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.     

Microencapsulation of probiotics by efficient vibration technology

The target site of action of probiotics is the intestine. They must be surviving the stomach acidic condition before reaching the target site. Three probiotic bacteria were microencapsulated in sodium alginate beads using a sophisticated microencapsulation technology provided by BÜCHI B-390. This study reports the tolerance of the different microencapsulated Lactobacillus at low pH using simulated gastric juice, comparing it with the tolerance of free bacteria. The three microencapsulated strains displayed time-dependent acid sensitivity at pH values under 3.0. At pH 2.0, a dramatic reduction in bacterial survival occurred after 5?min, with only L. casei surviving after 30?min, with 75% survival. At pH 2.5 microencapsulated L. casei survived for 90?, L. reuteri survived for 60?and L. bulgaricus survived for only 30?min, respectively. The microencapsulation technology used in this study may effectively protect Lactobacillus from gastric conditions and permit comparisons between strains.     

Novel solvent-based method for preparation of alginate beads with improved roundness and predictable size

Attempts to determine conditions or processes within alginate gel beads often suffer from inaccuracies due to an improper roundness of the analysed beads. Therefore, a novel solvent-based method for the preparation of alginate beads with improved shape was developed: An aqueous solution of 2% (w/v) alginate in water was injected into a solvent layering consisting of hexane, n-butanol, n-butanol with 1% (w/v) CaCl₂ and finally 2% (w/v) CaCl₂ in water. Beads of up to 3.5?mm in diameter obtained with this method had a roundness which was ～5% better than comparable beads prepared by dropping an alginate solution into a CaCl₂-hardening bath. This was determined by a software supported quantitative analysis of bead size and shape. Additionally, the novel solvent-based method allows for highly reproducible preparation of alginate beads with exactly predictable sizes. The biggest beads obtained with this method were 9?mm in diameter. Thus, with the solvent-based preparation of alginate beads it is now possible to easily obtain beads of exactly the type needed for a specific analytical purpose.     

High throughput encapsulation of murine fibroblasts in alginate using the JetCutter technology

The JetCutter technology originally developed for high-throughput encapsulation of particles and substances into small beads was applied in this study for the entrapment of mammalian cells in alginate beads. In contrast to other established techniques such as the air jet droplet generation or laminar jet break-up, the JetCutter is capable of working with highly viscous fluids necessary for the production of stable beads based on hydrogels. A 1.5% (w/v) sodium alginate solution containing 2.0 10 6 murine fibroblasts/ml was processed under good manufacturing practice (GMP) conditions to beads with a mean diameter of 320 #181;m. The production capacity of the JetCutter technology was 5200 beads/s or 330 ml bead suspension per h. Beads were coated with poly-L-lysine and with an additional alginate layer to produce hollow microcapsules containing living cells. The influence of this method of encapsulation on the cell viability and morphology was investigated by light microscopic techniques. Encapsulated cells showed unchanged rates of proliferation and preserved morphology. They were able to survive in culture for extended periods of time. In conclusion, the JetCutter technology seems to be well suitable for alginate bead encapsulation of living mammalian cells.     

High throughput encapsulation of murine fibroblasts in alginate using the JetCutter technology

The JetCutter technology originally developed for high-throughput encapsulation of particles and substances into small beads was applied in this study for the entrapment of mammalian cells in alginate beads. In contrast to other established techniques such as the air jet droplet generation or laminar jet break-up, the JetCutter is capable of working with highly viscous fluids necessary for the production of stable beads based on hydrogels. A 1.5% (w/v) sodium alginate solution containing 2.0 x 106 murine fibroblasts/ml was processed under good manufacturing practice (GMP) conditions to beads with a mean diameter of 320 microm. The production capacity of the JetCutter technology was 5200 beads/s or to approximately 330 ml bead suspension per h. Beads were coated with poly-L-lysine and with an additional alginate layer to produce hollow microcapsules containing living cells. The influence of this method of encapsulation on the cell viability and morphology was investigated by light microscopic techniques. Encapsulated cells showed unchanged rates of proliferation and preserved morphology. They were able to survive in culture for extended periods of time. In conclusion, the JetCutter technology seems to be well suitable for alginate bead encapsulation of living mammalian cells.     

Microentrapment of probiotic bacteria in a Ca2 +-induced whey protein gel and effects on their viability in a dynamic gastro-intestinal model

Entrapping probiotic bacteria in gels with ionic cross-linking is typically achieved with polysaccharides (alginate, pectin, carraghenan). In this study, whey proteins were used for this purpose by carrying out the Ca²⁺-induced gelation of pre-heated whey protein isolate (WPI). A Lactobacillus rhamnosus cell suspension was added in a denatured WPI solution in a 30?:?70 volume ratio. Gelation was carried out by extrusion of the cell suspension in a CaCl₂ solution. Beads of ～3?mm diameter were formed. The population in the beads was 8.0?×?10⁸?cells?g^?1. Entrapment efficiency in gel beads was 96%, with a survival level of 23%. Scanning electron microscopy of beads before freeze-drying showed a tight protein network containing encapsulated Lb. rhamnosus cells homogeneously distributed throughout the matrix. The survival to freeze-drying of the bead-entrapped cells was 41%. Viability of microentrapped cells in a dynamic gastro-intestinal (GI) model was studied and the results were compared to free cells freeze-dried in a milk-based cryoprotective solution, as well as in a pre-denatured WPI solution. Results showed that protein gelation provided protection against acidic conditions in the stomach after 90?min, as well as against bile after 30, 60 and 90?min in the duodenum. Moreover, the milk-based cryoprotective solution was equally effective after 90?min in the duodenum. It is concluded that the gelation of whey proteins induced by Ca²⁺ ions can protect the cells against adverse conditions of the GI system. However, certain stages in the entrapment process, particularly extrusion in the solution of CaCl₂, still need to be optimized in order to reduce the mortality of the cells during gelation.     

Formulation and stability evaluation of 3D alginate beads potentially useful for cumulus–oocyte complexes culture

Ovarian follicle encapsulation in synthetic or natural matrixes based on biopolymers is potentially a promising approach to in vitro maturation (IVM) process, since it maintains follicle 3D organisation by preventing its flattening and consequent disruption of gap junctions, preserving the functional relationship between oocyte and companion follicle cells. The aim of the work was to optimise physico-chemical parameters of alginate microcapsules for perspective IVM under 3D environments. On this purpose alginate and cross-linking agent concentrations were investigated. Alginate concentration between 0.75% and 0.125% w/w and Mg²⁺, Ba²⁺, Ca^2+?at concentration between 100 and 20?mM were tested. Follicle encapsulation was obtained by on purpose modified diffusion setting gelation technique, and evaluated together with beads, chemical and mechanical stability in standard and stressing conditions. Beads permeability was tested towards albumin, fetuin, pyruvate, glucose, pullulan. Results demonstrated that 0.25% alginate cross-linked in 100?mM CaCl₂ beads is suitable to follicle encapsulation.     

Novel solvent-based method for preparation of alginate beads with improved roundness and predictable size

Attempts to determine conditions or processes within alginate gel beads often suffer from inaccuracies due to an improper roundness of the analysed beads. Therefore, a novel solvent-based method for the preparation of alginate beads with improved shape was developed: An aqueous solution of 2% (w/v) alginate in water was injected into a solvent layering consisting of hexane, n-butanol, n-butanol with 1% (w/v) CaCl2 and finally 2% (w/v) CaCl2 in water. Beads of up to 3.5 mm in diameter obtained with this method had a roundness which was approximately 5% better than comparable beads prepared by dropping an alginate solution into a CaCl2-hardening bath. This was determined by a software supported quantitative analysis of bead size and shape. Additionally, the novel solvent-based method allows for highly reproducible preparation of alginate beads with exactly predictable sizes. The biggest beads obtained with this method were 9 mm in diameter. Thus, with the solvent-based preparation of alginate beads it is now possible to easily obtain beads of exactly the type needed for a specific analytical purpose.     

5-Fluorouracil encapsulated alginate beads for the treatment of breast cancer

Alginate beads containing 5-fluorouracil (5-FU) were prepared by the gelation of alginate with calcium cations. Alginate beads loaded with 5-FU were prepared at 1.0 and 2.0% (w/v) polymers. The effect of polymer concentration and the drug loading (1.0, 5.0 and 10%) on the release profile of 5-FU was investigated. As the drug load increased, larger beads were obtained in which the resultant beads contained higher 5-FU content. The encapsulation efficiencies obtained for 5-FU loads of 1.0, 5.0 and 10% (w/v) were 3.5, 7.4 and 10%, respectively. Scanning electron microscopy (SEM) and particle size analysis revealed differences between the formulations as to their appearance and size distribution. The amount of 5-FU released from the alginate beads increased with decreasing alginate concentrations.     

Synbiotic (probiotic and ginger extract) loaded floating beads: a novel therapeutic option in an experimental paradigm of gastric ulcer

Objective This study investigated the use of a bioactive phytochemical, namely ginger extract (GE), for its antioxidant and antiulcer effects, and also for supporting probiotic growth and activity. Use of probiotics is limited in therapy because of their transience and inability to survive the adverse physiological conditions of the gastrointestinal tract. Packaging probiotics in a suitably designed pharmaceutical system with GE may facilitate their establishment in the stomach mucosa. Methods A probiotic (Lactobacillus acidophilus) and GE were simultaneously and individually encapsulated/immobilized in alginate floating beads. The developed system was evaluated for diameter, buoyancy, entrapment, porosity, in‐vitro viability/release and pharmacodynamics in a cold restraint stress induced gastric ulcer model in rats. Key finding The developed floating beads stayed in the stomach for more than 10 h and both agents were released slowly and over a prolonged period from these beads. Significant and promising results were obtained for the combination (synbiotic) system in terms of ulcer index, mucus secretion, oxidative stress and histopathological parameters, as compared with the individual agents. The developed system could completely revert the damage induced in ulcerated stomachs at physiological (ulcer index and mucus secretion), biochemical (oxidative stress) and histological levels. Conclusion This study establishes that suitable packaging of GE and Lactobacillus acidophilus together in floating beads can help exploit their prospects as therapeutic curative agents rather than potential preventive agents.     

Release characteristics of brilliant blue from calcium-alginate beads coated with quaternized chitosan

Calcium-alginate beads coated with quaternized chitosan were prepared in a neutral environment, and morphologies were observed by SEM. Optimum conditions for the encapsulation and retention of a model drug (brilliant blue, BB) in acid were obtained from studies of preparation conditions, including alginate and quaternized chitosan concentration, calcium chloride (CaCl2) concentration in the gelling medium and by comparing one-step and two-step preparation methods. Results showed that very high BB encapsulation efficiency (99%, w/w) and low leakage in acid (8%, w/w) was achieved from dry beads when 2.0% (w/v) alginate was dropped into 1.0% (w/v) CaCl2 containing 0.3% (w/v) quaternized chitosan by a one-step method. The release of BB in 0.9% (w/v) NaCl was modulated by coating calcium-alginate with different weight average molecule weight (Mw) and degree of substitution (DS) of quaternized chitosan. A decreased of Mw accelerated the release of BB and a high DS value significantly decreased the release in 0.9% (w/v) NaCl.     

Mucoadhesive and floating chitosan-coated alginate beads for the controlled gastric release of amoxicillin

This work focused on the development of mucoadhesive and floating chitosan-coated alginate beads as a gastroretensive delivery vehicle for amoxicillin, towards the effective eradication of Helicobacter pylori, a major causative agent of peptic ulcers. Alginate was used as the core bead core polymer and chitosan as the mucoadhesive polymer coating. Amoxicillin-loaded alginate beads coated with 0.5% (w/v) chitosan (ALG/0.5%CHI) exhibited excellent floating ability, high encapsulation efficiency, high drug loading capacity, and a strong in vitro mucoadhesion to the gastric mucosal layer. In vitro, amoxicillin was released faster in simulated gastric fluid (pH 1.2, HCl) than in simulated intestinal fluid (phosphate buffer, pH 7.4). ALG/0.5%CHI could be prepared with a > 90% drug encapsulation efficiency and exhibited more than 90% muco-adhesiveness, 100% floating ability, and achieved sustained release of amoxicillin for over six hours in SGF.     

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In an attempt to fabricate floating beads of ciprofloxacin, drugloaded alginate beads were prepared by simultaneous external and internal gelation. The effect of blending of alginate with gellan, hydroxypropyl methylcellulose, starch, and chitosan on the bead properties were evaluated. Beads were spherical with incorporation efficiency in the range of 52.81 ± 2.64 to 78.95 ± 1.92%. Beads exhibited buoyancy over a period of 7–24 hr based on the formulation variables. In vitro release of ciprofloxacin from the alginate beads in simulated gastric fluid (SGF) (0.1 N HCl, pH 1.2), was influenced significantly (p < 0.001) by the properties and concentration of additives. Among the polymers incorporated into alginate beads. Hydroxy propyl methylcellulose (HPMC) provided an extended release over 7 hr. The drug release predominately followed Higuchi's square root model.     

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

Abstract

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.     

Sulindac loaded alginate beads for a mucoprotective and controlled drug release

Ionotropic gelation was used to entrap sulindac into calcium alginate beads as a potential drug carrier for the oral delivery of this anti-inflammatory drug. Beads were investigated in vitro for a possible sustained drug release and their use in vivo as a gastroprotective system for sulindac. Process parameters such as the polymer concentration, polymer/drug ratio, and different needle diameter were analysed for their influences on the bead properties. Size augmented with increasing needle diameter (0.9 mm needle: 1.28 to 1.44 mm; 0.45 mm needle: 1.04 to 1.07 mm) due to changes in droplet size as well as droplet viscosity. Yields varied between 87% and 98% while sulindac encapsulation efficiencies of about 88% and 94% were slightly increasing with higher alginate concentrations. Drug release profiles exhibited a complete release for all formulations within 4 hours with a faster release for smaller beads. Sulindac loaded alginate beads led to a significant reduction of macroscopic histological damage in the stomach and duodenum in mice. Similarly, microscopic analyses of the mucosal damage demonstrated a significant mucoprotective effect of all bead formulation compared to the free drug. The present alginate formulations exhibit promising properties of a controlled release form for sulindac; meanwhile they provide a distinct tissue protection in the stomach and duodenum.     

Microentrapment of probiotic bacteria in a Ca(2+)-induced whey protein gel and effects on their viability in a dynamic gastro-intestinal model

Entrapping probiotic bacteria in gels with ionic cross-linking is typically achieved with polysaccharides (alginate, pectin, carraghenan). In this study, whey proteins were used for this purpose by carrying out the Ca(2+)-induced gelation of pre-heated whey protein isolate (WPI). A Lactobacillus rhamnosus cell suspension was added in a denatured WPI solution in a 30 : 70 volume ratio. Gelation was carried out by extrusion of the cell suspension in a CaCl(2) solution. Beads of approximately 3 mm diameter were formed. The population in the beads was 8.0 x 10(8) cells g(-1). Entrapment efficiency in gel beads was 96%, with a survival level of 23%. Scanning electron microscopy of beads before freeze-drying showed a tight protein network containing encapsulated Lb. rhamnosus cells homogeneously distributed throughout the matrix. The survival to freeze-drying of the bead-entrapped cells was 41%. Viability of microentrapped cells in a dynamic gastro-intestinal (GI) model was studied and the results were compared to free cells freeze-dried in a milk-based cryoprotective solution, as well as in a pre-denatured WPI solution. Results showed that protein gelation provided protection against acidic conditions in the stomach after 90 min, as well as against bile after 30, 60 and 90 min in the duodenum. Moreover, the milk-based cryoprotective solution was equally effective after 90 min in the duodenum. It is concluded that the gelation of whey proteins induced by Ca(2+) ions can protect the cells against adverse conditions of the GI system. However, certain stages in the entrapment process, particularly extrusion in the solution of CaCl(2), still need to be optimized in order to reduce the mortality of the cells during gelation.     

In vitro and in vivo studies of ibuprofen-loaded biodegradable alginate beads

The irritation effects of ibuprofen, a widely used non-steroidal anti-inflammatory drug (NSAID), were evaluated on mouse gastric and duodenal mucosa when suspended in 0.5% (w/v) sodiumcarboxymethylcellulose (NaCMC) solution and loaded in alginate beads. The ionotropic gelation method was used to prepare controlled release alginate beads of ibuprofen. The influence of various formulation factors on the encapsulation efficiency, as in vitro drug release and micromeritic properties, was investigated. Other variables included the alginate concentration, percentage drug loading and stirring speed during the microencapsulation process. Scanning electron micrographs of alginate beads loaded with ibuprofen showed rough surface morphology and particle sizes in the range of 1.15 +/- 0.4 - 3.15 +/- 0.6 mm. The yield of microspheres, as collected after drying, was generally 80-90%. Formulation code H showing t50% value of 3.5 h was chosen for in vivo trials because of the appropriate drug release properties. For in vivo trials, free ibuprofen (100 mg kg(-1)), blank and ibuprofen (100 mg kg(-1)) loaded alginate beads (formulation code H) were suspended in 0.5% (w/v) NaCMC solution and each group was given to six mice orally by gavage. NaCMC solution was used as a control in experimental studies. In vivo data showed that the administration of ibuprofen in alginate beads prevented the gastric lesions.     

Calcium gluconate as cross-linker improves survival and shelf life of encapsulated and dried Metarhizium brunneum and Saccharomyces cerevisiae for the application as biological control agents

Calcium chloride (CC) is the most common cross-linker for the encapsulation of biocontrol microorganisms in alginate beads. The aim of this study was to evaluate if calcium gluconate (CG) can replace CC as cross-linker and at the same time improve viability after drying and rehydration, hygroscopic properties, shelf life and nutrient supply. Hence, the biocontrol fungi Metarhizium brunneum and Saccharomyces cerevisiae were encapsulated in Ca-alginate beads supplemented with starch. Beads were dried and maximum survival was found in beads cross-linked with CG. Beads prepared with CG showed lower hygroscopic properties, but a higher shelf life for encapsulated fungi. Moreover, we demonstrated that gluconate has a nutritive effect on encapsulated fungi, leading to increased mycelium growth of M. brunneum and to enhanced CO₂ release from beads containing Saccharomyces cerevisiae. The application of CG as cross-linker will pave the way towards increasing drying survival and shelf life of various, especially drying-sensitive microbes.     

Antifungal and anti-aflatoxigenic effect of probiotics against Aspergillus flavus and Aspergillus parasiticus

Aflatoxins are carcinogenic mycotoxin, produced by Aspergillus species. These molds infect food crops in warm humid conditions causing economic losses and affecting the consumers' health adversely. In this study, antifungal activity and aflatoxin inhibiting ability of four probiotic strains against Aspergillus flavus and Aspergillus parasiticus were studied. The aflatoxin secreted was analyzed and quantified by both UV spectrophotometer and HPLC. It was found that Lactobacillus delbrueckii subsp. lactis showed maximal antifungal (67.43% reduction) and anti-aflatoxigenic (94.33% reduction) activity against A. flavus whereas A. parasiticus was inhibited by Lactobacillus brevis with the antifungal reduction of 69.38% and anti-aflatoxigenic reduction of 96.12%.     

Lyophilised Vegetal BM 297 ATO-Inulin lipid-based synbiotic microparticles containing Bifidobacterium longum LMG 13197: design and characterisation

Abstract

This study aimed at the manufacturing and characterisation of Vegetal BM 297 ATO-inulin-Bifidobacterium longum LMG 13197 microparticles prepared by freeze drying. Emulsions containing 1%, 1.5%, 2%, 3.5% or 5% w/v inulin were prepared, with or without centrifugation before freeze drying. Morphological properties, particle size distribution, encapsulation efficiency of the microparticles and their ability to preserve viability of the enclosed B. longum LMG 13197 cells were evaluated. The microparticles produced from both formulations without a centrifugation step were irregular, porous with concavities and contained high number of bacterial cells. Formulations with or without inulin had average particle sizes of 33.4–81.0?μm with encapsulation efficiencies of 82% and 88%, respectively. Vegetal-inulin microparticles have the morphology and size that will enable their even distribution in final food products, and hence, they have the potential for use as a functional food additive because they are likely to deliver sufficient numbers of viable bacteria.