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Development of foods containing nanoencapsulated ingredient

Final Report Summary - NANOFOODS (Development of foods containing nanoencapsulated ingredient)

Executive summary:

NANOFOODS project adopted encapsulation technology to preserve food bioactive compounds during processing and storage, and to achieve their controlled release in the GI tract. Tailored nanocapsules, able to deliver omega-3 and silymarin complex into the lower gut, was designed and produced by an Israeli SMEs in collaboration with Technion.

Project Context and Objectives:

The overall objective of the project is to produce and validate the function of conceptually new healthy foods based on nanocapsules technology.

Nanotechnology and nanosciences stems from our ability to understand physical and chemical phenomena and processes at the molecular as well as atomic resolution. The preservation of bioactive food ingredients through product processing and storage, and their controlled release in the gastrointestinal tract is yet a major obstacle for the full exploitation of the health potential of many food bioactive components. In this framework the use of polysaccharide nanocapsules represent a new frontier in food formulation. Using appropriate polysaccharide it is possible to protect the bioactive compounds during processing and storage and release them after stomach transit or only in the lower gut. Moreover, the use of nanocapsules overcomes the problems related to the stability of the bioactive compounds and, using the appropriate conditions, does not impose any sensorial acceptance problems. In this way nanoencapsulation will offer the possibility to add bioactive ingredients also in foods undergoing thermal processing or long-term storage.

In summary NANOFOODS project set up the following objectives
-To translate pilot scale technology to industrial production of nanocapsules
-To provide physical and chemical data on the performance of the nanocapsules containing different bioactive compounds
-To develop functional dry pasta/nut cream/bread products by incorporating into the recipe the nanoencapsulated ingredient.
-To elaborate guidelines for the use of nanoencapsulated ingredients in the dry pasta/nut cream/bread products.
-To set up appropriate process for production of functional pasta, bread and nut cream by fine tuning of the conventional ones
-To provide a technological, chemical and sensorial characterization of the developed nanoencapsulated pasta, nut cream and bread products
-To perform a human randomized control trial to verify the efficacy of the functional pasta and bread to delivery bioactive compounds to the human gut.
At the end of the project all of them as been fully achieved

Project Results:

The S &T activity of the project were organized in 7 WPs as follows:
WP 1 Development of food nanocapsules
WP2 Characterization of food nanocapsules
WP3 Development of functional dry pasta using bioactive nanocapsules as ingredient
WP3 bis Development of functional nut cream/vegetable oil using bioactive nanocapsules as ingredient
WP4 Development of functional bread products using bioactive nanocapsules as ingredient
WP5 Characterization of food produced with nanoencapsulated ingredients
WP6 Human trials with IBD patients using pasta and bread produced with nanoencapsulated ingredients

Below the main results and achievement for each WP

Work package 1: Development of food nanocapsules
Objectives
To translate pilot scale technology to industrial production of nanocapsules
Development of prototype nanocapsules on a pilot scale

The aim of this step was to create the complexes in a pilot scale with a continuous process that will be suitable for scale up for the industry, as well as for the preparation of large samples for product formulations, testing, and in-vivo experiments. In order to form sub micron capsules, the process has been performed using dual feed jet homogenizer. To develop the prototypes of the nanocapsules different combination of core and capsule materials were used: HACS (coating), Flax seed oil, Omega-3 Ethyl Esters, Milk thistle seed (sylimarin)

Two techniques were examined:
1. Freeze-dried complexes preparation
Suspension was collected for the production of powder which was done by centrifugation (5500g, 20min, 0-10 °C), freeze dried at a constant controlled shelf temperature of -30°C and 0.1 mbar for 48 hours and grinded into a fine powder which was then used for further analyses.
2. Spray-dried complexes preparation
Suspension was collected for the production of powder which was done by Spray Dryer, operating at inlet temperature of 200°C, and outlet ranging from 70-90°C. Around 50gr of powder was collected and then used for further analyses. Batches were produced up to 500 gr per batch.

Development of industrial process for the production of nanocapsules
In this part of the project, the methodology used to produce the prototype nanocapsules was used to produce industrial batches of Kg's scale. This step was a straight forward step, as the scale-up in this case was linear. This was obtained by using a two step process, in which nanocapsules suspension is first produced using the Micro DeBee homogenizer, followed by spray drying to obtain a free flow powder.

Work package 2: Characterization of food nanocapsules

Objectives
To test and provide real time feedback on the performance of the nanocapsules
The complexes produced in the process described above were studied both structurally and functionally in order to test feasibility of the continuous process, and to verify complexes ability to serve as controlled delivery system for bioactive compounds. The scientific approach in this study composed of characterization of the physical and chemical properties of the complexes. The analysis and characterization were performed for at least one representative sample for each omega-3 source. This part of results is aimed to select the most promising encapsulation formulation and procedure.

Physical characterization of complexes
The following sections bring forth the results pertinent to complexes structure, morphology and physical state, particle size, and thermal properties.

Capsule stability under thermal and oxidative stress.
DSC was applied to test the thermal properties and dissociation temperatures of starch-omega-3 complexes. The melting temperatures of starch-omega3 complexes, as well as the enthalpies were calculated. The melting temperature (Tm) was defined as the temperature at the maximum of the peak, and the enthalpy (DeltaH) was calculated by integration the area under the peak. The enthalpies were calculated as J per g of dry sample. The onset temperature (Tonset) indicates beginning of the event. The DSC thermograms contained two endotherms. In the first event, the melting point of all the samples was higher than 90°C, and the enthalpies ranged from 1.9 to 2.71 (J/g). In the second event the melting point of all the complexes was approximately 144°C, and the enthalpies ranged from 1.64 to 3.77 (J/g). For HACS processed sample obtained melting point was slightly lower about 132°C.

The DSC thermograms of sylimarin and polyphenol complexes contained two endotherms. In the first event, the melting point of these samples was higher than about 90°C, and the enthalpies ranged from 0.7 to 2.7 (J/g). In the second event the melting point of all the complexes was about 141.4-146.5°C. The enthalpy of the complexes containing polyphenols (pH 4.9) and silymarin (spray dried) was 13 (J/g). The polyphenol (freeze dried) samples had a much lower enthalpy,about 0.01 (J/g). The HACS processed samples obtained a much lower melting point about 132°C and the enthalpy was about 1.7 (J/g).

The chemical properties of the molecular complexes in order to select the best formulation were measured. The hypothesis was that molecular complexes of ligand and starch can provide guest that is protected from oxygen and heat, and control its release in the digestive tract due to amylolitic activity. This part of the study was designed to measure the extent of protection during food processing, consumption and shelf life. Release of the nutrient was estimated under high temperatures, different pH and enzyme digestion treatments, simulating different conditions in processing foods and along the gastrointestinal tract. In addition, stability during shelf life was tested at ambient and elevated temperatures.

In order to find the best encapsulation formulation three different sources of omega-3 were used: Flax seed oil, Ethyl esters and Fish oil. To verify omega-3 content and compare these values to those declared by the manufacturer, samples of the oils were methylated (except for ethyl esters) and run by GC analysis. The only prominent difference could be observed for the ethyl esters sample, all other values were insignificantly different or slightly higher from the declared values.

Functionality at simulated GIT conditions: omega-3
In addition to stability under thermal treatment, functionality of the complexes is also expressed in the bioactive performance, retention and release in the stomach and GIT accordingly. It is suggested that the complexes are stable in the stomach and can provide control release of the guest in the digestive tract due to amylolitic activity. More detailed functional assessment was done by exposure of complexes to extreme pH conditions (HCI, pH=2, 2 hours, 37°C), imitating the stomach, and enzymatic digestion test (a-amylose 50U/ml in PBS, pH=6.9 3 hours, 37°C), simulating the gut conditions, PBS only was used as control. Chosen time frames are considered to spatially correspond to the stomach and small intestine.

Storage stability of starch-omega-3 complexes
Omega-3, encapsulated in starch complexes and integrated in a food product, should survive the storage until the consumption. In order to evaluate guest stability during storage, and to examine influence of different formulations, capsules were stored in three different temperatures: 4, 25 and 37°C. 'There was no significant change in the 'Core content' of 'Flax seed oil' sample. The 'Free core content' during storage at 4°C did not change drastically. There was a more significant drop in the 'Free core content' of omega-3 during storage at 37°C. Results for 'Free core content' 25°C located somewhere between those obtained from storage at 4°C and 37°C.

WP 3 Development of functional pasta using bioactive nanocapsules as ingredient (AMATO)
Objectives:
To develop functional pasta enriched with nanoencapsulated functional ingredients
Definition of parameters for pasta production at laboratory scale
To assess how nanoencapsulation may affect technological, sensorial and nutritional aspects in functional pasta, different experiments were performed at laboratory scale, preparing different pastas enriched with prototype nanocapsules, produced and provided by KARMAT, and containing various bioactive core materials. In this first experimental section, only omega-3 PUFA-rich ingredients by two sources (fish and flax seed oil), formed as complexes HACS-omega-3 fatty acids, were used.

Doughs were prepared using traditional recipe, modified for the enrichment with different amounts and kinds of functional ingredients; HACS-omega 3 nanocapsules (or free oils) were added directly to the flour and with other ingredients. Sensorial analysis for all experimental pasta was performed by a trained panel. As sensory acceptability was one of the outcomes to be evaluated for quality parameters of experimental pastas After laboratory experiments to assess dough behavior, pasta production with nanoencapsulated ingredients has been transferred to pilot plant to set the more suitable parameters for industrial process.

These activities, performed on a pilot plant were focused on:
1.Setting better processing parameters to transfer pasta production to the industrial scale
2.Assessment of physical integrity of nanocapsules and chemical stability of added PUFA omega-3.
3.Studying the parameters to produce functional pasta using nano-encapsulated silymarin.

Production of the prototype of pasta with encapsulated ingredients
Dough preparation was performed first mixing dry powders, in order to obtain a good dispersion of nanoparticles, and then adding water. Semolina containing approximately 14% of proteins was chosen among different products, provided by AMATO, in order to avoid the reduced strength of the gluten network due to the dilution effect derived from HACS-complexes addition. Water amount was increased, respect to the traditional recipe, because of higher water holding capacity due to nanocapsules enrichment, that lead to a reduced textural quality of the dough to be introduced into the drawing step. The dough is then mixed, until the obtainment of a homogeneous product to be introduced into the extrusion chamber.

A two-steps experimental plan was designed to verify the possibility to make extruded pasta using nanoencapsulated ingredient

1) To verify the feasibility of producing extruded nanoencapsulated functional pasta. To this aim "rigatoni" were produced from the same dough using two kind of dies. The traditional die made entirely in bronze and the one where Teflon insert were present. Different concentrations of encapsulated ingredient have been used and the entire pasta drying process were completed. The quality of the final products was evaluated using a training panel giving a score between 0 and 10 and measuring the amount of material released in the cooking water. Results showed that the bronze die was not suitable to produce pasta containing nanoencapsulated ingredients (at least with this type of ingredients). At 5% concentration of encapsulated ingredients pasta was definitively out of the commercial standard with unacceptable values of loss of material during cooking. The scores of PUFA containing ingredients was always lower than that of silymarin one because of the bad small of the product which is not acceptable for the panellists particularly at 5% concentration. On the other hand the quality of the products obtained using die having Teflon inserts were satisfactory up to a concentration of 2% particularly for silymarin samples that were not affected by low scores in the smell attributes.
2) On the basis of the first experiment the teflon die proved to be only suitable for the production of nanoencapsulated containing pasta. So using teflon die two type of experiments were run
2a) Modification of the extrusion pressure applied
2b) Modification of the formulation changing the content and temperature of the water

Final outcomes of WP3 activities
The aim of the overall activities described above in details, was to perform a scale-up to the industrial process for production of functional pasta enriched with nanoencapsulated ingredients. After laboratory and pilot plant experiments, several outcomes about sensorial quality and physico-chemical stability of the added ingredients were obtained, as accurately discussed in WP5. Based on these data, some guidelines were drawn and here discussed for the set-up of the industrial process for functional pasta production with nanocapsules containing bioactive compounds.

1.To prepare functional pasta enriched with PUFA omega-3, although encapsulation is able to mask to a certain extend off-flavours and off-tastes deriving from fish oil addition, the use of nanoencapsulated flax seed oil is a more suitable solution to obtain a more acceptable product.
2.Silymarin encapsulation shows good results in terms of colour and taste acceptability of enriched pasta, comparing with the enrichment by using free silymarin powder.
3.The use of semolina powder with higher protein content (approximately 14%) is advised to provide a stronger gluten network, which can be adversely affected by starch-based nanoparticles addition.
4.The addition of higher water amount is useful to balance the increased water holding capacity provided by nanocapsules and to obtain a less viscous dough that can be extruded without affecting pasta quality.
5.The use of refrigerated water avoid excessive elasticity of the dough to be extruded and allow to reach satisfactory results also with the addition of 5% encapsulated ingredients.
6.The use of Teflon inserts and low pressure (80-100 mbars) is preferred during the extrusion step to decrease damages that may occur in nanoparticles structure.
7.A shorter drying time, at 60°C, is advised in order to reduce the exposition of bioactive compounds to air current, preserving them from damages deriving from oxidation

Work package 3bis: Development of functional nut based pralines and creams using bioactive nanocapsules as ingredient

Objectives
To develop nut based creams enriched with nanoencapsulated bioactive ingredients.
The various nanoencapsulated ingredients that was developed in WP1 were provided by KARMAT and underwent to preliminary laboratory tests to define the behaviour (water activity, texture, physical parameters of the cream/vegetable oil) of the nanoencapsulated ingredient within the product matrix depending on the nanoencapsulated ingredient amount.

In a successive task formulation, mixing and conching: according to the indication of the previous phase different formula and processing parameters was tested by using a small laboratory equipment.

Conditions of mixing, temperature of conching have been considered to get a consumer accepted product. Finally the stability behavior and the sensorial quality of the nut cream/vegetable oil with the bioactive compounds encapsulated was tested for the selected formulations.

The results derived from laboratory scale trials suggest that:
1.The enrichment of nut creams with cocoa polyphenols may be implemented by the use of the nanoencapsulated ingredient, as an improvement of taste evaluation is observed.
2.An enrichment up to 15% of nanocapsules is possible, but some efforts should be done to achieve a good nanoparticles dispersion in the creamy matrix to avoid their perception in the mouth.
3.The use of a blend mixing procedure is required to reduce physical damage of nanocapsules coating material.
4.The chosen bioactive compounds show a good stability in the examined matrix.
5.Plastic viscosity model was found as the best model to describe the rheological behaviors of nut creams. Literature also confirms this kind of behavior. Increasing the amount of nanoparticles also increases the plastic viscosity. Nut creams with 5% or more nanoparticles were difficult to handle in rheometer instruments. The samples seem to behave as viscoelastic solid, instead of viscoelastic fluid.

Sensory analysis of experimental nut creams, containing 1.5% of cocoa polyphenols, in free and nanoencapsulated form, was performed by a trained panel. During this analysis, following attributes were evaluated:
Appearance, Colour, Smell, Taste, Texture, Overall rating

Scoring fore these attributes was based on 9 point scales, where minimum and maximum corresponded to "dislike extremely" and "like extremely", respectively.

Data about sensory analysis suggest that, for a polyphenols enrichment corresponding to 1.5% in the final product, a strong mixing procedure may lead on one hand to an acceptable nanocapsules dispersion, in order to avoid granules perception, but on the other hand is responsible of partial nanoparticles breakdown, with resulting astringency perception.

Work Package 4 : Development of functional bread products using bioactive nanocapsules as ingredient
Objectives
To produce functional bread products with novel nanoencapsulated bioactive compounds
The experiments in WP4 were structured into three successive parts

Dough making
To see the rheological behaviours of nano-encapsulated ingredients in dough formulation
-Bread making
To see the technological (fermentation, rising and baking) behaviours of nano-encapsulated ingredients in dough formulation
Testing of bread quality parameters
-To see the final quality of bread incorporated with nano-encapsulated ingredients
-To see the effect of high amylase activity on the integrity of nano-encapsulated ingredients during bread making process, especially the stability of core bioactive ingredient against processing conditions.

Dough Making
Basic rheological properties of the dough (water absorption, development time and stability) incorporated with different amount of HACS-Omega3 particles (1.0 2.5 5.0 and 10.0%) were determined together with the water absorption capacity of flour slightly increased as the amounts of particles increased in bread formulation.
Bread Making
Developed nano-encapsulated particles composed of HACS-Omega3 (from fish and flax seed) and HACS-Silymarins complexes were directly incorporated into the dough together with the other ingredients. Wheat flour, water, shortening, yeast, syrup (sugar and salt), ascorbic acid and nanoparticles were mixed in a mixer. After mixing the dough was fermented in two stages, hand-molded and put into rectangular pans. They were baked at 220oC for 24 min. Bread loaves were prepared for each baking formulation according to the processing flow sheet.

Testing of Bread Quality
To compare the differences in bread samples, some basic quality parameters were determined. Tests of breads were performed approximately 2 hours after baking. Following measurements were performed to determine the standard quality characteristics of breads;

Weight, Volume, Loaf specific volume, Loaf density, Loaf symmetry, Color and appearance (scoring by visual examination), Crust color, Crumb color, Crumb elasticity (scoring by non-instrumental examination), Crumb granulation and uniformity (scoring by visual examination)

Replacing normal wheat flour with high gluten wheat flour could significantly prevent the loss of loaf expansion in functional breads
The crumb porosity is another quality parameter of breads. Porosity index of breads incorporated with different amounts of the particles of nanoencapsulated omega fatty acids (HACS-Omega3 complex) ranged between 0.47 and 0.51 (dimensionless). The porosity index of crumb tended to decrease as the amount of particles increase in formulation. But the differences were not statistically significant (p less than 0.05).

Conclusion
The primary objective of the Work Package 4 was to develop a process for the production of functional bread incorporated with the particles of nano-encapsulated ingredients. The results revealed that the following points should be taken into account during the application of nano-encapsulated ingredients in bread at commercial scale;

1. In order to prevent fishy smell and taste, flax seed oil is useful alternative to fish oil as the source of omega fatty acids as a nano-encapsulated bioactive ingredient to be incorporated for the production of functional bread.
2. A 5.0-10.0% of encapsulated omega 3 fatty acid or silymarins is desired to achieve physiologically meaningful concentration in breads. This amount of encapsulated ingredient may significantly affect the rheological properties of the dough. Increased water holding capacity of dough and decreased expansion capability during baking are two important consequences of using the particles of nano-encapsulated ingredients in bread formulation. The use of high-gluten flour should be considered to improve bread quality when high amount of nano-encapsulated ingredients (5.0% or more) is desired at industrial scale process for the production of functional bread.
3. High amylose cornstarch (HACS) as the encapsulating material of omega 3 fatty acids or silymarins is subject to amylase attack during the fermentation process. Addition of amylase and excessive fermentation should be avoided to prevent any hydrolysis of starch based coating material, thus to prevent the oxidation/degradation of core bioactive materials during baking process.
4. Omega 3 fatty acids are sensitive to oxidation induced by the baking process at extreme temperatures. Encapsulation stabilizes omega 3 fatty acids against oxidation to a certain extend. In order to protect bioactive core material, baking temperature should be kept lower than 230oC for the production of functional bread at industrial scale. Silymarins are relatively stable during the baking process of breads.

Work package 5: Characterization of the food produced using nanoencapsulated ingredients
Objectives To provide a technological, chemical and sensorial characterization of the developed nanoencapsulated pasta and bread products
The experiments were designed for an in-depth characterization of developed functional foods in terms of technological behaviour, quality features, stability, associated risks and biological performance. Following foods were tested as the prototype products;
Bread, Hazelnut cream, Pasta

The measurements performed on prototype foods were as follows
-To assess basic quality characteristics of foods (bread and hazelnut cream) produced using the particles of nano-encapsulated ingredients (silymarins and cocoa phenolic compounds) in foods
Sensory analysis, Rheological behaviour analysis, Texture profile analysis, Color and porosity analysis by computer-vision, Scanning electron microscope analysis
-To assess risks associated with the use of the particles of nano-encapsulated ingredients (omega fatty acids, silymarins and cocoa phenolic compounds) in foods
Thermal stability of the particles of nano-encapsulated omega-3 fatty acids and silymarins in breads during processing, Formation of lipid oxidation products like hexanal, nonanal, Formation of acrylamide and hydroxymethylfurfural
-To assess the additional biological functionality in the final products incorporated with the particles of nano-encapsulated silymarins and cocoa phenolic compounds (to address the potential outputs for Task 5.3)

BREAD
The functional breads were produced with the particles of nano-encapsulated omega 3 fatty acids (from fish and flax seed oils) and silymarins (from milk thistle). The amount of the particles of nano-encapsulated bioactive ingredients was varied from none to 10.0% on flour basis.

Potential Risks/Benefits Associated with the Use of Encapsulated Ingredients in Breads
Baking is a complex process where a simultaneous heat and mass transfers occur which has certain consequences in the final product in terms of the chemical reactions involved. The Maillard reaction is one of the most significant reactions occurring in foods during baking. It is responsible for the formation of key aroma compounds, color and taste in bakery foods like bread. During the Maillard reaction, undesirable compounds like hydroxymethylfurfural (HMF) and acrylamide are also formed. This is relevant for bakery products including bread, because especially the crust layer is subjected to high temperatures for long time during the baking process. Breads prepared by incorporating different amounts of the particles of nano-encapsulated omega fatty acids were analyzed for their HMF and acrylamide contents. From a viewpoint of combined heat and mass transfer phenomenon, it is critically important to know distribution (or gradient) of undesirable compounds within the product. Since time-temperature profile is different for different parts of the product being heated, a concentration gradient of neo-formed compounds generally occurs within the product, especially for compounds being formed at temperatures exceeding 100oC. For bread, this means that crust and crumb parts will significantly differ from the viewpoint of HMF and acrylamide concentrations. Therefore, crust and crumb parts of the bread samples were analyzed separately. The crust and crumb parts of bread samples were carefully separated. After grinding, the samples were analyzed for their HMF and acrylamide concentrations.

HAZELNUT CREAM
With its composition, hazelnut cream can be considered as a concentrated suspension of solids in a Newtonian medium. However, this product is a yield stress fluid with non-Newtonian behavior. The rheological measurements of hazelnut creams incorporated with the particles of nano-encapsulated cocoa phenolic compounds were performed by a Brookfield cone and plate viscometer. The analysis of viscometer data is usually enhanced through the use of mathematical models. NCA/CMA Casson model was used for the determination of plastic viscosity and yield stress, Herschel-Bulkley model for consistency and flow index for hazelnut creams incorporated with the particles of nano-encapsulated cocoa phenolic compounds.

Four batches of hazelnut cream samples were produced to determine the effects of the particles of nano-encapsulated cocoa phenolic compounds on the rheological properties. Plastic viscosity values of hazelnut creams tended to increase as the amounts of particles increased

PASTA
The experiments were designed to deal with the characterization of functional pasta incorporated with the particles of nano-encapsulated ingredients. The major goal was to determine technological behaviour, quality features, stability and biological performance of the nano-encapsulated ingredients. KARMAT provided the particles of nano-encapsulated silymarins, and PUFA oil containing omega fatty acids to be mixed with semolina for pasta making. Experiments were performed on laminated pasta.

Four kinds of different experimental pasta enriched with PUFA omega-3 rich ingredients were prepared. The aim was to assess the effects of nanoencapsulated fish oil or flax seed oil, compared with corresponding free oils, on quality parameters.

WP6 Human trials with IBD patients using pasta and bread produced with nanoencapsulated ingredients

Objectives To perform a human randomized control trial to verify the efficacy of the functional pasta and functional bread in IBD patients
A protocol for randomized double-blind placebo-controlled study reporting the rationale of the study was submitted to Ethical Committee of University Hospital Federico II and approved.

A placebo-controlled study gives the possibility to evaluate the effects of encapsulated n-3 PUFA versus the not-encapsulated ingredient as well as versus a free-normal diet. Using IBD patients offers the unique possibility to evaluate, in a relatively short time period, the efficacy of nanocapsule on intestinal inflammation. Since it has been amply demonstrated by recent scientific advances that a compromised gut health through inflammation may play a fundamental role in the pathogenesis of metabolic syndrome, the findings from this study may be useful not only in the research for therapy of IBD but also for prevention of obesity and diabetes.

Patients with ulcerative colitis (UC) was recruited as they have an unbalanced systemic and intestinal inflammatory status and thus they are particularly suitable to investigate the potential physiological effect of encapsulated n-3 PUFA on inflammation.

The main end points of the study were: the percentage of cases of relapsers in the two study groups (evaluated by Mayo score); the inflammatory status (evaluated by systemic concentration of pro-inflammatory cytokines, mainly TNF-alpha and IL-6)

Biomarkers of intake: serum fatty acid composition
After treatment with n-3 PUFA enriched products an increase of serum n-3:n-6 PUFA ratio was found; on the contrary no chance in fatty acid composition was found in control patients.

This finding indicated that encapsulated n-3 PUFA enriched products guaranteed a good bioavailability of ALA. Anymore it confirmed data of compliance to the treatment obtained by 3-day food diaries auto-filled by subjects at baseline and over the study period.

In particular ALA-enriched products were able:
to counterbalance the increase of IL-1a, IFN gamma e TNF-alpha recorded in patients consuming control products, thus resulting the reduction from baseline value very significant (p less than 0.01) for TNF-alpha;
to determine a significant increase of IL-4 vs baseline and compared to variation recorded in patients consuming control products.

After follow-up period serum cytokines did not change in patients belonging to the control group while they returned to baseline value in patients treated with ALA-enriched products.

No significant difference was found in bacterial composition of feces from patients belonging to the treatment group over all the study period. Similarly no difference was found in control patients neither over the study period or compared to treated subjects at each time points.

This finding indicated that the slight amelioration of inflammatory status in patients who consumed n-3 PUFA products, was not accompanied with modification of gut microflora.

Results obtained from the analysis of serum carotenoids in two groups of patients showed that although serum β-carotene showed a trend to increase vs baseline values, it was not significantly different between the two groups after 3 months of treatment and after follow-up period. This finding confirmed data obtained by analysis of food diaries that indicated a high compliance to the dietary counsels in both groups and a trend to increase fruit and vegetable consumption over the study. Anyway the trend of increase of serum β-carotene during treatment compared to baseline, was found also in the follow-up thus it can be assumed that a similar antioxidant protection from diet was guaranteed over all the study period. It allowed us to exclude that diet (apart products we provided) could influence clinical and biochemical parameters monitored.

CONCLUSIONS
The present study demonstrated that the encapsulated n-3 PUFA enriched pasta and bread:
-were well accepted by patients also for long duration consumption and did not determine side effects;
-guaranteed a good bioaccessibility of n-3 PUFA;
-determined an amelioration of clinical conditions by reduction of relapse rate during the study period mainly associated to reduction of some pro-inflammatory cytokines and increase of the anti-inflammatory IL-4.

Potential Impact:

The potential impact of the project was mainly exploited by three different actions
1) The validation of the results obtained in laboratory scale producing prototypes of several encapsulated ingredients and functional foods
2) Demonstration activities towards markets
3) Market studies aimed at define the potentiality of the nanoencapsulated products in the different field
4) Indications for health and nutritional claim labelling of foods containing nanoencapsulated ingredients

1) Validation of the results obtained in laboratory scale trials
The validation activities of KARMAT were concentrated on the scale up of the production, which was necessary to cope with the needs of food producers for their demonstration activities. The research efforts were focused on the set up of processing conditions to optimize the content of bioactive materials in the capsules (to increase the core/coating ratio). Several large production batches with Flex seed oil and sylimarin were dispatched to the partner to allow the production of encapsulated foods and the feed back from food trials were useful for the fine tuning og the processing parameters.

2) Demonstration activities towards markets and consumers
During the project Karmat encapsulation presented the technology and products to potential consumers in various meeting and professional exhibitions. In 2009 a number of leading nutraceutical media from Europe and the US were exposed to the nanotechnology created in KARMAT. Among those distinguished journalists there were Elaine Watson who`s been writing on the food industry for the past 9 years and is currently a vice-editor of Food Manufacture magazine (see http://www.food manufacture.co.uk online). Stephen Daniells, a science editor for Decision News Media (see http://www.nutraingredients.com and http://www.foodnavigator.com online), and Kevin Robinson, who is the chief editor of Nutraceutical Business and Technology magazine (see http://www.nutraceuticalmag.com online).

Encapsulation technologies applied in foods
According to Frost and Sullivan (March 2009), food manufacturers are commercializing R&D activities due to innovative encapsulation technologies that will help achieve differentiation along with enhanced product value for food products. It is becoming increasingly important for food manufacturers to tap into the key and emerging consumer trends with innovative encapsulation techniques. Although there is a greater focus on micro encapsulation, research is discovering the potential of nano encapsulation technology. Research and Development (R&D) is primarily based on satisfying end users' demand to consume food products with enhanced nutritional value, quality, and safety without compromising on taste. 'The current trend is to consume natural and vegetarian products, fuelling the growth of plant based encapsulated products, especially in the functional foods and neutraceuticals sector'.

Nanotechnology applications for food ingredients and additives
A major focus of current nanotechnology applications in food is the development of nanostructured (or nanotextured) food ingredients and delivery systems for nutrients and supplements. For this, a variety of processes are being utilised, including nano-emulsions, surfactant micelles, emulsion bilayers and reverse micelles (Weiss et al. 2006). The nanostructured food ingredients are being developed with the claims that they offer improved taste, texture and consistency.

Nanotechnology applications for dry foods
Nano and micro-encapsulation technologies have been implemented in the dry food industry for the purpose of creating more healthy available food with potential benefits that will satisfy the consumer's needs. High energy and low nutrient density which characterize diet in developed countries are major targets that must be overcome. Nanotechnology may be able to engineer high energy dense foods abundant in the modern diet to maintain lower energy densities while looking and tasting the same as before modification to aid public acceptance and reduce any insulinotropic properties with potential positive effects on metabolism and health.

These novel nanoparticles are already being exploited commercially. For example, 140-180,000 nm micro-capsules has been used to mask the taste and odour of tuna fish oil added to bread for health benefits e.g. "Tip Top-up" brand bread from George Weston Foods, Australia. However to increase the Omega 3 potency and bioavailability, companies such as Aquanova and Zymes are now selling 30-40nm nano-forms or nano micelles of Omega 3. These capsules are 4000 times smaller than the Nu-Mega range. Another example of an encapsulated supplement is DriphormEvi, a dry powder form of fish oil produced by Nu-Mega. This Australian-based company advertises potential applications for their patented encapsulation technology to be implemented in bread and bakery products, breakfast products, and Cereals muesli bars. A fortified pizza blend with encapsulated Omega-3 and stable vitamin C are produced by the Wright Group (USA). Another example in the convenience food sector is in home-baked pizza products, where sodium bicarbonate can be encapsulated to prevent early release of the bicarbonate and delay the reaction of the leavening phosphate until the crust reaches a specific temperature in the oven (Frost and Sullivan). Nanosilver, is another feature using nanothechnology and is, being increasingly marketed as a health supplement. Although no food products containing nanosilver is currently available, its use as an additive to prepare antibacterial wheat flour and has been the subject of a recent patent application (WPI ACC NO: 2006-489267/200650, Preparation method antibacterial wheat flour by using silver nanoparticles by Park KH, South Korea).

The market for functional foods and nanoencapsulated food ingredients
The Functional Food market is being driven by a growing consumer understanding of diet/disease links, aging populations, rising health care costs, and advances in food technology and nutrition. Nanotechnology in recent years has developed into a wide-ranging, multibillion-dollar global industry. The global market impact of nanotechnology is widely expected to reach 1 trillion US$ by 2015, with approximately 2 million workers (Roco and Bainbridge 2001). It is also clear from a number of reports, reviews, patent applications and company products that applications of nanotechnology have also started to make an impact on different aspects of the food and associated industries (Chen et al. 2006). The nanofood sector is currently led by USA, followed by Japan, Australia and UK (table1). However, Asian countries (led by China) are expected to be the biggest market for nanofood by 2010

A recent report by Helmut Kaiser Consultancy has estimated that the nanofood market grew to 7 billion USD in 2006, and reached 20.4 billion USD by 2010 (Helmut Kaiser Consultancy 2004 [Internet]). Another report, by the consulting firm Cientifica (2006), has valued food applications of nanotechnologies at around 410 m USD (food processing 100 m USD, food ingredients 100 m USD, and food packaging 210 m USD). Thus, the nanofood fraction was a major part of this market sector. According to the report, the current applications are mainly for food packaging (improved barrier properties, etc), with some applications for delivery systems for nutraceuticals. The report estimated that by 2012 the overall market value would reach 5.8 billion USD (food processing 1303 m USD, food ingredients 1475 m USD, and food safety 97 m USD, and food packaging 2930 m USD). Generally, the market profile information and analysis from different sources revealed that Functional Food market is expanding and has a strong growth trend all over the world. It has been suggested that the number of companies currently applying nanotechnologies to food could be as high as 400. A number of major food and beverage companies are reported to have an interest in nanotechnology. These include Altria, Nestle, Kraft, Heinz and Unilever, as well as small nanotech start-up companies. It is also widely anticipated that the number of companies applying nanotechnologies to food will increase dramatically in the near future. Considering such rapid developments in this field, and the global setup of international food companies, it is not unreasonable to anticipate that more nanofood products will appear on the EU markets within the next few years.

In a global survey conducted on the Internet across 38 countries, ACNielsen (2005) asked consumers which of a list of foods promoting specific health benefits they purchased (table 2). As can be seen 'Cholesterol reducing oils and margarines' products (also including omega-3 related products) are present, and were approximately 30% of purchased products of Global Average. Bread with added supplements/vitamins products had slightly lower value of 18% of Global Average. Even though, in Europe the regularity value was the lowest, there is a growth potential of this sector of the market.

Estimation of the costs for the encapsulated ingredient produced with the activity of NANOFOODS project

The estimation of the costs of the various encapsulated ingredient i.e. different source of omerga-3 fatty acids, sylimarin and cocoa polyphenols was done using the market price during the period 2008/2010.

The price ranges from the 4,8 USD$/kg of Omega-3 Fish Oil to the 95 USD of Milk Thistle Seed Extract (containing 80% Silymarin)

It is important to underline that
1)The price of many bioactive ingredients are highly variable according to the market conditions
2)As a general rule and particularly for Milk Thistle Seed Extract (sylimarin) and cocoa polyphenols the price can be significantly lower if the production of commercial amount of encapsulated ingredient would be performed

As a consequence the calculate price for encapsulated ingredients realized in the frame of NANOFOODS project ranges from 10 USD/Kg of Fish oil to the 27 USD/Kg of Silymarin

The market for nut cream
Following both the present regulation and the market trends it is expectable that products rich on vitamins, minerals, sterols and stanols, unsaturated fatty acids (omega-3) and soluble fibre succeed in the market, taking profit of claims now approved or probably approved in the next future. This is the case of phenolic compounds which has already a negative opinion relating the phenolic compounds and the antioxidant effect claim, but in the next future could have a positive opinion in relation with other general health claims.

Consequently, one possible market approach is to make profit of the high content of some active compounds present nut-based ingredients. That means, to introduce in the market products with the required content of any of the mentioned compounds in order to have a claim. Appropriate target markets are bakery, confectionery and snacks; and some innovative products including nut and cocoa-based ingredients may consider spreadable creams, chocolate fillings, biscuits fillings, snack bars, etc. However, the main feature in NANOFOODS project is the use of nanoencapsulated ingredients in order to protect them once added to the final product, but also for technological and sensorial reasons.

The use of nanoencapsulated bioactive compounds in La Morella Nuts' creams is a very interesting way to boost the intrinsic benefits of this kind of creams but also to give an added value of the product due to the incorporation of compounds which can bear a nutritional and health claim. It is important to notice that this kind of products is widely consumed by different target population so the impact can be important.

We must consider the new results obtained related to bioavailability of some compounds found in nut and cocoa-based matrices, which have been added as nanocapsules, and also considering their synergistic effect. In this sense, but out of the scope of this project, we can choose a specific health target in order to develop a clinical study to finally submit a new dossier to the EFSA (article 13.5 way) in order to obtain a new claim based on proprietary data. This way would represent a new investment after the end of NANOFOODS project (probably not less than 1 million euros) and more time to design and execute it.

Functional foods have very high added values in comparison to quotidian products. Nevertheless, it must be taken into account that there's a risk in the final results of the translational study. So all the previous steps to substantiate the potential benefit of the whole product will be very valuable.

As said before, one must consider the advantage of having the protective effect over the active principles health properties provided by the nanocapsules. This fact means several advantages in terms of increased shelf-life of the product, increased synergies between active compounds and more stable sensory properties, among others.

The products under development have an added-value due to its high health component, and also due to the improved technological features above mentioned.

On the other hand, the nanoencapsulated ingredients developed in this project including several bioactive compounds with new proven health benefits. These new proven benefits would be related to the ones observed in previous in vitro and in vivo screening studies performed by la Morella Nuts and done under different project frameworks. These benefits deal with cardiovascular diseases and/or neurodegenerative/ageing effects disorders. These ingredients are very interesting due to their commercial potential. Nevertheless there is a wide gap between the in vitro and in vivo tests and how the human body recognises/assumes these benefits.

Technical papers in relation to the more generic dissemination of the benefits of these kinds of ingredients.

Presentations within the departments in La Morella Nuts. Before the commercialization of the products derived from the present project it is important to explain to the commercial department the concept of these new products, their key benefits, as well as their possible applications. These explanations could be performed by means of internal presentations turning into well-suited costumer presentations in case of their success.

Estimation of market cost of cocoa cream containing nanoencapsulated ingredients
La Morella Nuts developed a cocoa cream mix formulated with the particles of nano-encapsulated polyphenols originated from cocoa and a cocoa cream mix formulated with nanoparticles of nanoencapsulated vitamin D. Therefore a only type of cocoa cream was formulated with nanoencapsulated particles that contained cocoa polyphenols or vitamin D.

The market for pasta
Italy represents a huge market for pasta products. With almost 27 Kg/year of intake Italians consume more than five times the average amount of the other EU people. Italian consumers are very conservative about pasta products as a consequence more than 95% of the market is made of traditional pasta products. This slice of the market is quite stable with minor modification regarding only the share of the various pasta shape (in the last 10 years the so called short pasta such as penne, and rigatoni overcome the consumption of spaghetti) and the commercial battle among the various brands with the new entry of the pasta having the supermarket brand (Auchan, Coop, Carrefour, ecc)

From the innovation product standpoint the most relevant trend of the last five years in the pasta market regarded the area of the dietetic and healthy products. The intake of whole grain pasta "integrale" raise of more than 20% in the last five years mainly as consequence of heavy market campaign of the Italian leader marked, Barilla. The success of whole grain pasta demonstrated that it is possible to take advantage from the idea of pasta as pillar of the Mediterranean diet to develop pasta based functional products.

The concept to use this national staple food to deliver healthy components is appealing for all pasta producers. On the other hand, there are two main hurdles that have not been solved yet
1)consumers can not admit any decrease in the sensorial quality of pasta,
2)the standard pasta cooking procedure (i.e. boiling in abundant water) causes the lost of all added hydrophilic bioactive compounds

Encapsulation technologies as shown by the activities carried out in the frame of NANOFOODS project can solve both these problems.
1)It can mask the negative sensorial impact of the various bioactive ingredients (such as the bitterness of silymarin and the off flavour of PUFA)
2)It blocks the bioactive ingredients into the starch and gluten matrix avoiding the loss in the boiling water.

Estimation of the market cost of functional pasta containing nanoencapsulated ingredients
Amato developed many prototypes of pasta containing encapsulated fatty acids (fish oil and flex seed oil) or sylimarin at different concentration and four formulations were used for the demonstration activities as listed in the Table: pasta with flex seed oil (rich in ALA) and pasta containing milk Thistle Seed Extract. According to the trials run in WP3 two concentrations of encapsulated ingredients (2% and 5%) were selected. Considering the cost of the ingredients above mentioned a price per Kg of final product was 0,25 and 0,60 Euro for pasta enriched with Flax seed at 2% and 5% respectively; while it was 0,5 and 1,3 Euro for pasta enriched with Silymarin at 2% and 5% respectively.

The market for bread
While consumers are generally receptive towards health and wellness products, some functional foods have been more easily accepted than others. Those products, in particular fortified bread or biscuits, continue to be more niche positioned than others, most notably functional confectionery. Bread in theory shows more potential, as it is generally regarded as a healthy product (Atkins notwithstanding) which is more geared towards the addition of nutrients. However, although the category has shown growth due to considerable innovation, product failures have proved to be rife here. This weak performance was exacerbated by the fact that marketing efforts to communicate the products' benefits to consumers were only minimal at best.

Estimation of market cost of bread containing nanoencapsulated ingredients
MERK company developed 6 different type of bread mix formulated with the particles of nano-encapsulated omega-3 fatty acids originated from flax seed oil. The types of breads formulated with nano-encapsulated omega-3 fatty acids are wheat bread, whole wheat bread, rye bread, oat bread, seven grain bread, and corn bread. The bread mixes are formulated with the nanoparticles to enable a piece of load after baking contains 1 g of bioactive ingredient. This corresponds to 2.5 kg of the particles of nano-encapsulated omega-3 fatty acids per 100 kg flour mix used to prepare bread.

The change of the cost of 100 kg of bread mix with the particles of nano-encapsulated omega-3 fatty acids. As given in this table, the cost of bread mixes increases by a ratio ranging between 19% and 28% by the addition of the particles of nano-encapsulated omega-3 fatty acids.

Addition of nanoparticles into bread formulation increases the market price by a factor of approximately 23%.

For the case of functional breads added with the particles of nano-encapsulated silymarins, the cost of bread mixes is higher due to increased cost of the nano-encapsulated silymarins in comparison to nano-encapsulated omega-3 fatty acids. The cost of bread mixes increases by a ratio ranging between 33% and 46% by the addition of the particles of nano-encapsulated silymarins. Addition of nanoparticles into bread formulation increases the market price by a factor of approximately 39%.

MERK prepared technical papers for consumers describing the potential benefits of these kinds of nano-encapsulated ingredients in breads as a daily-consumed food product. Kind of "how to" technical papers for local bakeries are also under preparation to describe the basic processing conditions of these new generation functional breads.

4) Indications for health and nutritional claim labelling of foods containing nanoencapsulated ingredients
Nanotechnology applications for the food sector have raised a number of safety, environmental, ethical, policy and regulatory issues. The main concerns stem from the lack of knowledge about the potential effects and impacts of nanomaterials on human health.

Regulatory framework
The EU Regulation EC/1924/2006 on Nutrition and Health Claims made on Foods is not yet completely deployed. Whereas Nutritional Claims list was published as annex of the Regulation and widened in February 2010 with 5 more approved claims, in the case of Health Claims only few of them are approved until now. For the Health Claims approval it is necessary the previous opinion of the European Food and Safety Agency (EFSA) in terms of scientific substantiation of the submitted claims. These claims are examined following different procedures depending on the category of the claim: general function claims (article 13.1) reduction of disease risk claims (article 14), new claims based on proprietary data (article 13.5).

The biggest list of claims, corresponding to the so-called "General Health Claims list" or "Article 13.1 list" had to be published in the beginning of 2010 but, due to the big quantity of proposals submitted and the complexity of the process, it seems that finally will be published in the beginning of 2012.

In the case of article 14 and 13.5 claims it is necessary to present an individual dossier to EFSA that can be evaluated in an average of 9.1 months and 4.1months respectively (Brookes, 2010).

The market of health and wellness foods (among them those developed inside NANOFOODS activities) is highly dependent of the deployment of the before mentioned regulation. In 2009 sales of foods with "disease reduction claims" and "general function claims" represented the 53.1% and 29.1% of the global sales of health and wellness foods (Euromonitor, 2010). Consequently it is difficult to foresee a precise market impact of a specific functional food or ingredient before to know if particular claims will be approved or not.

The new products developed under the framework of articles 13.1. and 14 would represent a broad palette of ingredients in the functional food sector. These ingredients are useful for companies interested in new developments related to nutrition and health benefits and currently bearing positive opinions from the EFSA. This type of ingredients (Article 13.1. Nutrition and Health claims) will allow these companies to launch new increased-value products into the market in a short period of time and bearing these kinds of claims. Moreover, a protective effect over the active principles is assured due to the nanoencapsulation, thus translated in an increased shelf life of these kind of cocoa and nut creams which is a distinctive point due to the liability of certain bioactive compounds.

The 13.5. New claims strategy implies several phases/steps before launching to the market the products with a claim in their labelling. The steps are explained below, considering a general situation:
In order to support the observed benefits at in vitro/in vivo level more research must be done. This is important to keep in mind for the returned investment in the ingredients that bear new claims (article 13.5).

Minimum of two independent human studies must be conducted to substantiate the health benefits observed. Moreover, it is important to conduct this intervention study with the product with the claims in the label.

The cause-effect relation must be well defined, as well as the characterization of the product and the bioactive compound or mix.

The benefit for the health, in relation with the claimed message must be consistent and understandable. The human design study must be statistical strong and independent statistical specialist must analyzed the data from double-blind studies (as preference).

These points must be taken into account when performing a study market. The investment on this kind of studies is not negligible, as an approximation on study of the above mentioned characteristics could represent a 1,5 M EUROS if the benefit is cardiovascular and up to 3 M€ if the benefit is neuronal (Alzheimer's disease).

The results of the study could be presented in a dossier and presented to the EFSA NDA panel in order to evaluate if the product is able to bear the proposed claim.

These steps, may represent a one year enlargement of the procedure before launching the product to the market. An extra point is the product promotion needed through a strong marketing campaign.

The contribute of NANOFOODS project
Nanoparticles only during ingredient preparation in food only microparticles are present
The use of nanoparticles in food is often compromised as the food companies have not a clear picture of the regulation regarding the safety issues and they are wondered about the consumers' perception of this technology. This fear can be faced by the fact that in the case of material used in the frame of NANOFOODS project (as in various other cases) a commonly used and consumed food ingredient - starch was adopted. One may therefore ask: since when starch molecules are harmful nanoparticles? This is true, particularly since the fear of nanoparticles stems primarily from instances where inhalation may occur - clearly this is not the case for this technology and application, and probably in all cases where nanocapsules are incorporated into food matrixes.

Another concern is the spontaneous uptake of particle sized lower than 50 nm. Here again, the complexes commonly formed by the method used in this project have a size of approximately 400nm. To ensure compliance with this encapsulation system, the spray drying produced aggregates with the size of approximately 20 micron (20.000 nm).

Encapsulation allow protecting the bioactive compounds during food processing and storage
One of the potential benefits offered by nano-encapsulation is considered as the protection of bioactive ingredients intended for use in food formulations. Foods form very complicated matrices with varying chemical composition and physical structure. Both chemical composition and physical structure are subject to change upon processing and during storage. Thermal treatments among others are among the most detrimental from the viewpoint of chemical reactions occurred during processing, and possible involvement of bioactive compounds in these reactions.

NANOFOODS project successfully exemplified that processing stability of bioactive ingredients including silymarins and PUFAs could be improved by nano-encapsulation. In the case of functional bread and pasta products, the stability of silymarins was investigated against baking and drying processes, respectively. In functional bread, the use of the particles of nano-encapsulated silymarins resulted in approximately 7.0% more silymarins in comparison to the use of free silymarins powder. In functional pasta, there were 15%, 59%, and 55% of more taxifolin, silybins, and isosilybins in the final product after the drying process. This data was considered as a clear evidence of that nano-encapsulation significantly increased the stability, thus the availability of bioactive silymarins in the final bread and pasta products.

Encapsulation allow prevention of lipid oxidation
Bread data indicated that the formation of nonanal and hexanal are more pronounced in the bread containing the oil in the free form than when the same amount of PUFA were coated with starch nanocapsules. In other words project data demonstrated that nano-encapsulation reduces the formation of lipid oxidation markers. This is not only ad advantage per se, but it can contribute to the reaction of these oxidation products with amino acids thus contributing to the formation of potentially harmful Maillard reaction products.

The modulation of bioactive compounds reactivity during processing is a starting point for setting up mild formulation to reduce the concentration of process neo formed contaminants.

Encapsulation of the reactants can reduce the formation of potentially harmful compounds
Data on bakery products showed a decrease in acrylamide concentrations as the amount of nanoparticles increased in bread formulation.

This evidence was related to physical parameters: during the bread making experiments that the water holding capacity of dough formulated with 5.0% or more of encapsulated material was noticeably high. This prevented rapid loss of moisture in breads during baking, so the rapid increase of temperature in the crust regions. This phenomenon is worth of further investigation: the possibility that starchy nanocapsules can prevent the formation of thermal contaminant whose formation is dependent from water activity (i.e. all the Maillard Reaction products) can open the door to many applications combining the advantage of hydrocolloids to that of the encapsulation strategy.

List of Websites:
http://www.nanofoods.hacettepe.edu.tr