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Use of Microorganisms for Carotenoids Delivery
Next Generation of Probiotics for Cardiovascular Disease
The CaroDel project lasted 2 years, ended in January 2016 and received €2 million in financial support from the EU. The aims of the project were to develop an efficient oral delivery strategy for highly active carotenoids and to evaluate the potential beneficial (probiotic) effect on health of a Bacillus delivery vehicle, with the ultimate aim of improving biomarkers associated with cardiovascular disease. The CaroDel consortium of eight partners (including five SMEs) from six European countries brought together complementary expertise and was coordinated by ProDigest BVBA, Belgium, a spin-off of Ghent University.
Characterized by their orange, yellow and red pigments, carotenoids are mainly synthesized in plants but also have been isolated from other organisms, including some bacteria and fungi. Humans, like other animals, are unable to synthesize carotenoids but absorb them from their diet. Over 700 different carotenoids have been described, but only a few have been studied in relation to their impact on human physiology. Carotenoids act as antioxidants within the body, protecting against cellular damage, ageing and even some chronic diseases (including cardiovascular disease). Plant-derived carotenoids are widely associated with cardiovascular benefits, yet their low stability and poor bioavailability (absorption in the human body) hamper successful product development.
Several bacteria from the Bacillus species were shown to produce carotenoids which are highly stable throughout the gut and show higher antioxidant activity and bioavailability than common dietary carotenoids.
These discoveries provided strong and compelling reasons for supporting further development and commercialization of these bacteria-derived carotenoids.
By combining in vitro gut models and in vivo animal studies, the CaroDel project developed an efficient oral delivery system for these highly active carotenoids, in the form of Bacillus spores which can also exert probiotic effects.
CaroDel aimed to valorize the results of the earlier FP7 COLORSPORE project, in which initial isolation and characterization of Bacillus strains producing gastric-stable carotenoids was carried out. As particular Bacillus carotenoids were shown to have better stability in the gastrointestinal tract (GIT), antioxidant activity and bioavailability than common dietary carotenoids, the COLORSPORE project provided strong and compelling evidence to support further development and commercialization of these bacteria-derived carotenoids.
The CaroDel project therefore focused on developing an efficient oral delivery strategy for these highly active carotenoids, and on evaluating the potential direct (probiotic) beneficial effects on health of the Bacillus delivery vehicle, with the ultimate goal of improving biomarkers associated with cardiovascular disease (CVD).
The study compared the effective delivery of the carotenoids in the human body following administration as (i) vegetative Bacillus cells, (ii) Bacillus spores or (iii) extracted bacterial carotenoids. In parallel, the ability of the Bacillus strain to exert bona fide effects (i.e. effects on the host microbiota, metabolism and immunity) was investigated using in vitro gut models and in vivo rat studies. Based on the results, the best delivery strategy was selected and validated in a human study, in which carotenoid bioavailability was assessed as well as endpoints related to CVD biomarkers and potential probiotic activity. In combination with a full safety assessment, a proof-of-concept production strategy and an exploitation plan, the scientific evidence compiled in this project provides a framework for efficient further commercialization of a well-characterized Bacillus carotenoid product.
The main objectives of the project were:
1. To optimize the pilot scale production process of the strain and its carotenoids;
2. To determine the best delivery approach for the carotenoids in the human GIT by means of validated in vitro gut models and in vivo rat studies;
3. To determine the effect of the carotenoids and the Bacillus strain on host endpoints and the composition/activity of the gut microbial community;
4. To demonstrate that the selected Bacillus strain is safe for human consumption, which will allow future registration under quality and patient safety (QPS) regulations;
5. To model the scale-up of the production process in order to bring the product to market;
6. To evaluate the effect of the Bacillus strain in humans in relation to CVD biomarkers, modulation of the intestinal environment and host health endpoints; and
7. To generate the necessary knowledge to develop an intellectual property (IP) protection strategy and a business model to commercialize and sustain the product.
To address these objectives, the CaroDel project was divided in two phases: Phase 1 (selection of the best formulation) and Phase 2 (translational phase for taking the product to market), by combining five research work packages (WPs).
During Phase 1 of the CaroDel project, WP2, WP3 and WP4 were run in parallel (WP1 was a management work package).
WP2 (‘Production’) was designed to optimize the production of the different carotenoid formulations that were tested in the other research WPs.
WP3 (‘Evaluation of the carotenoids’ delivery and effect in the GIT’) was designed to elucidate – through in vitro research and animal studies – the intestinal fate and bioavailability of the carotenoids when administered as purified carotenoids extracted from the Bacillus strain, when contained in the vegetative cells, or when contained in the spores of the strain. The effects of the Bacillus strain on the intestinal environment were also assessed upon administration as spores or vegetative cells. The aim of this part of the project was to determine the optimal formulation to be used in the human trial, in terms of carotenoid bioavailability profile and, if possible, potential to modulate the intestinal environment.
In parallel, in WP4 (‘Evaluation of the safety of the carotenoid-producing Bacillus strain’), all the steps necessary to demonstrate the safety of the carotenoids and the carotenoid-producing strain in relation to novel food and QPS regulations, respectively (e.g. toxicology, antibiotic resistance, genome sequencing, and characterization of the carotenoids) were performed.
In Phase 2 of the project, the best formulation identified in WP3 and supported by the data on safety determined in WP4 was tested in a human study within WP5 (‘Human intervention trial’). Analyses were conducted by the different partners to determine the bioavailability of the ingested carotenoids, the effects on the human host in terms of CVD biomarkers, and the impact on gastrointestinal microbial composition and metabolic activity.
Finally, in WP6 (‘Regulatory and life cycle assessment’), the consortium defined the regulatory strategy, evaluated consumer perception of the novel concept, identified the necessary additional R&D steps and, finally, prepared a life cycle assessment plan. In WP7, an IP strategy was developed to protect the outcomes of the project and exploit the results.
In summary, successful achievement of the above objectives has brought the Bacillus carotenoid product close to commercialization and exploitation as a unique health ingredient.
Main results achieved
In year 1, the CaroDel consortium devoted its efforts to the production of the different formulations of carotenoids and the carotenoid-producing Bacillus strain, and to the determination of the optimal formulation to be used in the human trial. As the carotenoid-producing Bacillus strain did not encounter viability problems, the extracted bacterial carotenoids were excluded from the trial. In order to decide whether the spores or the vegetative cells of the Bacillus strain were the best option, the bioavailability and probiotic activity of both formulations were compared using in vitro gut models and in vivo animal studies. As both spores and vegetative cells exhibited probiotic activity, selection of the optimal formulation was primarily based on the best carotenoid bioavailability profile. It could be concluded that an optimized spore formulation was the better formulation in terms of bioavailability. Therefore, it was decided to use this optimized formulation of the Bacillus spores in the human trial.
In silico genomic screening and in vitro toxicity assessments suggest that the specific Bacillus spores used in this product are safe for human consumption and have an even better safety profile than other Bacilli.
Additionally, in vivo safety assays conducted in mice showed no signs of toxicity. Furthermore, the safety of oral intake was confirmed in two phase I safety studies conducted in healthy individuals, who were and were not overweight. No treatment-related adverse effects occurred with repeated intake over 2 weeks.
In year 2, a 6-week phase II efficacy study was performed in healthy, but overweight individuals. The study was designed as a randomized, placebo-controlled, double-blind, parallel study. Carotenoid analysis showed accumulation of bacterial carotenoids in the plasma of individuals treated with the Bacillus spores throughout the study period. This indicated that the bacterial carotenoids were absorbed in the human GIT and can therefore exert a systemic effect. Additionally, analysis of faecal samples showed that the bacterial strain was able to survive transit through the human GIT, potentially exerting probiotic effects. Indeed, beneficial effects were observed on some biological endpoints after intake of the spore formulation. As this was the first time that such effects were seen in humans, the results provide compelling evidence for the further development and commercialization of the CaroDel product.
A life cycle assessment showed that the CaroDel product would be environmentally competitive when compared to other products with a claimed positive effect on CVD biomarkers, making it a sustainable business. The most environmentally friendly delivery option would be to sell the product as a supplement (in capsules). Moreover, assessment of market trends, current heart health products, consumer attitudes and competitive products, showed that the CaroDel product could be successfully introduced on to the market.
CaroDel was designed to fill existing gaps between the discovery phase and the translation of those findings into a marketable product. The probiotic activity of the Bacillus strain and its effect on cardiovascular endpoints were investigated in parallel with an in vivo study of the absorption, safety and mechanism of action of the biological compound. The result was CaroDel, a new health ingredient that is different from all probiotics and carotenoids currently on the market.
ProDigest in a nutshell
ProDigest is a product leader in the development of unique laboratory models of the human and animal gastrointestinal tract. Without the need for animal or human studies, these models allow a unique insight into gut processes associated with the intestinal fate, metabolism and bioavailability of active ingredients and facilitate study of the complete gut microbiota under controlled conditions and its link with human and animal health. ProDigest is globally active as a service provider for food and pharmaceutical companies and since 2014 also installs its technology in selected R&D facilities around the world. Furthermore, ProDigest has set up a number of in-house product development projects related to microbial biotechnology and gut microbiota management and the development of novel biotherapeutics.
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Nutrafoods 3 – 2016