaluó el efecto del pH utilizando una solución de HCl, pH=2,0. Las cápsulas obtenidas fueron uniformes y se obtuvo una concentración final de AP7121 de 29,7±0,3 mg/L con una CIMAP7121: 0,8 mg/L para LM. Frente a enzimas proteolíticas, no se observó descenso de actividad, permaneciendo inalterable en las cápsulas (CIMAP7121 de LM: 0,8 mg/L). Luego de la exposición a pH=2,0, se observó pérdida significativa de actividad a las 4 h de exposición. Los resultados obtenidos habilitarían la utilización de AP7121 en cápsulas para su administración por vía oral, dada su resistencia al pH ácido estomacal y enzimas proteolíticas, factores limitantes para su uso sin protección de su actividad.

The antimicrobial peptide AP7121, produced by Enterococcus faecalis CECT7121, presents bactericidal activity on Gram-positive pathogens. However, its administration via oral route is inhibited by gastric acidity and proteolytic digestive enzymes. The objective of this work was to analyze the effectiveness of the encapsulation of AP7121 for oral administration. In order to do this, the encapsulation of the antimicrobial peptide was performed by forming sterile 2.2% sodium alginate drops containing AP7121 (30.0 mg / L) and circulating in an extruder device. The inhibitory activity of the obtained capsules was evaluated by determining the Minimum Inhibitory Concentration of AP7121 (MICAP7121), with Listeria monocytogenes ATCC 19111 (LM, MICAP7121: 0.8 mg / L). Likewise, the stability of the antimicrobial peptide was investigated against trypsin, α-chymotrypsin, proteinase K and pronase E (1 mg / mL) proteolytic enzymes, and the effect of pH was evaluated using an HCl, pH = 2.0 solution. The capsules obtained were uniform and a final AP7121 concentration of 29.7 ± 0.3 mg / L with a MIC AP7121: 0.8 mg / L for ML was obtained. Against proteolytic enzymes, no decrease in activity was observed, remaining unchanged in the capsules (MICAP7121 of LM: 0.8 mg / L). After exposure to pH = 2.0, a significant loss of activity was observed after 4 h of exposure. The results obtained would enable the use of AP7121 in capsules for oral administration given its resistance to stomach acid pH and proteolytic enzymes, factors that limit the use without protection of its activity.

Long-term conservation of Tarenaya rosea (Cleomaceae) root cultures: histological and histochemical analyses during cryopreservation using the encapsulation-vitrification technique.

Adventitious root cultures of Tarenaya rosea were successfully cryopreserved using the encapsulation-vitrification technique. Histological analysis revealed useful information on the successive steps of cryopreservation. Coupled with complementary histochemical approaches, these studies provided cellular and tissue descriptions of T. rosea root cultures during cryopreservation and contributed to an understanding of cellular stress responses, as well as characterization of the anatomical pattern of root regeneration. The effects of exposure duration to PVS3 solution (0-120 min), unloading treatment (direct and gradual), and recovery medium (liquid and solid) on recovery of cryopreserved roots were investigated. The highest recovery (91%) after cooling in liquid nitrogen (LN) was reached with PVS3 treatment for 90 min, gradual rehydration in unloading solution, and recovery on solid MS medium. The cryopreserved roots showed high multiplication capacity, which was maintained for up to four subcultures. The effect of cryopreservation on root structure was investigated by histological and histochemical studies. Plasmolysis intensified during exposure to loading and PVS3 solutions, but decreased after unloading treatment. The proportion of intercellular spaces increased progressively throughout the cryopreservation protocol, culminating in root cortex disruption. Histochemical analyses revealed polysaccharides, proteins, and both lipidic and pectic substances in intercellular spaces. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing that proliferative capacity of cryopreserved roots had not diminished.

Encapsulation of Lactobacillus plantarum ATCC 8014 and Pediococcus acidilactici ATCC 8042 in a freeze-dried alginate-gum arabic system and its in vitro testing under gastrointestinal conditions.

The aim of this study was to investigate the viability of Pediococcus acidilactici ATCC 8042 and Lactobacillus plantarum ATCC 8014 in a freeze-dried capsules system prepared with sodium alginate and gum arabic using the extrusion technique. The capsules made with alginate 2% (w/v)/gum arabic 2% (w/v) showed higher hardness (7.12 ± 0.71 N), with highly cohesive (0.81 ± 0.02) and elastic (0.99 ± 0.00) features on the Texture Profile Analysis (TPA), as well as higher sphericity with 1.75 ± 0.12 mm y 1.73 ± 0.13 mm diameter axes and regularity in their surface by Scanning Electron Microscopy (SEM). The use of skimmed milk at 10% as a cryoprotector in the freeze-drying process allowed the obtention of high viability percentages (88% a 96%) for both strains. Best results of viability for P. acidilactici encapsulated was with the use of alginate 2% (w/v)/gum arabic 2% (w/v) (92%±2.65), and L. plantarum with the use of alginate 2% (w/v) (84.71%±10.33) during the gastrointestinal environment challenge.

Encapsulation of Lactobacillus fermentum K73 by Refractance Window drying.

The purpose of this work was to model the survival of the microorganism and the kinetics of drying during the encapsulation of Lactobacillus fermentum K73 by Refractance Window drying. A whey culture medium with and without addition of maltodextrin were used as encapsulation matrices. The microorganism with the encapsulation matrices was dried at three water temperatures (333, 343 and 353 K) until reaching balanced moisture. Microorganism survival and thin layer drying kinetics were studied by using mathematical models. Results showed that modified Gompertz model and Midilli model described the survival of the microorganism and the drying kinetics, respectively. The most favorable process conditions found with the mathematical modelling were a drying time of 2460 s, at a temperature of 353 K. At these conditions, a product with 9.1 Log CFU/g and a final humidity of 10% [wet basis] using the culture medium as encapsulation matrix was obtained. The result shows that Refractance Window can be applied to encapsulate the microorganism probiotic with a proper survival of the microorganism.

Tridimensional alginate disks of tunable topologies for mammalian cell encapsulation.

We have developed a protocol to produce three-dimensional matrices based on alginate hydrogels for mammalian cell encapsulation. Based on the gelation properties of this polysaccharide, we implemented a calcium ion-based diffusion method where the designed hydrogels can be obtained with well-defined mechanical properties and replicable 3D topologies. The developed protocol can be extended to different types of alginates and an ample range of concentrations. This makes it very attractive for various biomedical applications where strict control over structure-function relationships is desirable.

Alimentos microencapsulados para el cultivo de larvas de pejelagarto (Atractosteus tropicus) / Microencapsulated diets for tropical gar (Atractosteus tropicus) larviculture

Resumen El pejelagarto es una especie importante para la acuicultura en el sureste de México. Se han realizado varios estudios sobre nutrición y fisiología digestiva; sin embargo, el uso de microcápsulas para la alimentación de larvas que permitan mejorar el crecimiento y la supervivencia no ha sido realizado. En este sentido, se evaluaron cuatro alimentos microencapsulados en el crecimiento y supervivencia de larvas de pejelagarto (Atractosteus tropicus). Los tratamientos consistieron en cuatro dietas experimentales y un control: 1) Microcápsula con base en harina de pescado (DP), 2) Microcápsula con base en una combinación de harina de cerdo y pollo (Re), 3) Microcápsula con base en Nannochloropsis gaditana (Ng), 4) Microcápsula con base en un preparado enzimático, pancreatina (PE) el tratamiento control 5) Nauplios de Artemia (Na). Los resultados de las dietas indican que las larvas alimentadas con Nauplios (Na) tuvieron el mayor crecimiento y supervivencia (3.93 cm, 0.19 g y 82 % respectivamente). Mientras que en el caso de las larvas alimentadas con las dietas microencapsuladas, los mejores valores de supervivencia se observaron con las dietas Nannochloropsis gaditana (Ng) y preparado enzimático (PE) (20.0 y 19.2 % respectivamente). Nuestros resultados demuestran que el uso de microencapsulados para la alimentación de larvas de A. tropicus es factible, aunque se requieren más investigaciones para optimizar su diseño y elaboración para mejorar el crecimiento y supervivencia de los organismos.

Abstract Tropical gar is an important species for aquaculture purposes in Southeast Mexico. Several studies regarding nutrition and digestive physiology have been done; however, the use of microcapsules for larvae feeding to improve growth and survival has not been conducted. In this sense, four microencapsulated diets were evaluated with respect to the growth and survival of tropical gar Atractosteus tropicus larvae. The treatments consisted of four experimental diets and one control diet 1) fish meal, 2) a combination of pork and poultry meals, 3) Nannochloropsis gaditana meal, 4) enzyme preparation and the control treatment 5) Artemia nauplii. The evaluation indicated that the larvae fed the Artemia nauplii obtained the greatest growth and survival (3.93 cm, 0.19 g y 82 % respectively), which justifies a correct culture system operation. While larvae fed microencapsulated diets best values in survival were observed with diets Nannochloropsis gaditana and enzyme preparation (20.0 and 19.2 %). Our results showed that microencapsulated could be used to feed A. tropicus in feasibly form. However, more information concerning to optimize the design and manufacturing are required to improve the growth and survival of organisms. Rev. Biol. Trop. 66(3): 1298-1313. Epub 2018 September 01.