Dimitris Charalampopoulos

Microencapsulation of probiotics for gastrointestinal delivery

The administration of probiotic bacteria as nutraceuticals is an area that has rapidly expanded in recent years, with a global market worth

Evaluation of the effect of malt, wheat and barley extracts on the viability of potentially probiotic lactic acid bacteria under acidic conditions

32.6 billion predicted by 2014. Many of the health promoting claims attributed to these bacteria are dependent on the cells being both viable and sufficiently numerous in the intestinal tract. The oral administration of most bacteria results in a large loss of viability associated with passage through the stomach, which is attributed to the high acid and bile salt concentrations present. This loss of viability effectively lowers the efficacy of the administered supplement. The formulation of these probiotics into microcapsules is an emerging method to reduce cell death during GI passage, as well as an opportunity to control release of these cells across the intestinal tract. The majority of this technology is based on the immobilization of bacteria into a polymer matrix, which retains its structure in the stomach before degrading and dissolving in the intestine, unlike the diffusion based unloading of most controlled release devices for small molecules. This review shall provide an overview of progress in this field as well as draw attention to areas where studies have fallen short. This will be followed by a discussion of emerging trends in the field, highlighting key areas in which further research is necessary.

Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices.

In this work, the effect of cereal extracts, used as delivery vehicles for potentially probiotic lactic acid bacteria (LAB), on the acid tolerance of the cells was evaluated under conditions that simulate the gastric tract. More specifically, the effect of malt, barley and wheat extracts on the viability of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri during exposure for 4 h in a phosphate buffer acidified at pH 2.5 was investigated. In the absence of cereal extracts all strains demonstrated a significant reduction in their cell population, particularly L. plantarum. The viability of L. plantarum was improved by approximately 4 log(10) cycles in the presence of malt and 3 log(10) cycles in the presence of wheat and barley. The survival of L. acidophilus and L. reuteri was increased by more than 1.5 and 0.7 log(10) cycle, respectively, upon addition of cereal extracts. In order to evaluate the contribution of the cereal constituents on cell survival, the individual effect of glucose, maltose and free amino nitrogen (FAN), which were added at concentrations that correlated to the reducing sugar and FAN content of the cereal extracts, was examined. The viability of L. plantarum was progressively improved as the maltose or glucose concentration increased; an increase by approximately 2 log(10) cycles was observed in the presence of 8.33 g/l sugar. The survival of L. acidophilus increased by more than 1 log(10) cycle, even at very low concentrations of maltose and glucose (e.g., 0.67 g/l), while L. reuteri stability was enhanced in the presence of maltose but no appreciable effect was demonstrated in the presence of glucose. Sugar analysis indicated that glycolysis was inhibited in all cases. Addition of tryptone and yeast extract, used as sources of FAN, enhanced L. acidophilus acid tolerance, but did not affect L. reuteri and L. plantarum. The results presented in this study indicate that malt, wheat and barley extracts exhibit a significant protective effect on the viability of L. plantarum, L. acidophilus and L. reuteri under acidic conditions, which could be mainly attributed to the amount of sugar present in the cereal extracts.

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

Due to the fact that probiotic cells need to be alive when they are consumed, culture-based analysis (plate count) is critical in ascertaining the quality (numbers of viable cells) of probiotic products. Since probiotic cells are typically stressed, due to various factors related to their production, processing and formulation, the standard methodology for total plate counts tends to underestimate the cell numbers of these products. Furthermore, products such as microencapsulated cultures require modifications in the release and sampling procedure in order to correctly estimate viable counts. This review examines the enumeration of probiotic bacteria in the following commercial products: powders, microencapsulated cultures, frozen concentrates, capsules, foods and beverages. The parameters which are specifically examined include: sample preparation (rehydration, thawing), dilutions (homogenization, media) and plating (media, incubation) procedures. Recommendations are provided for each of these analytical steps to improve the accuracy of the analysis. Although the recommendations specifically target the analysis of probiotics, many will apply to the analysis of commercial lactic starter cultures used in food fermentations as well.

Production and evaluation of dry alginate-chitosan microcapsules as an enteric delivery vehicle for probiotic bacteria.

Aims: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate.

Prebiotics in foods.

This study investigates the production of alginate microcapsules, which have been coated with the polysaccharide chitosan, and evaluates some of their properties with the intention of improving the gastrointestinal viability of a probiotic ( Bifidobacterium breve ) by encapsulation in this system. The microcapsules were dried by a variety of methods, and the most suitable was chosen. The work described in this Article is the first report detailing the effects of drying on the properties of these microcapsules and the viability of the bacteria within relative to wet microcapsules. The pH range over which chitosan and alginate form polyelectrolyte complexes was explored by spectrophotometry, and this extended into swelling studies on the microcapsules over a range of pHs associated with the gastrointestinal tract. It was shown that chitosan stabilizes the alginate microcapsules at pHs above 3, extending the stability of the capsules under these conditions. The effect of chitosan exposure time on the coating thickness was investigated for the first time by confocal laser scanning microscopy, and its penetration into the alginate matrix was shown to be particularly slow. Coating with chitosan was found to increase the survival of B. breve in simulated gastric fluid as well as prolong its release upon exposure to intestinal pH.

Survival of Lactobacillus plantarum in model solutions and fruit juices.

A wealth of information has been gathered over the past 15 years on prebiotics through experimental, animal and human studies, with the aim to understand the mechanism of actions and elucidate their beneficial health effects to the human host. Significant amount of evidence exists for their ability to increase the bioavailability of minerals and stimulate the immune system, although there is less clear evidence so far for their prophylactic or therapeutic role in gastrointestinal infections. Moreover, the effect of the food delivery vehicle on the efficacy of prebiotics is an area that has been hardly investigated. Besides their beneficial effects, prebiotics influence the textural and organoleptic properties of the food products, such as dairy and baked products. To do this however, they need to be stable during food processing, in particular under conditions of high temperature and low pH.

Chitosan coated alginate beads for the survival of microencapsulated Lactobacillus plantarum in pomegranate juice

The aim of the work was to study the survival of Lactobacillus plantarum NCIMB 8826 in model solutions and develop a mathematical model describing its dependence on pH, citric acid and ascorbic acid. A Central Composite Design (CCD) was developed studying each of the three factors at five levels within the following ranges, i.e., pH (3.0-4.2), citric acid (6-40 g/L), and ascorbic acid (100-1000 mg/L). In total, 17 experimental runs were carried out. The initial cell concentration in the model solutions was approximately 1 × 10(8)CFU/mL; the solutions were stored at 4°C for 6 weeks. Analysis of variance (ANOVA) of the stepwise regression demonstrated that a second order polynomial model fits well the data. The results demonstrated that high pH and citric acid concentration enhanced cell survival; one the other hand, ascorbic acid did not have an effect. Cell survival during storage was also investigated in various types of juices, including orange, grapefruit, blackcurrant, pineapple, pomegranate, cranberry and lemon juice. The model predicted well the cell survival in orange, blackcurrant and pineapple, however it failed to predict cell survival in grapefruit and pomegranate, indicating the influence of additional factors, besides pH and citric acid, on cell survival. Very good cell survival (less than 0.4 log decrease) was observed after 6 weeks of storage in orange, blackcurrant and pineapple juice, all of which had a pH of about 3.8. Cell survival in cranberry and pomegranate decreased very quickly, whereas in the case of lemon juice, the cell concentration decreased approximately 1.1 logs after 6 weeks of storage, albeit the fact that lemon juice had the lowest pH (pH~2.5) among all the juices tested. Taking into account the results from the compositional analysis of the juices and the model, it was deduced that in certain juices, other compounds seemed to protect the cells during storage; these were likely to be proteins and dietary fibre In contrast, in certain juices, such as pomegranate, cell survival was much lower than expected; this could be due to the presence of antimicrobial compounds, such as phenolic compounds.

Layer-by-layer coating of alginate matrices with chitosan-alginate for the improved survival and targeted delivery of probiotic bacteria after oral administration

This work studied the effect of multi-layer coating of alginate beads on the survival of encapsulated Lactobacillus plantarum in simulated gastric solution and during storage in pomegranate juice at 4°C. Uncoated, single and double chitosan coated beads were examined. The survival of the cells in simulated gastric solution (pH 1.5) was improved in the case of the chitosan coated beads by 0.5-2 logs compared to the uncoated beads. The cell concentration in pomegranate juice after six weeks of storage was higher than 5.5logCFU/mL for single and double coated beads, whereas for free cells and uncoated beads the cells died after 4 weeks of storage. In simulated gastric solution, the size of the beads decreased and their hardness increased with time; however, the opposite trend was observed for pomegranate juice, indicating that there is no correlation between cell survival and the hardness of the beads.

In Vitro Fermentation of Oat Bran Obtained by Debranning with a Mixed Culture of Human Fecal Bacteria

The oral administration of probiotic bacteria has shown potential in clinical trials for the alleviation of specific disorders of the gastrointestinal tract. However, cells must be alive in order to exert these benefits. The low pH of the stomach can greatly reduce the number of viable microorganisms that reach the intestine, thereby reducing the efficacy of the administration. Herein, a model probiotic, Bifidobacterium breve, has been encapsulated into an alginate matrix before coating in multilayers of alternating alginate and chitosan. The intention of this formulation was to improve the survival of B. breve during exposure to low pH and to target the delivery of the cells to the intestine. The material properties were first characterized before in vitro testing. Biacore™ experiments allowed for the polymer interactions to be confirmed; additionally, the stability of these multilayers to buffers simulating the pH of the gastrointestinal tract was demonstrated. Texture analysis was used to monitor changes in the gel strength during preparation, showing a weakening of the matrices during coating as a result of calcium ion sequestration. The build-up of multilayers was confirmed by confocal laser-scanning microscopy, which also showed the increase in the thickness of coat over time. During exposure to in vitro gastric conditions, an increase in viability from <3 log(CFU) per mL, seen in free cells, up to a maximum of 8.84 ± 0.17 log(CFU) per mL was noted in a 3-layer coated matrix. Multilayer-coated alginate matrices also showed a targeting of delivery to the intestine, with a gradual release of their loads over 240 min.