Dimitrios Charalampopoulos

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying.

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.

Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG.

Aims:  To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco‐2 cells.

A Laminated Polymer Film Formulation for Enteric Delivery of Live Vaccine and Probiotic Bacteria

Live bacterial cells (LBCs) are administered orally as attenuated vaccines to deliver biopharmaceutical agents and as probiotics to improve gastrointestinal (GI) health. However, LBCs present unique formulation challenges and must survive GI antimicrobial defenses including gastric acid after administration. We present a simple new formulation concept, termed polymer film laminate (PFL). LBCs are ambient dried onto cast acid-resistant enteric polymer films that are then laminated together to produce a solid oral dosage form. LBC of a model live bacterial vaccine and a probiotic were dried directly onto a cast film of enteric polymer. The effectiveness at protecting dried cells in a simulated gastric fluid (SGF, pH 2.0) depended on the composition of enteric polymer film used, with a blend of ethylcellulose plus Eudragit L100 55 providing greater protection from acid than Eudragit alone. However, although PFL made from blended polymer films completely released low-molecular-weight dye into intestinal conditions (pH 7.0), they failed to release LBCs. In contrast, PFL made from Eudragit alone successfully protected dried probiotic or vaccine LBC from SGF for 2 h, and subsequently released all viable cells within 60 min of transfer into simulated intestinal fluid. Release kinetics could be controlled by modifying the lamination method.

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria.

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.

Evaluating and optimizing oral formulations of live bacterial vaccines using a gastro-small intestine model

Gastrointestinal (GI) models that mimic physiological conditions in vitro are important tools for developing and optimizing biopharmaceutical formulations. Oral administration of live attenuated bacterial vaccines (LBV) can safely and effectively promote mucosal immunity but new formulations are required that provide controlled release of optimal numbers of viable bacterial cells, which must survive gastrointestinal transit overcoming various antimicrobial barriers. Here, we use a gastro-small intestine gut model of human GI conditions to study the survival and release kinetics of two oral LBV formulations: the licensed typhoid fever vaccine Vivotif comprising enteric coated capsules; and an experimental formulation of the model vaccine Salmonella Typhimurium SL3261 dried directly onto cast enteric polymer films and laminated to form a polymer film laminate (PFL). Neither formulation released significant numbers of viable cells when tested in the complete gastro-small intestine model. The poor performance in delivering viable cells could be attributed to a combination of acid and bile toxicity plus incomplete release of cells for Vivotif capsules, and to bile toxicity alone for PFL. To achieve effective protection from intestinal bile in addition to effective acid resistance, bile adsorbent resins were incorporated into the PFL to produce a new formulation, termed BR-PFL. Efficient and complete release of 4.4×10(7) live cells per dose was achieved from BR-PFL at distal intestinal pH, with release kinetics controlled by the composition of the enteric polymer film, and no loss in viability observed in any stage of the GI model. Use of this in vitro GI model thereby allowed rational design of an oral LBV formulation to maximize viable cell release.

Upgrading Food Processing Side Streams

Wheat-derived Distillers’ Dried Grains with Solubles (DDGS) and wet solids (in-process sample) were utilised as raw material for the production of biopolymers and oligosaccharides. The protein content of samples was extracted under aqueous ethanol conditions (70%, v/v) at 70 °C, in the presence of 1% (w/w) sodium metabisulfite as reducing agent. DDGS protein extracts had a protein content of ∼45% (w/w) and the wet solid-derived extracts ∼58% (w/w). The achieved protein yield for DDGS was 30.1% (w/w) whereas 55.3% (w/w) of the total protein was recovered from wet solids. The lower protein extractability from DDGS could be attributed to the decreased solubility of protein aggregates formed during the intensive thermal treatment of the drum drying stage. Protein extracts from both samples were utilised for the development of biodegradable films, with DDGS protein-derived films exhibiting a darker colour compared to wet solid ones, possibly due to heat-induced Maillard reactions during the DDGS production process. DDGS and wet solids protein films showed higher moisture sensitivity, compared to films produced using commercial gluten. This feature could be advantageous for using the films for agricultural and horticultural applications, e.g. as fertiliser release matrices and soil conditioners. Moreover, the solid residues of DDGS and wet solids after protein extraction had high carbohydrate content (49%, w/w) consisting primarily of water unextractable arabinoxylans. Enzymatic treatment of the residues with a food-grade endo-xylanase led to the production of potentially prebiotic xylo-oligosaccharide (XOS) mixtures with a degree of polymerisation (DP) of up to 7, and a XOS purity (DP≥2) of 70.2% in the DDGS hydrolysates and 51.8% in the wet solids hydrolysates.