Service Communautaire d'Information sur la Recherche et le Développement - CORDIS

Final Report Summary - CARODEL (Use of Microorganisms for Carotenoids Delivery: Next Generation of Probiotics for Cardiovascular Disease)

Executive Summary:
Characterized by their orange, yellow and red pigments, carotenoids are mainly synthesized in plants, but they also have been isolated from other organisms, including some bacteria and fungi. Humans, like other animals, are unable to synthesize carotenoids, but absorb them through 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 diseases). Plant-derived carotenoids are widely associated with cardiovascular benefits, yet their low stability and poor bioavailability (i.e. the 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 to support further development and commercialization of these bacteria-derived carotenoids.
By combing in vitro gut models and in vivo animal studies, the CARODEL project resulted in an efficient oral delivery strategy of such highly active carotenoids, in the form of Bacillus spores, with the additional ability to exert probiotic effects (i.e. effects on gut microbiota, metabolism and beyond). This delivery strategy was validated in a human study. As probiotic activity of the Bacillus spores and its effect on cardiovascular endpoints were investigated in parallel, whilst absorption, safety and mechanism of action of the carotenoid compound were studied in animal and human studies, CARODEL resulted in a new health ingredient that is different from all probiotics and carotenoids that are currently on the market by combining its dual action. Therefore, the product will be attractive for customers who want to improve their cardiovascular and/or intestinal health.

Project Context and Objectives:
CARODEL aimed to valorize the results from the previous FP7 COLORSPORE project, in which initial isolation and characterization work was performed on Bacillus strains producing gastric-stable carotenoids. As the stability in the gastrointestinal tract (GIT), antioxidant activity and bioavailability of particular Bacillus carotenoids was shown to be higher than those of common dietary carotenoids, the conclusions from COLORSPORE provided strong and compelling evidence to support further development and commercialization of these bacteria-derived carotenoids.
CARODEL therefore focused on the development of an efficient oral delivery strategy of such highly active carotenoids, and evaluation of potential direct health-beneficial (probiotic) activity of the Bacillus delivery vehicle, with the ultimate goal to improve biomarkers associated with (the prevention of) cardiovascular disease (CVD).
In practice, effective delivery of the carotenoids in the human body was compared upon 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 beyond) was investigated using in vitro gut models and in vivo rat studies. Based on this, 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 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 therefore:
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 gastrointestinal tract 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 in the gut microbial community;
4. To prove that the selected Bacillus strain is safe for human consumption, which will allow future registration under the QPS regulation;
5. To model the scale-up of the production to bring the product on the market;
6. To evaluate the effect of the Bacillus strain in humans with respect to CVD biomarkers, and modulation of the intestinal environment and host health endpoints;
7. To generate the necessary know-how for IP protection strategy, business model for commercialization and sustainability of the product.
To address these objectives, the CARODEL project was divided in two phases, i.e. Phase 1 (the selection of the best formulation) and Phase 2 (translational phase to the market), by combining 5 research work packages (WP2-6) supported by a management work package (WP1) and a dissemination work package (WP7).
During Phase 1 of the CARODEL project, WP2, WP3 and WP4 were run in parallel.

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 - by means of in vitro research and animal studies - the intestinal fate and bioavailability of the carotenoids when administered either as purified carotenoids extracted from the Bacillus strain, when contained in the vegetative cells or when contained in the spores of the strain. Furthermore, the effects of the Bacillus strain on the intestinal environment were 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 necessary steps in order to prove the safety of the carotenoids and the carotenoid-producing strain in relation to the 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 WP 5 (“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 the gastrointestinal microbial composition and metabolic activity.
Finally, in WP6 (“Regulatory and life cycle assessment”) the consortium defined the regulatory strategy, evaluated the consumer perception of the novel concept, identified the necessary additional R&D steps and, finally, prepared a life cycle assessment plan. Moreover, the results of WP7 are an IP strategy to protect the outcomes of the project and an exploitation plan of all the results generated in the project.
Summarized, the successful achievement of the objectives above brings the Bacillus carotenoid product close to commercialization and exploitation as a unique health ingredient.

Project Results:
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 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 use of the extracted bacterial carotenoids could be excluded from the selection. In order to decide whether the spores or the vegetative cells of the Bacillus strain were the best option, 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 activities, the 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 as compared to other Bacilli. Additionally, in vivo safety assays conducted in mice revealed no signs of toxicity. Furthermore, safety of oral intake was confirmed in two phase I safety studies conducted in healthy individuals, both with and without overweight. No treatment-related adverse effects occurred upon repeated intake during 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 an accumulation of bacterial carotenoids in plasma of individuals treated with the Bacillus spores throughout the study period. This indicated that the bacterial carotenoids were absorbed in the human gastrointestinal tract and can therefore exert a systemic effect. Additionally, analysis of fecal samples showed that the bacterial strain was able to survive the transit through the human gastrointestinal tract, potentially exerting probiotic effects. Indeed, beneficial effects were observed on some biological endpoints after intake of the spore formulation. As it was the first time that such effects were shown 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 selling 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 the market.
With the extensive strain characterization and safety assessment of the Bacillus spores, the main elements of the required safety data package for regulatory submission are available. This safety data package can be used both for GRAS submission in the US and Novel Food submission in the EU. Since the effects of the Bacillus spores on intestinal and cardiovascular health were studied in a double-blind, placebo-controlled and randomized human intervention study, certain Structure/ Function claims may be possible in the US without further regulatory approvals. Therefore, it is recommended to first launch the product in dietary supplements in the US with health claims based on the results of the first clinical study. In the meantime, marketing in the EU should focus on health practitioners, as health claims can be communicated to professionals and additional clinical data can be obtained. Based on the data of these additional clinical studies, a decision can be made whether or not to submit a health claim dossier in the EU.

Potential Impact:
CARODEL was designed to fill the existing gaps between the discovery phase and the translation of those findings to a marketable product. By investigating in parallel the probiotic activity of the Bacillus strain and its effect on cardiovascular endpoints, whilst absorption, safety and mechanism of action of the biological compound were studied in vivo, CARODEL resulted in a new health ingredient that is different from all probiotics and carotenoids that are currently on the market.

Health benefits of carotenoids
The recent interest in carotenoids has been brought about by the health benefits of a diet rich in fruits and vegetables (Krinsky et al., 2005). Indeed, epidemiological evidence shows that carotenoid-rich fruits and vegetables may decrease the risk of various diseases, including eye diseases, cancers and CVD. The protective effects are thought to be related with their potent antioxidant activity, which is well demonstrated in in vitro assays. However, the relevance of antioxidant properties in vivo remains subject of debate (Rice-Evans et al., 1997) and other biological activities are most likely involved in the health protective effects. One particularly interesting activity of carotenoids relates to the effect on multiple biomarkers of CVD. Driven again by epidemiological evidence on CVD prevention and carotenoid-rich foods, a link between carotenoid intake and protection from LDL oxidation, atherosclerotic progression, hypertension, inflammation and endothelial dysfunction has been proposed (Hozawa et al., 2009; Hozawa et al., 2007 & Rao et al., 2007).
Among the different carotenoids, lycopene (extracted from tomatoes) has been widely studied among which its effect on CVD biomarkers. This extensive scientific evidence has recently resulted in a first positive EFSA opinion for carotenoid products and CVD prevention (O’Kennedy et al., 2006). Indeed, a 13.5 EFSA health claim was accepted for Fruitflow®, a water-soluble tomato concentrate, in helping to maintain normal platelet aggregation, thereby contributing to a healthy blood flow. Such scientific evidence, combined with the positive EFSA opinion, clearly warrants further scientific research towards carotenoids and CVD prevention.
Currently, carotenoids are consumed as a food or as a dietary supplement. No official recommendations exist on daily intake of carotenoids, but in the case of -carotene a daily intake of 2-4 mg has been suggested (Biesalski et al., 1997). Interestingly, most supplements carry a recommended daily allowance (RDA, 800 mg), far exceeding the daily intake. The reason for this is the degradation of carotenoids in the stomach. Indeed, previous studies have shown that when -carotene is incubated in simulated gastric juices, it is almost completely degraded in as little as 10 minutes with no detectable amounts after 20 minutes. The typical transit time for food to pass the stomach is about 60 minutes, which means that, unless carotenoids can evade the action of gastric fluids (e.g. by being protected within a food matrix), a large proportion of the ingested dose will be degraded by the time it reaches the small intestine. The superior gastric stability of Bacillus-derived carotenoids, therefore, represents a clear competitive advantage over plant-based carotenoids.

Bacillus probiotics
Bacillus species are Gram-positive bacteria able to form endospores, i.e. uniquely robust entities that are able to survive extremes of temperature, irradiation and long-term storage (Nicholson, 2002). They have been studied extensively in the past as a model system to improve knowledge on unicellular differentiation. Several species of Bacillus are even consumed as food ingredients (Hong et al., 2005). This includes B. subtilis, B. licheniformis, B. clausii, B. pumilus and B. coagulans. Typical examples of dietary sources include fermented food products, such as the traditional Japanese natto, containing soybeans fermented by B. subtilis, which has been used for hundreds of years in Japan and is consumed in large quantities by most of the population (Hosoi et al., 2004). Similarly, B. clausii spores are the sole component of Enterogermina, an over-the-counter (OTC) medicine for the treatment of childhood diarrhoea (Hong et al., 2005). In this particular case, B. clausii is used as a licensed medicine and has specific claims attached to it. Other examples are the use of B. subtilis and B. licheniformis in the animal feed product known as BioPlus 2B™ which is manufactured by Chr. Hansen and Clostat™ by Kemin Industries. Together, there is sufficient evidence indicating that Bacillus species can be safe for human consumption (Hong et al., 2005).
In addition to the safety, there is substantial data indicating potential health benefits of Bacillus strains, such as stimulation of innate immunity (Fiorini et al., 1985; Caruso et al., 1993; Ciprandi et al., 1986; Ciprandi et al., 2004; Ciprandi et al., 2005). In the case of B. subtilis var. Natto a serine protease known as Nattokinase is secreted by the strain, which has been shown to reduce blood clotting by fibrinolysis (Sumi et al., 1987; Sumi et al., 1995). Such alkaline protease is produced by most Bacilli and most probably, many if not all Bacilli carry this same potential health benefit.
Compared to other live bacteria that are used as probiotics, Bacillus spores are particularly attractive for technological/functional reasons. Indeed, the robust spores are not only able to pass though the gastric barrier without loss of function, they are also stable in many food processing steps and can be stored at room temperature.
With this ‘dual action’ of the carotenoids and the Bacillus strain, the CARODEL product is therefore a very promising candidate ingredient which will contribute to cardiovascular health. This health area is life style-related and its importance for public health is only increasing with an ageing population in Europe and beyond. Hence, work on the prevention of cardiovascular diseases is supported by the increasing interest of consumers, life science companies, health insurers and health authorities. There is no doubt that a new health ingredient that addresses these health concerns and is safe and affordable for daily preventive use will stand a good chance to be taken-up by the market throughout Europe. Additionally, sustainable production, by means of microbial fermentation, will allow the product to compete against similar products derived from alternative natural sources.
During the course of the project huge efforts were made to disseminate information concerning the existence of the project and its progress to an audience as wide as possible. The scientific data and related information will still be disseminated beyond the project as it will serve as a pre-marketing strategy with the aim of identifying potential partners for future developments.
Clearly, none of the participating SMEs would have been able to setup and execute single handed such a large-scale translational research project. Being part of the CARODEL-consortium allowed the SMEs to deploy their developed expertise and technological skills in a concerted manner. This deepened and broadened the research network, product development skills set and regulatory/IPR understanding of all SMEs involved, and hence improved their innovation potential and competitiveness. Moreover, as the novel food ingredient will have the potential of improving heart health, the SMEs involved in the CARODEL project will have access to the markets of both carotenoid and probiotic dietary supplements.

List of Websites:

Possemiers, Sam (Project Coordinator)
Tel: +32 9 241 11 90
Fax: +32 9 241 11 91


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