Community Research and Development Information Service - CORDIS

FP7

BAKE4FUN Report Summary

Project reference: 606476
Funded under: FP7-SME

Final Report Summary - BAKE4FUN (Innovative biotechnological solutions for the production of new bakery functional foods)

Executive Summary:
There are between 1.5 and 2 billion people in the world who have problems related to poor nutrition or bad eating habits. On the other hand, consumers are increasingly concerned about origin of foods, and attentive to wellness and sustainability. The European funded project BAKE4FUN investigated on innovative food formulations. BAKE4FUN aimed to bridge the technological gap between SMEs and health-promoting bakery foods. These products, all made with naturally rich ingredients, are functional, sustainable and commonly available in the marketplace. Specifically, thanks to their high content of good elements such as antioxidant compounds and iron, they are recommended for everyday diet. The project has designed, validated and developed 4 innovative health-promoting bakery products, ingredients and technologies: a) iron-fortified bakery products suitable to provide an intake of bioavailable iron overcoming the negative effect of fortification caused by free form of iron; b) iron microencapsulation technology used for iron-fortified bakery, improving digestibility of iron-fortified bakery products with affordable costs; c) iron-fortified shelf stable flour mixes, combining standard flours with micro-encapsulated iron, saleable in bakeries; d) ancient grains bakery products, based on the use of einkorn, a non-conventional whole grain flour rich in antioxidant compounds, with anti-inflammatory properties, positively influencing the glycemic index.
BAKE4FUN provided to an opportunity to increase SMEs turnover in the short, as well as mid-long term by the production, outsourcing or licensing of the newly developed products. In fact, einkorn flour var Monlis and einkorn bakery products rich in bioactive compounds are already for the online PROMETEO market. Moreover, a low cost iron microencapsulation protocol to include bioaccessible and bioavailable microencapsulated iron (EPSAFERRO microcapsules) in new iron fortified bakery products was delivered. Finally, a high cost-efficient experimental in vivo model enabled SMEs to asses newly iron fortified foods and to plan for further cost efficient clinical trials. Finally, the low cost iron microencapsulation protocol, besides its application to iron, could be exploited by SMEs to microencapsulate other different high added value bioactives.

Project Context and Objectives:
Nutritional iron deficiency (ID) is estimated to affect 1.5–2 billion people worldwide (WHO, 2010). In developing countries this is usually due to a limited food supply, but ID represents a public health problem also in some industrialized countries where consumers, trying to consume a preventive diet i.e reducing food intake or the consumption of specific foods, may face to a decrease of micronutrient intake and status. Although iron fortified wheat flour exists since many years in the market, and the market of functional bakery foods is continuously increasing, to date the efforts of industries devoted to innovative formulations/technologies did not overcome the negative effect of the added iron on bakery products sensory quality. Consequently iron-fortified foods are usually rejected by consumers due to unacceptable changes of their organoleptic characteristics. Moreover many questions remain still open on iron bioavailablity of fortified foods. A recent report of Scientific Advisory Committee on Nutrition (SACN) on Iron and Health (2010) evidenced that although iron-fortified foods make a substantial contribution to intake, the evidence from efficacy trials suggests that foods such as flour fortified with elemental iron are unlikely to make a valuable contribution to increasing iron stores (owing to low solubility and low intestinal uptake). As SACN recommended there is a need of research studies to study the extent to which foods fortified with iron, e.g., cereals and cereal products, contribute to the supply of absorbed iron and to achieving adequate iron status.

One of the most interesting trends of bakery products is related to consumers’ increasing awareness of the health benefits of whole cereals, and to consumers’ attraction toward the so-called ancient grains. In fact since whole grains have many healthful benefits, several companies are introducing them into products that consumers seek to incorporate into their diets (Black and Lewis 2009). In particular among the top 15 lauches in bread and bread products in Europe, “whole grain” accounted for the higher number of lauches, “no additives” the second and the third one is what consumers perceived as “traditional”. Although non conventional cereal grains lack some of the technological traits required by commercial bakeries, they face consumers’ preference due to their organoleptic characteristics, and their ethical trait and sustainability. Besides the fact that breads obtained entirely from whole grain flours of emmer, spelt, Kamut, and triticale are characterised by a range of pleasing flavours, the concentration of some bioactive compounds may be higher in ancient grains than in modern wheat.
In particular a cultivated einkorn (Triticum monococcum L. subsp. monococcum), a diploid hulled wheat closely related to durum and bread wheat, is a cereal with high protein, carotenoid, and tocol contents These considerations together with its pleasant organoleptic characteristics suggest a possible utilization of einkorn flour hold the potential of developing high-antioxidant bakery products, able to significantly raise the dietary intake of carotenoids. Hidalgo, A., Brandolini, A. (2011).

The main purpose of the BAKE4FUN project was to design, validate and develop innovative health-promoting bakery products.
In particular, 2 classes of bakery functional foods were formulated:
1. a class of iron-fortified bakery products which, by using a new iron microencapsulation technology for increasing iron stability and bioavailability, responds to the consumers’ need of counteracting iron deficiency by the consumption of common, widely consumed foods;
2. a class of innovative bakery products based on the use of non-conventional high-added value whole grain flours, able to respond to consumers’ demand of healthy, sustainable and ethically correct products, to increase antioxidant intake and to improve the gut microflora.

The main technological hurdle in the production of novel bakery products, either iron-fortified and based on the use of whole grain flours, is represented by giving the new products sensorial and palatability characteristics allowing them to be used by the general population.
In BAKE4FUN, the use of different fermentation process, including sourdough fermentation, were studied and employed in order to set the best food production technologies and processes in term of sensorial parameters at the basis of the acceptance and marketability of a product.
Additionally, the scientific substantiation of the possible functional properties ascribable to the newly developed food products is a striking regulatory demand at EU level after opportune clinical studies.
BAKE4FUN responded to the need of developing safe, effective and validated functional bakery products by extensively study in vitro which novel technologies (microencapsulation, novel industrial processes, novel breadmaking procedures) and which food formulation based on the use of non-conventional flours will potentially result in innovative bakery products with a selective advantage in term of beneficial properties and marketability.
Notably, from a technological point of view, the bioaccessibility of micronutrients in fortified products and the digestibility of novel flours are the main issues giving a possible added value in the development of new bakery products. Therefore, the study of the effect of selected formulation/technologies on iron retention in microcapsules and on its bioaccessibility, as well as the analyses of the formulations and fermentation processes of non conventional flours at high digestibility, will be central in the development of BAKE4FUN project.
Finally, in order to evaluate and validate in vivo the potential health promoting mechanism at the basis of the most promising bakery products selected among the bunch developed in BAKE4FUN, their functional characteristics were studied in an animal model (pigs). This model was used to study: i) the bioavailability of iron in fortified breads, their effects on the iron status, on biomarkers of oxidation and inflammation, and on the gut microbiota; ii) the effect of the innovative bakery products based on non-conventional flours on the gut microbiota, the glycaemic load, and the inflammatory and oxidative status.

SCIENTIFIC OBJECTIVES
The scientific objective of BAKE4FUN was to better understand the potential benefits and mechanisms of action of the innovative breads obtained by the improved formulation/technologies. This objective was reached by:
• Measuring the bioaccessibility and understanding of mechanism of action of iron in relation to the different formulation/technology adopted
• Measuring the antioxidative compounds of ancient einkorn flours (AF) and their fate in breadmaking process
• Measuring in animal model the bioavailability of iron in iron fortified flour breads (FFB) on the basis on physiologically relevant end-points related to haemoglobin repletion in anemic pigs
• Measuring the in vitro antinflammatory and digestibility characteristics of sourdough breads
• Measuring in animal model the effect of sourdough fermentation on glycemic index, antinflammatory effects, microbiota antinflammatory characteristics
• BAKE4FUN contributed to preliminary data substantiating further cost-effective clinical trials for health claim submission of iron fortified functional bread.

TECHNICAL OBJECTIVES
The technological objective of BAKE4FUN was the development of improved formulation/technologies to produce functional breads. This objective was reached by:
• Understanding the stability of microencapsulated iron (MI)
• Understanding the bioaccessibility of iron in the functional flours (FF)
• In vitro evaluation of the antinflammatory properties of functional breads
• In vivo evaluation of iron bioavailability, absorption and functionality in the new bakery products
• In vivo evaluation of the glycaemic index of the new bakery products
• In vivo evaluation of the impact of new bakery products on the intestinal microbiota and their metabolites
• In vivo evaluation of the impact of new bakery products on the oxidative and inflammatory status

TECHNOLOGICAL DEMAND OF THE SMES ADDRESSED BY BAKE4FUN
Increasing consumer awareness towards functional foods in combination with advances in various scientific domains, provides companies with unique opportunities to develop an almost infinite array of new functional food concepts. It should also be considered that functional foods are usually sold at higher prices than the conventional counterparts, thus they contain larger profit margins than conventional foods; this obviously makes the sector attractive for all the players in the food industry, including them representing the supply chain of bakery industries.
SMEs participating to the BAKE4FUN represent three different positions in food chain: bioactive compound ingredients (EPSA), production of flour (PROMETEO) or flour premixes (INDESPAN) and bakery (VINI). For each of them the development and commerce of potentially functional ingredients or foods is rather complex, expensive and risky, and would require not only a close interaction with the other players of the food chain, especially the end users (the bakeries), but also significant economic research efforts.
The SME participants outsourced research by investing in BAKE4FUN RTD performers in order to return the technological know-how they need to develop new products/processes. In particular BAKE4FUN provided:
a) know-how to produce stable microcapsules (resistant to food chain stresses but able to deliver free iron at intestine level),
b) research results to exploit the functional characteristics of sourdough bread obtained by einkorn wheat,
c) set up of a breadmaking process suitable to produce appealing iron fortified bakery products.
This involved selecting and identifying the different chemical forms of iron, screening and selecting cover material and technology for MI, screening and identifying potentially functional compounds in AF and their retention in breads. Moreover, within the BAKE4FUN project the in vitro and in vivo activities for assessing AF and FF physiological effects was performed, developing a breadmaking formulation/technology suitable to this food matrix, and taking into account iron bio-availability and its potential changes during processing and food preparation.


BAKE4FUN PROVIDED FORMULATION/TECHNOLOGICAL SOLUTION TO PROVIDE TO MARKET ATTRACTIVE FUNCTIONAL FOODS by using two formulation/technologies:

1) MICROENCAPSULATION OF IRON provided enhancement of iron absorbtion and mitigation of undesirable interactions between fortificant iron and food vehicles.
The iron microencapsulation is a thin coating of inert material used to prevent the iron from oxidizing the food. This thin coating protects the iron from the food (and food from the iron) and also masks the taste of the iron. The coating dissolves in the stomach, releasing the iron salt, to be absorbed along with iron contained in the foods that constituted the meal.
The encapsulated micronutrients recently developed provided iron to at-risk populations are added as powder form directly to food at the household level.
Microencapsulation technology was optimised in order to retain in bread the core material containing iron confined within capsule walls along both the breadmaking process, storage and towards the stomach to be released through the capsule walls in intestine. Thus, microencapsulation provided different benefits such as: the increase in the iron bioaccessibility achieved thanks to the protection of iron compounds that otherwise could be damaged, due to light, temperature, oxygen, etc..; the protection against interaction with other compounds (as phytates); to avoid the unpleasant flavour of bioaccessible form of iron (as ferrous sulphate). It also makes it possible to be released at intestine conditions, where iron is going to be absorbed.
In particular BAKE4FUN studied the microencapsulation of iron for the preparation of premixed flours for bakery uses. In this case one of the main challenges that microcapsules are subjected to will be the chemical and physical stresses occurring in breadmaking process. BAKE4FUN provided up protocols and scale up production of microcapsules not only homogeneously distributed within flour premixes but also resistant to:
a) premixes storage conditions (oxygen, umidity and temperature shifts);
b) breadmaking conditions: oxidases and lipoxigenases naturally occurring in flour and activated by the dough mixing process, redox potential, high water activity, fermentation, low pH, organic acids, baking.
c) chemical and physical changes induced by the incorporation of sourdough. In fact when sourdough is incorporated to the main dough for the last fermentation step, the bacterial metabolites such as organic acids (mainly lactic acid and acetic) together with sourdough microbiota enzymes may represent a strong threat to the integrity of microcapsules

2) INCORPORATION OF A WHOLE Triticum monococcum ASSOCIATED TO A FERMENTATION PROCESS, increases the sensorial, nutritional and healthy characteristics by also using a sourdough process.
Fermentation, and in particular sourdough fermentation performed by a mixed population of Saccharomyces cerevisiae and lactic acid bacteria has a key role in improving the flavour and the structure of bread. Whole cereal sourdough fermentations also show significant potential in improvement of the nutritional quality and health effects of foods and ingredients. In addition, sourdough can also actively retard starch digestibility leading to low glycemic responses, modulate levels and bioaccessibility of bioactive compounds, and improve mineral bioavailability (Poutanen et al. 2009). Since whole grains have many healthful benefits, several companies are introducing them into products that consumers seek to incorporate into their diets (Black and Lewis, 2009). New specialty foods based on grain blends has permitted the use of so-called ‘ancient grains’ as components that convey naturalness, unconventional, and nutritional properties. Many bread products varying in taste and texture can be developed utilizing alternative grain flour alone or in various proportions with common bread wheat flour. The main alternative cereals are Kamut, Spelt, Triticale, einkorn and emmer. In particular cultivated einkorn is T. monococcum often referred to as "the covered wheats," since the kernels do not thresh free of the glumes or the lemma and palea when harvested. In particular although non conventional cereal grains lack some of the technological traits required by commercial bakeries, they face consumer preference for their organoleptic characteristics, beside ethical and sustainable claims. In fact breads obtained entirely from whole grain flours of emmer, spelt, Kamut, and triticale are characterised by a range of pleasing flavours. Moreover the first scientific papers reporting their composition surprisingly evidenced that, in comparison to modern wheat, the concentration of some bioactive compounds may be higher ( Hidalgo, A., Brandolini, A., 2011).

BA4FUN provided application of microencapsulation technology to produce stable and highly bioavalable iron fortified functional product. At the same time BAKE4FUN used a breadmaking technology based on the synergistic combination with ancient grains to increase consumers acceptance of the fortified breads.
A multi step fermentation process was studied in order to incorporate einkorn sourdough fermentation as first step of industrial breadmaking processes of traditional (einkorn flour) or functional (iron fortified flour) breads with improved sensorial, nutritional and healthy characteristics.

The main technological know-how that was provided to SMEs is:
• Selection of novel functional ancient flours and the influence of breadmaking process on their nutritional and sensorial characteristics of the bakery products obtained
• Formulation of innovative iron microcapsules characterized by an increased iron bioaccessibility to be used for the development of more homogenous and stable fortified flour mixes,
• Homogenous and stable fortified flour mixes containing iron microcapsules
• Understanding of breadmaking process the nutritional, antioxidant and sensorial characteristics of the AFB
• Understanding the influence of breadmaking and food transformation procedures on stability of iron microcapsules, nutritional and antioxidant characteristics and sensorial quality of selected FFB.

Project Results:
The complex activity related to microencapsulation consisted in: identification and selection of core and cover substances in order to study which factors may condition iron release in food matrix and thereafter reduce the shelf life of fortified bread. Furthermore the encapsulation methods improving availability of microencapsulated iron were selected. Finally the microencapsulation protocols were optimized for obtaining the desired structural and textural properties as well as loading characteristics and the microcapsules to optimise particle size. The release profiles of encapsulated actives from coated carrier particles and powders were evaluated by suitable analytical techniques. In parallel, experimental approaches aimed at characterising non-conventional flours and their utilization for the production of novel functional bakery products were performed to select a set of fortified bakery products and non-conventional wheat functional bakery products. Iron bioaccessibility, absorption and transportation were studied as discriminant parameter for further selecting a fortified bakery products to be produced at industrial scale for the in vivo evaluation of its efficacy in pig model. Similarly, the digestibility, anti-inflammatory and anti-oxidative effect of the selected non-conventional bakery products produced from einkorn flours were evaluated and the best functional bakery products will be selected and tested in pig model for their ability to modulate the gut microbiota and the main health parameters.

In order to summarise the Scientific and Technological results of the whole project and to better explain the delivered foregrounds a list of the main points is here reported followed by their respective description:
a) PROCESS FOR EXPLOITATION OF EINKORN FLOUR FOR PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS
b) KNOW HOW ON HEALTHY AND NUTRITIONAL PROPERTIES OF EINKORN SOURDOUGH
c) METHOD FOR MICROENCAPSULATION OF BIOACTIVE IRON
d) PROCESS FOR EXPLOITATION OF ENRICHED FLOUR FOR PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS.

e) METHOD FOR STABILIZATION AND HOMOGENEOUS DISTRIBUTION OF MICROENCAPSULATED BIOACTIVE IRON IN STANDARD FLOURS


EINKORN FLOUR BAKERY PRODUCTS
BAKE4FUN results provided different scientific and technological results suitable to enable SMEs to select novel functional ancient flours and understand their interactive effects with breadmaking process on nutritional, sensorial and healthy characteristics of the bakery products obtained. Specifically those foregrounds delivered as consequence of two main results: a) PROCESS FOR EXPLOITATION OF EINKORN FLOUR FOR PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS; b) KNOW HOW ON HEALTHY AND NUTRITIONAL PROPERTIES OF EINKORN SOURDOUGH
a) PROCESS FOR EXPLOITATION OF EINKORN FLOUR FOR PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS
a.1 EINKORN FLOUR CHARACTERISTICS ( fully described in D 2.1)
where studied in order to evidence the total content of polyphenol (TPC), carotenoid and antioxidant activity determined in wheat flours (standard flour – STDF) and einkorn flour samples. The highest content of the bioactive compounds and antioxidant activity were observed in wholegrain einkorn flours. As a consequent result, the choice of the whole grain einkorn flour was done and einkorn flour breads (AFB) prototypes available for the in vitro tests were produced. Process and formulation to prepare 5 experimental AFBs (einkorn flour bread) were selected. The number of experimental breads was increased from 3 up to 5 but this change in relation to the Annex I was already indicated in the interim report. The sourdough starters based on selected lactic acid bacteria and yeasts set up in Subtask 2.4.1 were used also for AFB. Experimental AFB breads were prepared by flour selected in task 2.1 adopting a two-step fermentation to improve organoleptic characteristics and shelf life. In fact selected flours were used in the studies on bread making process (Task 2.4) and resulting bakery products were characterised for their concentration of antioxidant compounds and phytates (as phytic acid). Baking process decreased content of bioactive compound as carotenoids content, however it was still higher than in the bread prepared using exclusively wheat flour. Sourdough fermentation brought to significant higher polyphenols content compared to conventional fermentation. Fermentation with lactobacillus brought also reduction in phytic acids, increase radical scavenger activity.
Additional analysis, not foreseen in the Annex I, were performed by TUL since their importance for the nutritional profile of bread. Specifically carbohydrates, lipids, proteins was determined in the aim to be able define to nutritional facts of experimental bread.

a.2 SENSORIAL AND FUNCTIONAL CHARACTERISTICS OF AFB (Einkorn Flour Bread) were evaluated in experimental breads in terms of sensorial characteristics in SubTask 2.4.3 activities and exptensively reported in D 2.2). In particular, in the panellist evaluation, samples made with sourdough of the standard flour sourdough fermented were characterized by good acceptance, while their alveoli were negatively evaluated. The better crumb structure and consistency was obtained in breads combining AF (einkorn flour) with CONV process (baker’s yeast fermentation). On the other hand, sample made by conventional fermentation were better accepted because of their good aroma and flavour. The sensorial profile of bread made exclusively with STDF (standard flour) or AF, or flours combination showing that the panellists were able to discriminate the type of used flour. Among AFB the panellists gave the higher rate to the AFCF (Bread made with Einkorn Flour + Conventional Fermentation) and AFSSF (Bread made with Ancient Flour + Sourdough Standard flour Fermentation) samples from the sensorial profile of the ancient flour but all of them seemed to be acceptable as overall impact of sensorial attributes.

a.3 A PROTOCOL FOR THE PRODUCTION OF AF AT INDUSTRIAL LEVEL was provided to SMEs (fully described in D5.3). In fact, Prometeo is a company cultivating and collecting crops by other producers already producing spelt flour. For the new product, einkorn flour (var. Monlis), the first step is to check the crop production and the harvest in order to verify the compliance of the quality parameters specified in this contract (moisture, purity, presence of pathogens/pest, etc.). The processing of einkorn Monlis is finalized with the milling to obtain a whole flour. The process begins from cleaning, de-stoning (stone clearance) stone cleaning to the de-husking. Einkorn is an hulled wheat and, as all the other species of “farro”, before the use it’s necessary to de-husk the “spikelets”(kernel covered by the glumes) to free the kernels (grains). The naked and whole grains of einkorn are obtained from this process. The whole grains are milled by natural stones. The use of only natural stone ground processes is a procedure that fully protects the germ and aleurone layer, both of great importance in terms of nutrition and taste. It also contributes to improving the quality and taste of the product. The einkorn seeds are finally stored at 16°C and at controlled humidity in order to maintain the grain humidity at 12%. A high amount of samples of einkorn grain have been selected and labeled in order to use the same lot for the experimental activities. The whole flour of einkorn Monlis necessary for experiments (totally around 200 Kg) is periodically milled and supplied to the Research and Demonstration activities of Bake4Fun project.

a.4 PROTOCOL SCALE UP AFB
The protocol to scale up the AFB production represents the consequent result of the previous result in a step by step approach for research activities reported in D5.4. Specifically, to release protocols for the production of AFB and FFB at industrial scale very fine tuning activities were performed by VINI in collaboration with UNIBO and PROMETEO since the lab scale process fitted almost perfectly the industrial plant conditions. “Cottage bread” (Chleb wiejski) recipe has been chosen at the beginning of the project for testing because it contains no additives (improvers, fats, sugars etc.). This approach was adopted to be sure to produce an innovative bread based on a reliable industrial process. The protocols for the production of AFB at industrial scale were set up and the resulting breads appeared very promising. VINI’s plant equipment suitable for the production of AFB experimental bread were: fermenters, mixers, leavening room (Temperature and humidity controlled), baking ovens (both shelf and trolley-chamber ones) and cutters.


b) KNOW HOW ON HEALTHY AND NUTRITIONAL PROPERTIES OF EINKORN SOURDOUGH
b.1 BIOACCESSIBILITY ANTIINFLAMMATORY AND PROTEIN DIGESTIBILITY IN EINKORN BREAD
These research results represent two very important characteristics in whole grain foods. In fact, as reported in D3.2, the antinflammatory effects were performed on the 6 AFB prototypes produced in SubTask 2.3.1. AINIA carried out gastrointestinal digestion of all AFB pro totypes for the planned analyses. For the in vitro study the anti-inflammatory effect of AFB in Caco-2 cells, AINIA carried out the anti-inflammatory bioassay on the digested samples to evaluate the activity of the 5 AFB prototypes in Caco-2 cells. UNIBO performed the analysis of inflammatory biomarkers on the biological samples provided by AINIA. Although all digested breads were evidenced to counteract the inflammatory response induced in Caco-2 cells by treatment with IL-1#, differences among breads were not huge. Anyway, the most relevant parameter in the determination of bread effect seemed to be the type of flour, breads made with ancient flour having a higher anti-inflammatory potential than those made with standard flour. Anti-inflammatory activity carried out by IL6 production evidenced a lower inflammatory status in HEK cells exposed to the supernatant derived from Caco-2 supplemented. The supernatant from AFCF and AFSAF supplemented Caco-2 cells showed the lowest inflammation status. However results regarding the impact of conventional, sourdough ancient and sourdough standard fermentation on the anti-inflammatory potential of breads were not univocal.
The in vitro study of the protein digestibility (IVPD) was performed by UNIBO. In summary, using both Bradford assay and SDS-PAGE no significant differences were detected in protein bio-accessibility of the different breads after in vitro digestion. Protein content in the blank digestion, due to digestive enzymes added during in vitro procedure, was high. Since the same amount of digestive enzymes was added during sample digestion than in blank digestion, this high amount could have masked differences among samples. Due to the uncertainty of in vitro data on antinflammatory activity, it was impossible to reduce the number of type of AFB. In fact all the 6 AFB instead of 5 analysed in vitro were also used for in vivo trials (as already reported in the First Report).

b.2 IN VIVO AFB EFFECTS
These results represent the final most important and complex experiment on AFB as fully reported in D.7. Specifically, the in vivo trial covered the evaluation of the impact of Einkorn flour bakery products on the intestinal microbiota, oxidative and inflammatory status. UNIBO carried out the in vivo trials and all the subsequent bioassays on the obtained pig samples (feces, urines, blood and tissues). The trial was completed correctly, the number of animals used was higher than the planned one (36 instead of 25 since and 6 AFB instead of 5) to be better able to distinguish between the effect of the Einkorn flour and of the sourdough fermentation. According to Task 4.1 results, the sourdough fermentation was more able than the conventional one to sustain the growth of pigs showing an higher nutritional content. As effect on gut microbiota ecosystem, the results indicated that the dietary treatments were able to modulate the swine gut microbiota structure, resulting in both common and unique patterns of variations. Such variations may be related to functional differences in the microbiome with a different impact on host physiology. Anyway, the different diet did not modify the level of antioxidant defences in pigs, apart from plasma GSH concentration that increased upon the dietary treatment in pig fed the standard flour bread with conventional fermentation, and TBARS level which evidenced a significant decrease in the group fed with the ancient flour bread with ancient flour sourdough fermentation. These findings seemed to suggest for a reduced lipid peroxidation. Moreover a NMR (Nuclear Magnetic Resonance) approach, not foreseen in the Annex 1, was utilized to investigate the evolution of the animal metabolome as a consequence of the diet, best followed by focusing on the fecal metabolome. The concentration of 8 molecules was found to be influenced by the flour, and 9 by the leaving agent. Overall our know-how on the healthy and nutritional properties of einkorn flour and einkorn sourdough bread have been significantly increased and, even if a low number of statistically significant differences were found, a consistent number of tendency have been detected. Results regarding the impact of ancient flour regard anti-inflammatory potential were not univocal, further studies will be necessary to better identify the effects of such as modifications in a challenging condition (e.g. after an inflammatory stimulus) to detect a different level of competence in the response of the host after a different alimentary regime. Moreove in Task 4.2 was performed by UNIBO the evaluation of the impact of novel ancient flour bakery products on the glycaemic index. Even if no statistically significant differences were detected, breads baked with ancient flours seemed to induce a lower and delayed glycemic peak. Moreover breads baked with ancient flours and/or sourdough fermentation did not stimulate insulin release. However results regarding the impact of conventional, sourdough ancient and sourdough standard fermentation on the glycaemic index of breads were not univocal.

IRON FORTIFIED BAKERY PRODUCTS

BAKE4FUN allowed SMEs to formulate innovative iron microcapsules characterized by an increased iron bioaccessibility to be used for the development of more homogenous and stable fortified flour mixes. The main project scientific and technological results attained to reach this goal are:
c) METHOD FOR MICROENCAPSULATION OF BIOACTIVE IRON that included c1) THE STUDY OF IRON BIOACCESSIBILITY IN DIFFERENT TYPES OF MICROCAPSULES, c2) SCALING UP OF PRODUCTION OF MI AT INDUSTRIAL SCALE

c.1 STUDY OF IRON BIOACCESSIBILITY IN DIFFERENT TYPES OF MICROCAPSULES
The methodology for microencapsulation and the prototypes for different types of microencapsulated iron were available and tested with the objective of creating novel prototypes of microencapsulated iron (as fully reported in D3.1). A microencapsulation process using spray drying was developed to produce microparticles containing iron confined within capsule walls. Protection against challenges induced by temperature, acidity and oxidation. In order to avoid reactions with other components in food, wall integrity of microcapsules treated at 180°C has been tested by SEM that evidenced no damages. In the first nine months the selection of different matrix for wall and core composition was performed in order to obtain microencapsulated iron (MI) suitable to resist to gastrointestinal challenges. A Dynamic Gastrointestinal Digestor (DGD) was used to simulate the gastrointestinal resistance of MI provided the information to identify and select the chemical form of iron, and covered material suitable to add in flour mixes to prepare bread at pilot plant scale. The results of the trials carried out in WP 3, in particular Task 3.1 were complemented with those obtained in WP2 as above described. On the basis of bioaccessibility of iron (FeII + FeIII) and relation Fe III/FeII in the soluble fraction, three MI were selected to fortify bread for the in vitro studies. After the first period it was decided to use microcapsules consisting of: 25% Ferrous sulphate, 25% ascorbic acid, and 50% thermoresistant modified starch in the following studies. The chosen MI were prepared using spray drying microencapsulation technology.

c.2 PRODUCTION OF MI AT INDUSTRIAL SCALE
After the lab scale study an implementation by EPSA procedure of MI production was performed in collaboration with AINIA, in order provide any fine adjustements required by the industrial scale production. As described in D5.1, high viscosity of the feed solution turned out to be a problem. The problem was overcame by increasing the temperature of feed solution in the mix tank, this modification allowed to lower starch solution viscosity. However this change in the procedure was associated with increase costs of drying. On the other hand application of higher temperature of feed preparation allowed to increase starch concentration that compensated costs of evaporation. The estimated cost of production MI5 at industrial scale is 11€/kg.


Furthermore, in order to produce homogenous and stable fortified flour mixes containing iron microcapsules BAKE4FUN provided to SMEs the following result:
d) PROCESS FOR EXPLOITATION OF ENRICHED FLOUR FOR PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS.
This BAKE4FUN result was accountable to two different foreground knowledge:
d.1) THE KNOWLEDGE OF FLOUR CHARACTERISTICS (see the above results “FLOUR CHARACTERISTICS” D2.1);
d.2) THE SELECTION OF MICROENCAPSULATION TECHNOLOGY based both on the organoleptic impact of iron fortified bread and to the effect of the different iron microcapsules and breadmaking process on iron bioaccessibility (in vitro). Specifically, after a first selection of a standard flour characteristics based on price, composition and technological properties, sensorial and functional traits of the related breads, the protocol for their production at industrial scale and their shelf life was provided including the eventual effects of the addition of selected microcapsules. These results enabled the SMEs to produce iron enriched flours stable at certain storage conditions and provided technological solution to support iron microcapsules avoiding negative effects on the sensorial characteristics as here described:

d.2a SELECTION OF MICROENCAPSULATION TECHNOLOGY: SENSORIAL AND FUNCTIONAL FFB
As reported in D2.2, in each sensorial analysis the panel was composed of more than 25 adults aged from 18 to 50. For bakery products 6 attributes to constitute a descriptive profile for the bread were used to evaluate the panel performance: colour, aroma, flavour, consistency, alveolation, and total acceptance. Intensity of the sensory attributes was rated on a discontinuous, structured scale from 0 (very low) to 7 (high). And for iron the perception of other flavour was also include on the sensory profile, it was rated on a discrete scale (Yes/No). A multi-dimensional profile was then drawn up to view the products’ sensory differences. The sensorial panel evaluation did not indicate significant differences between different breads in case of flavour, texture or odour. With regards to colour, slight differences have been detected with breads enriched with microencapsulated MI5 and microencapsulated MI9 in comparison to control bread however this change of the parameter did not implies any rejection of the product. According to the panellists, the breads from the conventional fermentation were well accepted. Iron fortification had a significant impact on the aroma and alveolation of bread samples. However, samples fortified with microencapsulated M5 presented the better acceptance, similar to the control.
d.2b SELECTION OF MICROENCAPSULATION TECHNOLOGY: IRON BIOACCESSIBILITY IN FFB
To achieve this goal, in Task 3.2 a gastrointestinal model (Dynamic Gastrointestinal Digestor, DGD) mimicking the digestive tract was used to digest 15 bread prototypes produced for subtask 2.4.1 was used with the same scientific approach adopted to study the iron bioaccessibility in different types of microcapsules (see before) and fully described in D3.1. This model is a dynamic computer controlled multi-compartmental system with adjustable parameters that simulates the physiological conditions of the stomach and intestine. This system was used to simulate the human gastrointestinal digestion of bread samples , its process during transit through the stomach and the small intestine, in order to:
- Evaluate of the IRON BIOACCESSIBILITY of iron in standard flour breads enriched with different microencapsulated iron
- Evaluate of the IRON BIOAVAILABILITY of iron by cell culture model to evaluate the intestinal absorption of enriched breads with different microencapsulated iron

IRON BIOACCESSIBILITY
The bioaccessibility and bioavailability of iron in standard flour breads enriched with different microencapsulated iron (M4, M5, M7 and M8) and produced with different fermentation processes (conventional and sourdough) were compared. Bread samples were digested with the final aim of selecting the enriched bread with the highest iron bioaccessibility/bioavailability. These breads were produced following the same baking process but with different amount of microencapsulated iron according to their iron content and the final amount expected in bread per 100g. The best bread in terms of bioaccessibility/bioavailability of iron was the one used in the in vivo studies in WP4.
In vitro digestion of each bread was performed using a single independent in vitro procedure to simulate the human digestive system via a three-step digestion (mouth, stomach and small intestine); furthermore, a blank digestion (without any food) was performed as control sample.
Digested breads were used to carried out the iron bioaccessibility study and bioavailability with Caco-2 cells, analysing the iron uptake. Specifically cellular assays were performed to study the iron uptake as ferritin, with differentiated cells 14-16 days after seeding. Cells were incubated 2h with the samples, and later samples were removed and culture medium was added for additional 22h (to ferritin formation). Then cells were harvested for ferritin analysis. Furthermore for iron transport, a CacoReadyTM kit was used in cells treated with the digested breads . Cells were also collected to gene expression analysis by Real time PCR measure of DMT1 and IREG1 mRNA expression. Finally for the ferritin analysis. an immunocolorimetric assay (Ferritin Assay RAMCO) was used to measured Caco-2 cell ferritin content. Total iron and its species (FeII and FeIII) were carried out by the partner TUL (University of Lodz). The iron content was analysed at the beginning and at the end of the simulated gastrointestinal process and in cell samples (in the apical and the basal media). In order to preserve all the characteristics and quality of the samples and avoid any possible change of the iron content, the samples were frozen and sent in dry ice to TUL (University of Lodz)

IRON BIOAVAILABILITY
Bioaccessibility refers to the amount of a bioactive compound that is soluble and can be absorbed in the intestinal tract, expressed in percentage. Thus,

% Bioaccessibility (BA) = soluble iron (FeII + FeIII) in the intestinal digested (mg/100g bread) / total iron in bread (mg/100 g bread) x 100

In the trials, the aim was to evaluate the bioaccessibility of the Iron (as total content and FeII+ FeIII) after the gastrointestinal digestion in order to estimate the amount of iron available to be absorbed at the small intestine. Thus, iron (total and FeII and FeIII) was analysed in bread before digestion and after digestion (digested samples)
The bioaccessibility of iron (FeII+FeIII) obtained in bread produced with the three different fermentation processes (conventional, sourdough with standard flour and sourdough with ancient flour) after the gastrointestinal digestion was compared. The highest values of bioaccessibility of iron (% of Iron available to be absorbed in the small intestine) correspond to breads produced with conventional fermentation, namely FFCFM4, FFCFM5, FFCFM7 and FFCFM8 ranged between 75 and 95%.
In addition, amongst the bread samples produced with conventional fermentation, those with the best bioaccessibility results (over 90%) were FFCFM4 and FFCFM7. In fact, breads enriched with M4 (35% ferrous sulphate+65% TMS) and with M7 (15% ferrous lactate+85%TMS) evidenced the highest bioaccessibility.
The results showed that the bioavailability of the iron depends on the bakery process and the formula of the microcapsule. Among the FFBs developed with conventional fermentation (FFCFs) the prototype FFCFM5 presented the highest % iron transport efficiency (10,63%), followed by FFCFM4 and FFCM7 (2.7 and 2.4 respectively), being the FFCM8 the one with the minor transport. Regarding the ferritin in these prototypes, no ferritin was detected.
The FFBs prototypes with sourdough fermentation (FFSF) presented less efficiency of iron transport than the other type of bakery production with less than 1.5%. Among the microcapsules used, FFSFM4 and FFSFM5 showed more iron transport than FFSFM7 and FFSFM8. About the iron uptake, FFSFM5 was the only sample where ferritin was detected which it could explain the less iron transport because the iron is mainly stored.

The comparison among the three types of bakery production showed that those breads enriched with microcapsules M4 and M5 were more efficient in iron transport than M7 and M8. Among all FFBs, basing on the data of % of iron transport efficiency, the FFCFM5 (Bread made with Standard Flour + Conventional Fermentation+ Microcapsule M5), FFCFM4 (Bread made with Standard Flour + Conventional Fermentation+ Microcapsule M4), FFCFM7 (Bread made with Standard Flour + Conventional Fermentation+ Microcapsule M7), and FFAFM4 (Bread made with Standard Flour + Sourdough Fermentation with Ancient Flour+ Microcapsule M4) showed the highest efficiency (more than 2%). Finally, FFSFM5 (Bread made with Standard Flour + Sourdough Fermentation with Standard Flour + Microcapsule M5), the highest uptake (as ferritin storage).
According to the above results and taking into account sensorial analysis (detailed in deliverable D2.2) THE SELECTED BREAD FOR IN VIVO TRIALS WAS FFCFM5 (bread produced with standard flour and conventional fermentation enriches with M5)
Due to the uncertainty of in vitro data on antinflammatory activity, it was impossible to reduce the number of type of AFB. In fact all the 6 AFB instead of 5 analysed in vitro were also used for in vivo trials (reported in the First Report).


e. METHOD FOR STABILIZATION AND HOMOGENEOUS DISTRIBUTION OF MICROENCAPSULATED BIOACTIVE IRON IN STANDARD FLOURS
The protocol for their production at industrial scale and their shelf life was provided including the eventual effects of the addition of selected microcapsules. These results enabled the SMEs to produce iron enriched flours stable at certain storage conditions and provided technological solution to support iron microcapsules avoiding negative effects on the sensorial characteristics. The stabilisation and homogeneous distribution of microencapsulated bioactive iron in standard flours was standardised and a protocol was delivered to produce them at industrial level, on the basis of the following findings:
e.1 PROTOCOL FOR FF PRODUCTION AT INDUSTRAL LEVEL (reported in D5.2) describing the protocol for the production of the FF (Functional Flour mix) for the manufacturing of breads at industrial scale. This has been developed on the basis of the main following aspects: e.1a) STANDARDISATION OF PRODUCTION and e.1b) the SET UP OF STORAGE CONDITIONS in order to obtain the particle size distribution of flour similar to the other ingredients.
e.1a PROTOCOL FOR FF PRODUCTION AT INDUSTRAL LEVEL
Included the set up of formulation according to final product and the physical-chemical characteristics of the ingredients, mixing phase (minor and major ingredients separately) in order to avoid segregation, to obtain a homogeneous mixture of the components of the formula; finally a Ribbon bleder of 1000 L of volume (ingredients 2/3 of total volume) was used at 25rpm of speed for 25 minutes.

e.1b STANDARDISATION OF PRODUCTION
The conditions of FF storage are the same than the flour. In fact relative humidity, temperature and time of storage are the principal conditions we should consider.
During storage, changes occur in flour by its enzyme content and micro-organisms. Starch is decomposed into soluble sugars. The protein and lipids are decomposed and the acidity increases. Although some of the lost moisture will eventually be reabsorbed during storage at higher relative humidity. The rate and intensity of the changes vary with different grades of flour and normally occur more rapidly and to a great extent as the flour’s moisture content is increased above 12 %. Increasing relative humidity and ambient temperature, the growth of microorganisms, insects, and the degree of physicochemical changes in the flour increases.
Excessively prolonged storage periods, ultimately lead to a deterioration of the flour’s baking quality due to reactions taking place during storage.
The required conditions for a good storage along 90 days was low relative humidity (60%), temperature of 20-24°C, good ventilation and high standard of hygiene conditions.


Finally, BAKE4FUN resulted in providing to SMEs a
f. PROTOCOL TO EXPLOIT THE SELECTED ENRICHED FLOURS TO PRODUCE HIGH COST-EFFICIENT IRON FORTIFIED BAKERY PRODUCTS
It provides to the knowledge of the iron intake necessary to counteract iron depletion by the iron fortified bakery products newly developed. This result was attained by two main research activities focused on understanding the effect of iron fortified bread (FFB) produced at industrial scale on safety and in vivo functional properties and specifically: f.1) SET UP OF A PROTOCOL FOR THE PRODUCTION OF AFB AT INDUSTRIAL SCALE described in D5.4; f.2) IN VIVO STUDY ON THE IMPACT OF FFB ON OVERALL HEALTH PARAMETERS fully reported in D4.8.

f.1 SET UP OF A PROTOCOL FOR THE PRODUCTION OF AFB AT INDUSTRIAL SCALE
To release protocols for the production of FFB at industrial scale very fine tuning activities were performed since the lab scale process fitted almost perfectly the industrial plant conditions. Cottage bread” (Chleb wiejski) recipe has been choosen at the beginning of the project for testing because it contains no additives (improvers, fats, sugars etc.). This approach was adopted to be sure to produce an innovative bread based on a reliable industrial process. The protocols for the production of FFB at industrial scale were set up and the resulting breads appeared very promising. Plant equipment suitable for the production in bakery VINI of FFB suitable to produce experimental bread were: fermenters, mixers, leavening room (Temperature and humidity controlled), baking ovens (both shelf and trolley-chamber ones) and cutters.

f.2 IN VIVO STUDY ON THE IMPACT OF FFB ON OVERALL HEALTH PARAMETERS.
f.2a An evaluation of iron bioavailability and impact of novel bakery products enriched by iron on the intestinal microbiota, oxidative and inflammatory status was performed. For the evaluation of iron bioavailability and impact of novel bakery products enriched by iron on the intestinal microbiota, oxidative and inflammatory status UNIBO carried out the in vivo trials (Task 4.3). The subsequent bioassays on the obtained pig samples (feces, urines, blood and tissues) were performed in part by UNIBO and in part by TUL (iron content). The trial was completed, 24 animals were used instead of 28. This was related to the increased number of animals utilized in task 4.1 and to the necessity to don’t exceed the total number of animals in the Italian Ministry Authorization. Moreover, in order to be able to control precisely the quantity of administered Iron, it has been decided to avoid the use of a regular iron-enriched diet in our positive control group.
Whereas the three groups of anemic piglets had similar initial blood Hb and HCT concentration, only piglets eating iron enriched breads showed important increases in variables such as HCT, Hb and RBC when compared to piglets fed with standard diet and shows statistically significant differences between T0 and T7 (iron replacement period).
The iron content of intestinal mucosa (iron absorption site) and liver (iron storage site) were higher in the groups eating enriched bread without statistically significant differences between microencapsulated and free iron. The iron transporters (IREG1, DMT1) at duodenal mucosal level did not show significant statistical differences among groups but showed a relatively higher value in pig of group eating bread with microcapsules. Fecal iron content showed similar levels at T0 among groups and a significant difference between T0 and T7 in the two groups that received iron integration, so not all the supplementation was absorbed by the animals. The Hepcidin gene expression in liver, that positively correlate with plasmatic iron saturation, did not show significant statistical differences among groups, but a progressive decreasing trend starting from anemic pig eating bread with microencapsulated iron, group with free iron and anemic group eating standard bread is detected. Finally ferritin, that closely reflects the size of iron body stores, did not show statistically significant differences among groups but anemic pig eating bread with microencapsulated iron seems to have the highest values.
The results indicated that the dietary treatments were able to modulate the swine gut microbiota structure, Principal Coordinates Analysis of both unweighted and weighted UniFrac distances resulted in a segregation between the piglet gut microbiota profiles at T0 and T7, the different treatments were able to modulate the piglet gut microbiota structure, resulting in both common and unique patterns of variations. Such variations may be related to functional differences in the microbiome with a different impact on host physiology.
Overall our know-how on the healthy effects of microencapsulated iron fortified bread and iron bioavailability have been significantly increased and, even if a low number of statistically significant differences were found, a consistent number of tendency have been detected. The microencapsulated iron effects are not distinguishable to those of the same amount of free iron avoiding the negative effects of free iron supplementation.

SAFETY CONCERNS
Experimental bread resulted safe against the main pathogen bacteria suitable to survive and develop in bread were analysed one day after their baking (fresh consumption). Moreover also a “Challenge test“ was performed on the last period by inoculating a cocktail of different molds to bakery products (Aspergillus, Fusarium and Penicillium genus) in order to test if selected AFB and FFB formulations and processes may represent a good basis for longer shelf life products. With no further preserving factors, just einkorn sourdough bread evidenced a good potential as a longer shelf life bread, in the other cases any preservatives (sorbate, packaging, ethanol in head space of package, etc.) or further strategies are recommended to prolong the shelf stable similar to other benchmark products present in the marketplace.
Potential Impact:
For the SMEs partners of the BAKE4FUN consortium, there are clear and demonstrable benefits to be gained from the successful implementation of the successfully developed technology. These benefits are applied to food producers and retailers, as well as firms within food sector. The overall impact of this consortium was to place SMEs within the European food industry in a highly competitive position in both the national, European and international markets thereby creating jobs, increasing revenues to allow further investment and technological developments, while, improving the health of citizens/consumers. The impacts evidenced are on: food ingredients companies, food bakery industries, and other adjacent food industries will profit manufacturing healthier products for a particular group of consumers, the elderly because of their iron depletion or anemic disease. Functional foods (FFs) represent one of the most interesting areas of research and innovation in the food industry (Jones and Jew, 2007). Although a generalised positive public perception of FFs in general, there is evidence that consumers differ in the extent to which they buy specific food products with functional ingredients, showing a marked preference for bakery products. Indeed, bread and bakery products have recently become one of the main vehicles for functional ingredients. In addition, whole grains and other ancient flours naturally contain functional ingredients, including phytochemicals (phytic acid, glutathione, and phytosterols), as well as dietary fibre (Inulin and beta-glucan), which yield health benefits, which could not be consumed in white bread alternatives. In the light of this scenario, BAKE4FUN project tackles the need of developing novel functional bakery food formulations, which could be more easily adopted in the everyday diet, since the importance of facilitating the inclusion of healthy foods in consumers eating habits is well demonstrated (JPI Healthy Diet for Healthy Life, SRA). BAKE4FUN result represent a significant potential impact on the bakery and functional food industry, which forms an integral part of the multi-billion dollar food and beverage industry. In particular, well-established European bakery industry is mature and characterised by a high degree of penetration and consolidation, thus resulting in a competitive landscape composed by numerous large companies and SMEs that vary widely in terms of their product offerings and technology platforms. A strong emphasis on new product developments and innovation in the bakery industry has also compelled ingredient manufacturers to constantly invest in R&D in order to better respond to the needs of bakery manufacturers. In particular, some of the main factors that have been demonstrated to determine the competitive success of ingredient manufacturers include innovation/differentiation, product portfolio, availability, functionality and biological activity of FFs, to mention a few. In this perspective, the implementation of BAKE4FUN results provided an opportunity for SME participants to obtain increased benefits in terms of their market position and competitiveness. Each SME is the owner of some project results suitable to increase its turnover in the short, as well as mid-long term. With that aim different strategies may be used: i) direct selling of the newly developed products studied within BAKE4FUN; ii) selling of the newly developed products studied within BAKE4FUN to other food transformers and manufacturers; iii) licensing of the new technologies developed by BAKE4FUN to other food transformers and manufacturers; iv) in-house development of new bakery products based on the innovative technologies developed by BAKE4FUN. Each separate strategy, or their integration, provided a mean to consolidate the domestic market share for the SMEs composing the BAKE4FUN partnership. Additionally, the BAKE4FUN results can facilitate the expansion of the SMEs partners into the wider EU market. Before the BAKE4FUN starting, only INDESPAN and EPSA had already acceded to international market commercialisation of their ingredients, whilst PROMETEO and VINI had not yet marketed their bakery products outside their own domestic markets. The adoption of breakthrough technologies for the innovation of functional products with a relevant predicted marketing potential will allow INDESPAN and EPSA to consolidate their European positioning and, at the same time, will give the opportunity to PROMETEO and VINI to profit from new bakery products entering in the EU market.

DISSEMINATION
The primary target population of end users for the B4F project are general consumers and professionals influencing purchase option and healthy habits adoption in the population as nutritionists, dieticians, medical professionals, but also food distributors, retailers and food suppliers. As a consequence, the BAKE4FUN consortium aims at disseminating and exploiting the new knowledge generated within B4F to national and international Food and Health Forum, Networks, Stakeholders and the other target groups reported in section 2.1.1.The Bake4Fun partners also plan to place advertorials and technical articles in industry journals or industrial associations newsletters.
Considering the target populations listed above, the following types of dissemination media have been identified to ensure most effective communication with end users and other stakeholders: website, press releases, newsletters, events, publications and media.

- WEBSITE
A project website (http://www.bake4fun.eu) was designed and implemented during the first months of the project and updated at least every 3 months with more content added as the project develops. This public web site provided general information on the project, including the project cycle and the project objectives, the partners’ pro files and the general framework in which the project is set both from a research perspective and a policy point of view as well as links to partners’ websites. The website is an information point for the work of the project itself (including details of partners, objectives, work areas, results, interim results and working papers, public delive rables, etc.) and for related fields of work (links to other projects, services, collaborative efforts, etc., relevant to each part of the project). The website is projected to give specific information on the project advancement and results. The public web site is one of the main channels for wider public information dissemination and may contain some of the information also available on the partners' site, subject to partners' agreement. Additionally, abstract translation (Italian, Polish, Spanish and Chinese) are downloadable. The site has three levels: the first one is the public website frontend where visitors will find information, media and other documents. A reserved area is provided for all collaborators. In this part of the site it is possible to find reserved information.

- NEWSLETTER
Four different issues of B4F newsletter was prepared and distributed by email to the mailing list of stakeholders or subscribers from the website. It included information about the project structure. Information and newsletters on the project were sent to an increasing number of contacts. A list of associations for dissemination purposes was pro vided. This activity resulted very useful to participate to different UE project forums (Healthgrains, Tradeit, TrafooN).

- EVENTS
To reach the widest audience as well as the general public specific many actions were undertaken (website, logo, Expositions, Conferences, Newsletter, press rel eases, audio/video media etc.). In particular a big effort has been devoted by the SMEs and RTD involved in B4F to promote the project results at international fairs, expositions, conferences in the field of bakery products and functional foods also to increase the opportunity to find new potential partners interested in know-how transfer. Moreover someone of these events (EFFOST 2014 and EFFOST 2015) represented an occasion to meet other UE project Coordinators during some special session of project result presentation (Healthgrains, Tradeit, TrafooN, LEO) and to have contact for future UE grant applications.
During the two year more than 40 events including fairs, exhibitions, conferences and trade shows the RTD and SME members spent big efforts to promote the project results among the stakeholders in the field of bakery products and functional foods. The importance of fairs and tradeshow is constantly increasing, as fairs are communication tools, having a large commercial impact in terms of creating opportunities to enhance the distribution of new products or services. Participation to food fairs also increased the opportunity to find new potential partners interested in know-how transfer (e.g. by buying license). To enhance the effectiveness of the participation to international fairs, B4F SME Participants developped specific marketing tools, including presentations and publications. Similarly, to strengthen the presence and the exploitability of the project results on the web, social marketing tools were developed.

- PUBLICATIONS
Moreover around 15 publications and press releases were used to disseminate the project results through publications in specialised journals at national and international level.

- VIDEO
Finally a short video (cartoon) including the main results was presented during the final meeting and uploaded on the website. The video is in English but subtitled in Spanish, Polish and Italian. In the website is available also a summary in Chinese, Spanish, Polish and Italian.

ECONOMIC IMPACT
The economic impact of the was assessed. In fact, the participant SMEs expcted the project results impact their business according to their respective business plans reported in Del 6.4.

- PROMETEO benefit from the selection of novel enriched ancient flours on the basis of the presence and activity of functional compounds with beneficial properties for human health. This allowed PROMETEO to scientifically substantiate general function health claims to be used in the EU market. Furthermore, the study of the influence of food transformation procedures on the nutritional and sensorial characteristics of the bakery products obtained by the selected ancient flours, allowed PROMETEO to select the best process parameters to be adopted at industrial scale for the development of innovative bakery products. This enabled PROMETEO to strengthen its knowhow, at the light of the eventual investment for the set up of internal laboratory for the production of bakery products. PROMETEO basing on the future harvest yield data previewed to increase of about 170 Tons their production with a further increase of about 20% for the second and third years generating a total of around 87000 euro of profit increase in the next 3 years.

- INDESPAN: benefit from formulation of innovative microcapsule characterized by an increased mineral bioaccessibility, used for the development of more homogenous and stable fortified flour mixes. This allows INDESPAN to strengthen its positioning in the current food ingredient market, by exploiting newly developed ingredients at higher added value and with novel scientific-technological characteristics giving INDESPAN an industrial competitive advantage. Furthermore, BAKE4FUN will allow INDESPAN to enlarge its industrial portfolio, with the possibility to expand from the food ingredient market to the nutraceutical and bakery one. INDESPAN after 5 years of producing and commercialising the new ingredient will achieve 180.000 euros of profit by increasing progressively the incomes from 10% up to 30% per year with a gross margin between 20 and 25%. The first year a commercial launching cost of around 80.000 euros is planned.


- EPSA benefit from formulation of innovative microcapsule characterized by an increased mineral bioaccessibility, used for the development of more homogenous and stable fortified flour mixes. EPSA is currently active in the production of ingredients and food additives to be included in food formulations, including bakery products. The results of BAKE4FUN allows EPSA to enlarge the industrial portfolio, with the inclusion of innovative and more bioaccesible microencapsulated iron among the commercialised food ingredients. Furthermore, the acquired knowledge in relation to the technologies for the innovation in classical microencapsulation procedures might allow EPSA to adopt these new technologies for the development of novel ingredients, based on the microencapsulation of other minerals, bioactive compounds, and ingredients with high added value. EPSA estimated stotal profit of the project results in 1.180.000 euros after 5 years producing and commercialising the ingredients. The profit will start during the second year of the commercialisation of the new ingredients.

- VINI: benefit from the study of the influence of food transformation procedures on the nutritional and sensorial characteristics of the bakery products obtained by the selected ancient flours. These results provided new know-how suitable to select the best process parameters to be adopted at industrial scale for the development of innovative bakery products and specifically related to the breadmaking procedure, to be applied to innovate and improve the characteristics of the currently produced healthy breads and bakery products. Furthermore, the study the influence of breadmaking and food transformation procedures on stability of iron microcapsules, nutritional and antioxidant characteristics and bread sensorial quality obtained from fortified flour mixes is going to improve the VINI industrial portfolio, producing novel bakery products characterized by the inclusion of microencapsulated minerals and, eventually, other functional molecules or bioactive compounds. VINI after 5 years of producing and commercialising the new products planned for a total profit of around 180.000 euros progressively achieved from the second half of 2016.

PROJECT RESULTS
The project results being prototypes and the exploitation principle being ownership, the SME participants are given the full freedom to exploit the prototypes for further business prospect with customers, social acceptance studies, development of specific applications, etc. This scheme exactly corresponds to the request of the SME participants involved in the project.
A. PROCESS FOR EXPLOITATION OF EINKORN FLOUR FOR PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS
As a consequence of results acquired on functional compounds of einkorn flour, it was confirmed its healthy characteristics such as highest polyphenol content and high antioxidant activity due to the high amount of lutein and zeaxanthin). Moreover a new process for the exploitation of einkorn flour for production of functional bakery products was developed. The einkorn flour and bread prepared using wholegrain einkorn satisfy the conditions for a healthy product. On the basis of these results Prometeo launched a new line of flour and bakery products based on einkorn flour studied in BAKE4FUN (var. Monlis) available in the online market (http://shop.prometeourbino.it/collections/linea-farro-monococco)
Impact: PROMETEO scientifically substantiated general function health claims to be used in the EU market of Monlis einkorn flour. Furthermore, the study of the influence of food transformation procedures on the nutritional and sensorial characteristics of the bakery products obtained by einkorn flour, allowed PROMETEO and VINI to select the best process parameters at industrial scale. In particular bakery products, based on the use of einkorn flour rich in antioxidant compounds, evidenced anti-inflammatory properties and positively influencing the glycemic index. PROMETEO and VINI to strengthen their know- how in order to directly produce (VINI), or to invest in an internal laboratory or to outsource the production (PROMETEO). On the basis of these results Prometeo launched a new line of bakery products based on einkorn flours studied in BAKE4FUN available in the online market (http://shop.prometeourbino.it/collections/linea-farro-monococco).


B. KNOW HOW ON HEALTHY AND NUTRITIONAL PROPERTIES OF EINKORN SOURDOUGH BREAD
Overall the SMEs know-how on the healthy and nutritional properties of einkorn flour and einkorn sourdough bread have been significantly increased not only in their composition as previously described. From in vitro results a lower inflammatory potential of einkorn and sourdough fermentation was evidenced. On the other hand, by in vivo results, even if a low number of statistically significant differences taking into consideration the pig variability of response to a diet, a consistent number of diet-related tendencies was found, mainly as consequence of the interaction of einkorn flour and sourdough process such as: 1) reduction in insulin release; 2) better ability to sustain the swine growth; 3) modulation of gut microbiota and of the fecal metabolome. Impact: these aspects represent a significant scientific baseline that could be exploited by SMEs to implement better focused clinical trials aimed to substantiate a health claim.


C. METHOD FOR MICROENCAPSULATION OF BIOACTIVE IRON
The project results on microencapsulation of bioactive iron will allow EPSA to reinforce that new health line in its business and the access to new markets that may demand this type of ingredients in different ways. Therefore the exploitable foregrounds are:
1) A new iron microencapsulating protocol for bakery products based on a core containing ferrous sulfate and ascorbic acid encapsulated by a thermoresistant starch by means a spry drying technology process. The new method allows Epsa to produce microcapsules by a specific formulation and technology selected on the basis of their highest iron availability and lowest negative impact on sensorial properties. The selection included also breadmaking process in order to optimise all the whole chain factors contributing to the iron availability and sensorial impact.
2) the protocol for the production of encapsulated iron provides EPSA with a technical basis to innovate in the microencapsulation of other minerals and bioactive compounds also due to the low price of the selected type of iron microcapsule ( the estimated cost of production MI5 at industrial scale is 11€/kg) that will be exploited as a basis to further tests with other ingredients with high added value such as other minerals, bioactive compounds and ingredients.
Impact: the formulation of innovative iron microcapsules characterized by an increased bioaccessibility, allowed EPSA and thereafter INDESPAN to include in their porfolio innovative and more bioaccessible microencapsulated iron launched with the commercial name of EPSAFERRO based on a cost- efficient and therefore competitive technology .



D. PROCESS FOR EXPLOITATION OF ENRICHED FLOUR FOR PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS
Enriched flours will be exploited to produce new functional bakery products on the basis of the following obtained results: highly bioavailable microencapsulated iron, stable flour mixes containing bioactive iron and baking process optimized to maximize sensorial impact and iron bioavalability. In fact, the in vivo trials delivered some important results: 1) experimental model of spontaneous anemia was clearly established and useful to answer to the experimental questions about bioavailability, absorption and function of iron in the new bakery products. The higly cost-efficient approach could be suitable to asses different newly developed products by SMEs; 2) Iron from enriched breads was equally bioavailable and absorbed in comparison to free iron. That means that the microencapsulation technology, the enriched flours and the breadmaking process may guarantee a high iron availability (>60%) and the same ability of free iron to rescue the anemic condition of the bread fortified without the negative aspects related to free iron ingestion.
Impact: EPSA and especially VINI and INDESPAN acquired the knowledge to increase their turnover from the pre-mixed iron fortified flours optimized to functional bakery products and the breadmaking parameters aimed to retain bioactive iron stability and consequently bread functionalities.


E. METHODS FOR STABILISATION AND HOMOGENEOUS DISTRIBUTION OF MICROENCAPSULATED BIOACTIVE IRON IN STANDARD FLOURS
Different iron microcapsules were studied (9 different types) ranging between 0,20%-0,55% of standard flour depending on the MI composition. The adjustment in enriched flours was made to be able to obtain 20 mg of iron in 100 g of bread. The protocol for the production stable and homogeneous fortified flours has been set up on the basis of the scaling up and validation of Task 2.3 results reported in Del 5.2. According to tests carried out at the pilot plant, the conditions of production at industrial scale are adequate to obtain a homogeneous mixture to ensure adequate iron content in the final bread. Results obtained by a storage test, to assay the stability of FFS along a 3 months period assayed the stability of shelf life in terms of microbiological and pests concerns, sensorial and chemical characteristics (i.e., rancidity) and moisture. Moreover also the colour was retained in the storage period of standard flour enriched by the 9 microencapsules. The protocol for the production of the FF (Functional Flour mix) for the manufacturing of breads at industrial scale includes the following main aspects: formulation, mixing parameters and storage conditions. The required conditions for a good storage along 90 days was low relative humidity (60%), temperature of 20-24°C, good ventilation and high standard of hygiene conditions. In these conditions enriched flour mixes may be commercialised for at least 3 months.
Impact: EPSA and especially INDESPAN included in their portfolio an innovative and more bioaccesible microencapsulated iron optimized for functional bakery products. This technology is suitable also for the development of novel ingredients, based on the microencapsulation of other minerals, bioactive compounds, and ingredients with high added value and to use those to provide new flour mixes for INDESPAN.


F. PROCESS FOR EXPLOITATION OF A COMBINATION OF EINKORN FLOUR + ENRICHED FLOUR FOR THE PRODUCTION OF FUNCTIONAL BAKERY PRODUCTS
Microencapsulation technology, stability of enriched flours and the breadmaking process were validated also for bakery products combining einkorn and fortified flour. However, to produce a functional bread maintaining both the healthy characteristics of einkorn flour sourdough processed and the iron bioavailability further implementation activities are necessary to improve sensorial impact. In fact the acidity produced by sourdough process seem to induce a negative perception of added iron. Two main solutions need to be tested: a) to reduce the acidity of dough by a shorter fermentation time or a lesser percentage of sourdough; b) to mask off flavours by the addition of flavouring additives that must be shaped on the basis of the final bakery product.
Impact: PROMETEO, INDESPAN and VINI strengthen their know-how related to functional foods by facing a generalised positive public perception of functional bakery foods by exploit results on functional properties of einkorn flour in combination to iron fortification to obtain a class of multifunctional bakery products as vehicles for functional ingredients.

List of Websites:
http://www.bake4fun.eu/

Contact

Gianotti, Andrea (Researcher)
Tel.: +39 0512096577
E-mail
Record Number: 188000 / Last updated on: 2016-08-18