Jean-Michel Cardot

Coated whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast

Viable Saccharomyces boulardii, used as a biotherapeutic agent, was encapsulated in food-grade whey protein isolate (WP) and alginate (ALG) microparticles, in order to protect and vehicle them in gastrointestinal environment. Yeast-loaded microparticles with a WP/ALG ratio of 62/38 were produced with high encapsulation efficiency (95%) using an extrusion/cold gelation method and coated with ALG or WP by a simple immersion method. Swelling, yeast survival, WP loss and yeast release in simulated gastric and intestinal fluids (SGF and SIF, pH 1.2 and 7.5) with and without their respective digestive enzymes (pepsin and pancreatin) were investigated. In SGF, ALG network shrinkage limited enzyme diffusion into the WP/ALG matrix. Coated and uncoated WP/ALG microparticles were resistant in SGF even with pepsin. Survival of yeast cells in microparticles was 40% compared to 10% for free yeast cells and was improved to 60% by coating. In SIF, yeast cell release followed coated microparticle swelling with a desirable delay. Coated WP/ALG microparticles appear to have potential as oral delivery systems for Saccharomyces boulardii or as encapsulation means for probiotic cells in pharmaceutical or food processing applications.

Effect of a New Probiotic Saccharomyces cerevisiae Strain on Survival of Escherichia coli O157:H7 in a Dynamic Gastrointestinal Model

ABSTRACT Survival of Escherichia coli O157:H7 was investigated using a dynamic gastrointestinal model. A high bacterial mortality was observed in the stomach and duodenum. In contrast, bacteria grew in the distal parts of the small intestine. The coadministration of Saccharomyces cerevisiae CNCM I-3856 led to a significant reduction of bacterial resumption, maybe through ethanol production.

Investigation of the Biopharmaceutical Behavior of Theophylline Hydrophilic Matrix Tablets Using USP Methods and an Artificial Digestive System

This work aimed to investigate the biopharmaceutical behavior of hydrophilic matrix tablets of theophylline using different in vitro methods: USP II, USP IV, and a novel in vitro system simulating the gastrointestinal tract in man called the artificial digestive system (ADS). The potentiality of each method was evaluated by establishing in vitro/in vivo correlation. Using USP methods, the drug release was pH-independent and dependent on agitation intensity. Level A IVIVCs could be established using the different in vitro methods but one to one correlation was established only when the ADS method was used. For the prediction of in vivo drug dosage form behavior based on in vitro methods, the ADS showed a high predictability when compared to USP in vitro methods.

Efficacy of mucoadhesive hydrogel microparticles of whey protein and alginate for oral insulin delivery.

ABSTRACTPurposeTo evaluate the efficacy of mucoadhesive insulin-loaded whey protein (WP) /alginate (ALG) microparticles (MP) for oral insulin administration.MethodsInsulin-loaded microparticles (ins-MP) made of whey protein and alginate were prepared by a cold gelation technique and an adsorption method, without adjunction of organic solvent in order to develop a biocompatible vehicle for oral administration of insulin. In vitro characterization, evaluations of ins-MP in excised intestinal tissues and hypoglycaemic effects after intestinal administration in healthy rats were performedResultsThe release properties and swelling behaviors, investigated in different pH buffers, demonstrated a release based on diffusion mechanism following matrix swelling. Mucoadhesion studies in rabbits and insulin transport experiments with excised intestinal rat tissues revealed that encapsulation in microparticles with mucoadhesive properties promotes insulin absorption across duodenal membranes and bioactivity protection. In vivo experiments reinforced the interest of encapsulation in whey protein/alginate combination. Confocal microscopic observations associated with blood glucose levels bring to light duodenal absorption of insulin biologically active following in vivo administration.ConclusionsInsulin-loaded WP/ALG MP with high quantities of drug entrapped, in vitro matrix swelling and protective effect as well as excellent mucohadesive properties was developped. Improvement of intestinal delivery of insulin and increased in bioavailability were recorded.

Development and in vitro characterization of insulin loaded whey protein and alginate microparticles

Insulin was encapsulated into microparticles (MP) made of denaturized whey proteins (WP) and alginate (ALG) using an extrusion/cold gelation process with calcium ions. High encapsulation efficiency of 85% was obtained. Influence of insulin on polymeric viscosity and on microparticle behavior was evaluated. Insulin seemed to interact with WP chains by non covalent binding and steric hindrance. This influence was balanced by ALG addition. Nevertheless, insulin was released rapidly by diffusion at both acidic and intestinal dissolution media. Despite this fast in vitro release, WP/ALG MP showed an important enzymatic inhibition effect on trypsin and alpha-chymotrypsin. Thus, WP/ALG MP contributed to an effective insulin protection towards enzymatic degradation. The aforementioned results suggested that WP based microparticles are a promising carrier for improving oral delivery of insulin.

Investigation of coated whey protein/alginate beads as sustained release dosage form in simulated gastrointestinal environment

Aim: The biopharmaceutical behavior of new formulations based on both food-grade polymers, whey protein (WP) and alginate (ALG) was studied using different in vitro methods. The Biopharmaceutical Classification System (BCS) class I drug Theophylline was chosen as drug model. Method: Drug release was studied (i) at pH 1.2 (2 hours) followed by pH 7.5, and in simulated gastric fluid (SGF; 2 hours) followed by simulated intestinal fluid (SIF) using the paddle method and (ii) in an artificial digestive system. Results: Freeze-dried mixed WP/ALG (62/38) beads were coated with WP or ALG with encapsulation efficiency 34.9% and 18.3%, respectively. At pH 1.2, coated beads exhibited gastroresistant properties (< 10% of drug released after 2 hours) followed at pH 7.5 by a sustained release behavior (< 60% of drug released at 24 hours) controlled by an erosion mechanism. In SGF, despite enzyme hydrolysis, drug release was still controlled due to ALG shrinkage. After transfer in SIF, formulations were completely degraded in less than 2 h with total drug release. In an artificial digestive system, coated beads appeared gastroresistant, intestinal part sustained drug release was controlled by erosion. Conclusion: Combination of in vitro methods allowed prediction of the in vivo potentialities of WP- and ALG- coated WP/ALG beads as oral sustained release systems.

Three-stage continuous culture system with a self-generated anaerobia to study the regionalized metabolism of the human gut microbiota

The technical and ethical difficulties in studying the gut microbiota in vivo warrant the development and improvement of in vitro systems able to simulate and control the physicochemical factors of the gut biology. Moreover, the functional regionalization of this organ implies a model simulating these differences. Here we propose an improved and alternative three-stage continuous bioreactor called 3S-ECSIM (three-stage Environmental Control System for Intestinal Microbiota) to study the human large intestine. Its main feature compared with other in vitro systems is the anaerobic atmosphere originating directly from the microbiota metabolism, leading to different gas ratios of CO2 and H2 in each compartment. Analyses of the metabolic and microbiological profiles (LC-MS and a phylogenetic microarray) show different profiles together with a maintenance of this differentiation between the three compartments, simulating respectively a proximal, a transversal and a distal colon. Moreover, the last reactor presents a high similarity with the initial fecal sample, at the microbiological diversity level. Based on our results, this in-vitro process improvement is a valuable alternative tool to dynamically study the structure and metabolism of gut microbiota, and its response to nutrients, prebiotics, probiotics, drugs or xenobiotics.

Development and characterization of coated-microparticles based on whey protein/alginate using the Encapsulator device

The aim of this study is to prepare whey protein (WP)-based microparticles (MP) using the Encapsulator® device. The viscosity dependence of the extrusion device required to mix WP with a food-grade and less viscous polymer. Mixed WP/ALG MP were obtained with the optimized WP/alginate (ALG) ratio (62/38). These particles were further coated with WP or ALG using non-traumatic and solvent-free coating process developed in this study. Size and morphology of coated and uncoated MP were determined. Then, swelling and degradation (WP release) of formulations were investigated in pH 1.2 and 7.5 buffers and in simulated gastric and intestinal fluids (SGF, SIF) and compared to pure ALG and pure WP particle behaviours. At pH 1.2, pure ALG shrank and pure WP swelled, whereas the sizes of mixed WP/ALG matrix were stable. In SGF, WP/ALG MP resisted to pepsin degradation compare to pure WP particles due to ALG shrinkage which limited pepsin diffusion within particles. Coating addition with WP or ALG slowed down pepsin degradation. At pH 7.5, WP/ALG particles were rapidly degraded due to ALG sensitivity but the addition of a WP coating limited effectively the swelling and the degradation of MP. In SIF, pancreatin accelerated MP degradation but ALG-coated MP exhibited interesting robustness. These results confirmed the interest and the feasibility to produce coated WP-based MP which could be a potential orally controlled release drug delivery system.

Use of Artificial Digestive Systems to Investigate the Biopharmaceutical Factors Influencing the Survival of Probiotic Yeast During Gastrointestinal Transit in Humans

PurposeTo evaluate the influence of the main biopharmaceutical factors on the viability of a new probiotic yeast strain, using dynamic in vitro systems simulating human gastric/small intestinal (TIM) and large intestinal (ARCOL) environments.MethodsThe viability of Saccharomyces cerevisiae CNCM I-3856 throughout the artificial digestive tract was determined by microbial counting. We investigated the effects of galenic formulation, food intake, dose, mode and frequency of administration on yeast survival rate.ResultsIn both fasted and fed states, yeast viability in the upper digestive tract was significantly higher when the probiotic was administered in hydroxypropylmethylcellulose (HPMC) capsules compared to tablets. Food intake led to a delay in yeast release and a two-fold increase in strain survival. Whatever the dose, yeasts were particularly sensitive to the large intestinal environment. High concentrations of probiotic could only be maintained in the colon when it was inoculated twice a day over a 5-h-period.ConclusionsTIM and ARCOL are complementary in vitro tools relevant for screening purposes, supplying valuable information on the effects of galenic form, food intake and dose regimen on the viability of probiotics throughout the human digestive tract.

Development and Validation of a Continuous In Vitro System Reproducing Some Biotic and Abiotic Factors of the Veal Calf Intestine

ABSTRACT Following the January 2006 European ban of antibiotics used as growth promoters in the veal calf industry, new feed additives are needed in order to maintain animal health and growth performance. As an alternative to in vivo experiments in the testing of such additives, an in vitro system modeling the intestinal ecosystem of the veal calf was developed. Stabilization of the main cultured microbial groups and their metabolic activity were tracked in an in vitro continuous fermentor operated under anaerobiosis, at pH 6.5, and at a temperature of 38.5°C and supplied with one of three different nutritive media (M1, M2, or M3). These media mainly differed in their concentrations of simple and complex carbohydrates and in their lipid sources. In vitro microbial levels and fermentative metabolite concentrations were compared to in vivo data, and the biochemical composition of the nutritive media was compared to that of the veal calf intestinal content. All three nutritive media were able to stabilize anaerobic and facultative anaerobic microflora, lactate-utilizing bacteria, bifidobacteria, lactobacilli, enterococci, and Bacteroides fragilis group bacteria at levels close to in vivo values. The microbiota was metabolically active, with high concentrations of lactate, ammonia, and short-chain fatty acids found in the fermentative medium. Comparison with in vivo data indicated that M3 outperformed M1 and M2 in simulating the conditions encountered in the veal calf intestine. This in vitro system would be useful in the prescreening of new feed additives by studying their effect on the intestinal microbiota levels and fermentative metabolite production.