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Project Context and Objectives:
Many naturally-occurring compounds in plants and animal products, known collectively as bioactives, possess a variety of physiological functions promoting human health and wellbeing, and contributing to reduced risk of diet-related diseases. Identifying bioactives, assessing their health effects, and establishing their mechanisms of actions are all active areas of scientific inquiry and, through industrial exploitation, potential societal benefit. However, the scientific understanding of the role and mechanisms of dietary bioactive is still limited.
Bioactives are present in common foods at low concentration, usually far from the effective dose. To overcome this, the food and drink industry is developing new products containing high concentrations of selected bioactives, so-called “functional food products” or bioactive-enriched food (BEF).
The aim of PATHWAY-27 is to deliver a better understanding of the role and mechanisms of BEF. To this end, PATHWAY-27 uses a strategy developed to overcome the main gaps in the current knowledge for the demonstration of the healthy effects of BEF.
Gap # 1, Bioactives vs foods vs diet. Although dietary health effects are considered related to bioactives, bioactives are just one part of foods, and individual foods are just one part of the overall diet. To overcome this important gap, PATHWAY-27 evaluates foods enriched with selected bioactives, and not bioactives as discrete molecules.
Gap # 2, Weak markers of effectiveness. To effectively assess the health-promoting and risk-reducing activities of BEF, their effectiveness must be evaluated against measurable, physiologically relevant end-points. PATHWAY-27 evaluates the effect of BEF on metabolic syndrome (MetS), since all criteria of MetS diagnosis represent measurable, physiologically relevant end-points. By exploiting the parameters used for MetS diagnosis as primary and secondary end points, PATHWAY-27 bridges the gap associated with weak markers of effectiveness.
Gap # 3, Unclear mechanisms of action and contrasting human results. The mechanisms of action of bioactives are usually studied in vitro, whereas in vivo studies are very often focused on demonstrating an effect on specific end points, without considering the underlying mechanism. This creates a scientific dichotomy and the assumption, without any demonstration, that the in vivo effects are related to the mechanism/s of action observed in vitro represents an enormous, and unscientific, gap in our knowledge. In PATHWAY-27, in vitro and in vivo studies are integrated to establish scientific cross-talk and feedbacks allowing a better understanding of the clinical effects. The integration of genetic, epigenetic and metabonomics approaches is a powerful and innovative strategy in PATHWAY-27 for elucidating the role and mechanism of action of bioactives when administered to humans as ingredient of BEF.
Gap # 4, High cost. The high cost of human intervention studies is a significant barrier to SMEs performing them and, thereby, reduces their ability to develop new food products and gain a significant share of the healthy food market. To by-pass this gap, PATHWAY-27 has developed a strategy involving preliminary pilot studies.
Although PATHWAY-27 uses three model bioactives (DHA, beta-glucan and anthocyanins) and three model food matrices (bakery products, dairy products, and egg-based products) to deliver a better understanding of the role and mechanism/s of action of BEF, conclusion derived from the project activities will be widely applicable to other bioactives and other bioactive-enriched food.
The main PATHWAY-27 output is the development and dissemination of comprehensive guidelines for undertaking studies on BEF, and to support health claims. These guidelines will be helpful to SMEs producing BEF and interested in applying for health claims related to their products.

Project Results:
One of first objectives of PATHWAY-27 was to formulate the food products (one in each matrix, each one enriched with the five combination of bioactives) to be used for the development of BEF. Briefly, 45 prototypes were developed. In order to select the most promising BEF within each matrix, the following attributes of quality were considered: (1) the microbiological safety; (2) the fraction of bioactive compounds recovered after preparation of BEF; (3) the chemical stability of the bioactives during storage; (4) the sensory quality and consumer acceptability; (5) the fulfilling of a correct nutrient profile; (6) the bio-accessibility of bioactive compounds upon in vitro digestion of BEF; (7) ease of storage; (8) ease of home preparation. The integrated evaluation of results allowed the selection of the most promising BEF (one in each matrix) to be enriched with the five combinations of bioactives. The 3 selected products (biscuits, milkshake and pancakes) were produced and then used in pilot studies.
Three pilot studies were performed, each focusing on a specific food matrix. Three centers were involved in the recruitment, each one associated to a single food matrix. The studies were conducted for a period of 4 weeks. Pilots were designed as exploratory trials aiming at identifying the best matrix × enrichment combination that would result in the strongest effect on the designated endpoints. Pilot studies allowed the selection of the three most active enrichments (one in each matrix) to be used in the large intervention study (LIS).
Pilot studies had not only the meaning to select the best enrichment within each matrix, but also (and mainly) to solve possible problems arising before/during the LIS. Problems encountered in BEF production, sending and storage were successfully solved. Overall, all BEF were produced and delivered to recruiting centers in the required amounts during the pilot studies, and during the LIS so far. The concentration of bioactives was adequate in all production batches.
BEF selected in the pilot studies are used in the LIS, that is designed as randomized, double blind, placebo controlled trial, and involves four different centers. The LIS started on 29 February 2016 and it is still ongoing. The effects of BEF in the LIS volunteers will be evaluated not only on primary and secondary clinical endpoints, but also using different advanced omics techniques. At present, all available biological samples from human volunteers (urine, blood, adipose tissue and stool) have been sent to partners in charge for these analyses.
Investigation on the in vitro effects of bioactives are still ongoing. The two main cell model systems used are adipocytes and hepatocytes, due to the pivotal role adipose tissue and liver in development of the MetS. Since in vivo foods are digested and some bioactives are extensively metabolized, DHA, protocatechuic acid (PCA, the major metabolite of most anthocyanins) and propionic acid (PRO, produced in the colon from beta-glucan) were used for in vitro supplementation. Briefly, beneficial effects of bioactives, either alone or in combination, on fat cell metabolism and inflammatory phenotype were evidenced. As well, results obtained in hepatocytes contribute to the understanding of the various mechanisms supposed to be the basis of the potential preventive effect of the selected bioactives in the MetS. In hepatocytes, investigation performed using NMR identified a “DHA signature” in the cell lipidome and metabolome. In addition, the treatment with the bioactives resulted in time-dependent specific alterations of the transcriptome. Considering supplementation with single bioactives, DHA was the one inducing the bigger alteration of the cell transcriptome. PRO and PCA alone had no or very limited effect on transcription, but exerted synergistic effects when combined with DHA. The evaluation of the effects of bioactives in adipocytes transcriptome is ongoing, as well as epigenetic analysis.
The whole consortium has been involved in the preparation of the PATHWAY-27 Guidelines. After several internal revisions, the Guidelines for the Scientific Community have been sent to external experts. Based on their comments, the guidelines will be further revised, and specific points will be addressed during the guidance paper workshops in September 2017. The revised version of the PATHWAY-27 Guidelines for the Food Industry have been also sent to external experts for review. After a public consultation in summer 2017, they will be discussed at the guidance paper workshops in September 2017. Both Guidelines are expected to be submitted to the EC in due time (November 2017).

Potential Impact:
The scientific community considers that bioactives and related bioactive-enriched food (BEF) have a high potential for reducing health risks and improving health quality and eating habits. However, there is a pressing need of robust supporting science to underpin the efficacy of bioactives and BEF. Addressing this need is pivotal in establishing robust strategies for delivering beneficial effects on human health, responding to consumer demands, and creating an environment for future market success of BEF.
In Framework Programmes 6 and 7, approximately 66 million € were targeted to the identification of bioactives and establishing their mechanisms of action. Many nationally-funded projects in Europe also had these objectives. Whilst this funding led to a very substantial increase in knowledge on certain bioactives, the impact of this research in delivering novel food products has been limited. This is due to inefficient knowledge transfer to industry (which, in the food and drink sector, is dominated by SMEs) and to difficulties in convincing EFSA of the validity of the health claim dossiers on which the positive effects of these bioactives, considered not as pure compounds but as an integral part of a food contributing to the overall diet, are based.
The main expected, final results of PATHWAY-27 is the delivery of two specific set of guidelines, one addressed to the Scientific Community and the other to the Food Industry, that could greatly contribute to the scientific knowledge on BEF effectiveness and to innovation potential and competitiveness of the Food Industry, particularly SMEs.
The novelty of the PATHWAY-27 Guidelines is based on different points:
- The project crucially consider bioactives as ingredients of BEF rather than as discrete molecules. In this way, all technical problems related to the formulation, development, production of bioactive-enriched foods will be addressed in the Guidelines;
- The availability and activity of bioactive compounds when administered as integral parts of foods, not as supplements, will be considered, including the impact of digestion. This will clarify the poorly understood interactions between bioactives/food matrix, and the impact of technological processing needed to produce BEF, allowing the choice of the most suitable ones;
- The design of appropriate dietary intervention studies will be carefully considered, highlighting the differences between trials evaluating drugs and foods. PATHWAY-27 guidelines will provide best practice and suggestion for undertaking successful nutritional studies;
- The importance of development and validation of innovative biomarkers that are relevant to humans will be evidenced;
- The use of in vitro studies to elucidate the mechanism(s) of action of bioactive, connecting them to the in vivo ones, will be carefully considered.
In addition, the PATHWAY-27 Guidelines will be based on pure theory, but on the experiences developed during the project. All information from PATHWAY-27, especially difficulties encountered and how these were resolved, will be used to write comprehensive guidance documents.
While evidencing the activities needed to underpin health claims with appropriate scientific evidence, the PATHWAY-27 Guidelines for the Scientific Community will support the implementation of European legislation on health and nutrition claims by establishing a generic protocol for the choice of robust biomarkers and relative end points, their thresholds, and all the scientific parameters of primary importance for the effective substantiation of health and nutritional claims through provision of the necessary quantity and quality of scientific data.
In addition, the PATHWAY-27 Guidelines for the Food Industry will increase the innovation potential and competitiveness of SMEs. At present, it is mainly the large food companies that exploit the market of healthy foods due to the high cost of obtaining the necessary amount and quality of scientific data to support health claims submitted to EFSA. Documentation, developed, targeted and disseminated through PATHWAY-27 will guide and greatly assist SMEs through all the actions needed for marketing a BEF, not confined to PATHWAY-27 bioactives, from product design to submitting a dossier in support of a health claim.

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