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PROgress in Saving Proteins And Recovering Energy

Final Report Summary - PROSPARE (Progress in saving proteins and recovering energy)

Project context and objectives:

Food industry leftovers constitute an enormous cost and an unnecessary waste of food-grade material. In the production of meat for human consumption, up to 50% of the animal weight is discharged. In European Union (EU) only, the total leftover mass from the meat industry amounts to 15 million tons, formally residues, but actually potential raw materials rich in proteins and lipids. Although this potentiality, most of this material is incinerated and only 22% is converted into feed and a bare 3% becomes food. The traditional rendering technologies, based on prolonged heating of the leftovers, ensure microbial safety and increase digestibility, but use enormous amounts of energy and induce the degradation of high biological value components.

The European project PROSPARE, a joint European-Russian research project, addressed this issue through the application of a new technology able to recover poultry proteic leftovers and feather and to transform them, respectively, in Functional Animal Protein (FAP), intended for food and in Functional Feather Protein (FFP), intended for feed and to convert discharged fat into biodiesel. The aim of the project is also to fully characterise these new food, feed and fuel materials. The PROSPARE consortium includes eight research groups: four from the EU (two from Italy and two from Belgium) and four from the Russian Federation.

Project results:

Novel technologies of feather hydrolysis

Laboratory plant for feather high temperature short term treatment (HTST) was developed and technology of feather conversion was optimised taking protein yield as key parameter. Based on the results of multifactor experiment following optimal parameters of feather HTST treatment at laboratory scale were defined: initial humidity of raw materials of 50-55%, temperature of hydrolysis 190-200??, duration of high-temperature processing 90 sec. Digestibility of the resultant functional feather protein (FFP) exceeded 85 %, indicating its high feed value. It was also shown that applied conditions for hydrothermal processing of feather raw material allowed a full decontamination (in view of aerobic colony count) of protein concentrate, at the same time providing practically complete preservation of thermolabile amino acids (lysine, methionine and cystine).

The technology was scaled up to pilot level. A pilot plant with capacity 3.5 tons of feathers per day was constructed by Symbol nearby the Moscow. The pilot plant for feather treatment provides possibility to feather treatment on (semi)industrial scale. A complete set of equipment for HTST treatment of feathers was designed and installed, including unit for preliminary purification of feathers, dewatering press, sterilisation block, experimental hydrolyser-extruder, heating system, dryer, blender, block of product packaging and automatic system for control of technological operations. Multifactor experiment aimed at optimisation of feather hydrothermal hydrolysis conditions for obtaining of protein hydrolysate with advanced digestibility was carried out.

The developed technology of sequential HTST and enzymatic conversion of chicken feathers allowed obtaining completely digestible protein hydrolysates of high biological value. Functional properties and molecular weight distribution of fermented FFP can be altered by variation of hydrolysis parameters.

Novel technologies for bones&meat conversion to FAP and fat

The laboratory plant for poultry bone&meat trimmings conversion into functional animal protein (FAP) and fat has been developed and technology of protein hydrolysates (FAP) production from bone&meat trimmings has been optimised. The optimised technological parameters of bioconversion at laboratory scale were: enzyme composition - MEK-2 (Protamex, Alkalase, Neutrase and Flavourzyme) in concentration of 0.25 % to the weight of raw material, fermentation temperature - 55 °C, hydromodule 1:2, duration of fermentation - 2.5 h. FAP samples obtained at lab scale were shown to have high content (more than 83%) of completely digestible (in vitro digestibility more than 99%) protein with high nutritional value due to abundance in essential amino acids, particularly in lysine, threonine and tryptophan.

Development and deployment of the pilot plant for bioconversion of poultry meat&bone residues into FAP and fat was implemented. Pilot plant with capacity 2.1 tons/ day for poultry meat&bone trimmings (broiler necks, backs, carcasses, wings) bioconversion into FAP was constructed in the Moscow region. The pilot plant included facilities for complete technological process of poultry meat&bone residues bioconversion starting from the raw materials and leading to obtaining of FAP and some other by-products (chicken fat, bone cake, undigested meat fraction). The optimisation of technological process of FAP production at pilot scale was also carried out by multifactor experiment with Box-Benkhen design with dried FAP yield as a key parameter. The optimised hydrolysis parameters for FAP production at a pilot level were: duration of enzymatic hydrolysis 2.5h hydromodule 1/2.0 multienzyme composition dosage 0.20-0.25% of raw material weight. The optimised technological protocol developed provides more than 70% recovery of potentially available protein from poultry leftovers. Mild conditions of enzymatic hydrolysis ensure high retention of labile biologically active compounds (e.g. thermo labile amino and fatty acids). FAP samples produced at pilot scale complied with all Russian safety requirements. The hydrolysate obtained (FAP) was characterised as a valuable source of easily digestible protein.

Assuming that the highest yield of soluble protein (67%) was observed when enzyme blend MEK 2 (Alcalase, Neutrase, Protamex, Flavourzyme) was used, it was considered as the most perspective for further experiments at pilot scale.

FAP samples are rich in protein (total protein 85-88%). Ashes content of FAP samples is fairy stable comprising nearly 6%. FAP total fat and moisture content are characterised by the highest magnitudes of variation among all the physical-chemical parameters studied (0.4-1.1% and 4.0-8.0 % respectively).

FAP samples produced at a pilot scale were mainly (more than 55%) composed of low molecular weight constituents (M.w. less than 3 kDa). The highest magnitude of variation was observed for the relative contribution of high molecular weight constituents (43%). FAP produced at pilot scale is less intensively hydrolysed.

Therefore, FAP is a highly proteinaceous ingredient (total protein more than 83%), containing protein mostly in soluble form (91-97% of the protein). FAP is characterised by stable basic chemical composition, rich in free amino acids (20-28% of the bulk) with good nutritional value for Trp, Lys and apolar amino acids. FAP samples comply with all safety requirements of Russian Federation (RF) legislation as well as EU. The developed technology of meat&bone trimmings bioconversion is highly reproducible from laboratory to pilot level.

The FAP obtained at pilot scale was considered as FAP1 due to its molecular weight distribution and was subjected to further fractionation using micro - and ultra-filtration techniques. As a result the peptide fraction of FAP 1 was isolated and marked as FAP 2. The optimised technological protocol of FAP2 production included preliminary filtration, pasteurisation, sequential tangential micro- (800 nm pore-sized filter) and ultrafiltration (50 and 10 nm pore-sized filters), drying and packaging. FAP2 is a concentrate of FAP1 obtained on 10 nm pore-sized filter. The filtration protocol elaborated provided de-fatting of FAP1 along with partial de-ashing as well as separation of lipoproteins and low molecular weight contaminants (biogenic amines, antibiotics etc.). FAP1 and FAP2 were shown to be distinctly different in molecular weight distribution. FAP2 (ultra filtrated FAP) was shown to be mainly (more than 60%) composed of medium molecular weigh (M.w. 3-10 kDa) constituents, while FAP1 (FAP) - of low molecular weight (M.w.less than 3 kDa) compounds.

Sum of the free and total amino acids of FAP2 were found to be respectively 173 (±20) and 676 (±33) mg/g. The free amino acidic fraction of FAP2 was dominated by Gln, Glu, Lys, Arg and Leu, all at values of about 15-18 mg/g. Most of amino acids predominantly present in FAP2 in peptide form. Total amino acid composition of FAP2 is dominated by Gly and Glx (more than 100 mg/g of FAP2). Also quite high total content was observed for Ala, Asx, Pro, Lys and Arg.

The nutritional value of processed FAP2 was determined by calculation of the chemical score, using as reference egg white proteins. Quite interestingly, the nutritional score were more balanced as compared to FAP1: Thr and Lys were the most adequate (chemical score around 100%), aromatic amino acids (Phe + Tyr and Trp) were around 70%, sulphorated amino acids, the limiting ones in FAP1, were at a notable 60,6% and the limiting amino acids were the branched chain ones, anyway with a not negligible 50%. It appears from these data, that the ultrafiltration process yields a protein mixture of better nutritional properties. Both FAP1 and FAP2 exhibited very high digestibility values (more than 98%). FAP 2 exhibited 1.3-1.5 times lower antioxidant capacity values comparably to FAP1. On the basis of ORAC values, FAP 2 is comparable to other protein hydrolysates of animal origin.

Sensory evaluation of FAP1 and FAP2 was performed by a panel of 20 specially trained assessors. Eleven individual attributes were established to characterise the sensory quality of FAP1 and FAP2; these attributes were classified into two categories: appearance and odour. FAP2 is almost clear broth with faint odour of chicken, as well as with the feeble odour of dried mushrooms. The lack of dominant sensory characteristics both negative and positive makes the product very promising for using in food production.

Therefore, the technology of deep controllable enzymatic conversion of chicken meat & bone residues has been developed. Different poultry processing leftovers (carcasses, backs, necks, wings etc.) could be used as raw materials. As a result protein hydrolysate FAP was obtained. FAP is a highly proteinaceous ingredient (total protein more than 83%), mostly in soluble form that is characterised by stable basic chemical composition, rich in free amino acids (20-28% of the bulk), nearly absent racemisation, good nutritional value for Trp, Lys and apolar amino acids, high content in carnosine, anserine, creatine and creatinine. It has been demonstrated that FAP functional properties can be altered by variation of enzyme composition and dosage. The technology developed is highly reproducible from laboratory to pilot level.

Novel technology for fat conversion into Biodiesel

A new process for the transformation of the fat obtained by PROSPARE poultry processing into biodiesel was studied and developed. This new technology is based on a continuous production method with as main goals to enhance throughput allowing smaller reactors and requesting a less extensive purification/washing of the biodiesel. The process, called HTPM-process, is based on a heterogeneous base catalyst mounted in a fixed bed and operates at higher temperature and pressure to allow miscibility of the two basic ingredients of the process, being the fat or oil and the methanol.

In the first two years of the project, the quality of the fat and fat-rich streams obtained from PROSPARE poultry processing was analysed on several parameters, either relevant to the biodiesel conversion process or relevant to the biodiesel quality. The European norms were taken into account as a base for the quality determination. In particular, the fat samples were analysed on their major constituents (lipid and water content), a detailed lipid analysis was performed and the presence of minor contaminants was determined.

The main conclusions, also considering further work, were that the fat samples had a good quality, although the water and free fatty acids, present as main contaminants, makes conversion with classical biodiesel technology more difficult, due to side reactions. Anyway, it was expected that the new HTPM biodiesel process would be flexible enough to allow for these contaminants, even if present at higher concentrations. Lipid analysis showed that the lipids mainly consisted of triglycerides, as expected, although the lipid profile obviously differed from vegetable oils. Additional analysis of phospholipids was also performed. The phosphorus content was found to be reasonable. A detailed analysis of the minor contaminants showed that the fat sample obtained at more optimised conditions still contained a too large amount of suspended particles. In the last year of the project, it was investigated whether additional pre- or post purification steps were needed to remove these contaminants.

In the first year of the project, a laboratory procedure to convert the fat samples to biodiesel, using conventional technology as well as HTPM-technology, was also set up. The laboratory set-up was adjusted to treat smaller fat samples, both for the classical as the HTPM-technology. Then, the procedure to treat the fat samples, both using classical technology based on a homogeneous catalyst and the HTPM-technology was fixed.

In the second year, the biodiesel set-up was successfully adapted to be able to treat viscous fat streams. The optimal process window was determined, able to convert the fat samples from PROSPARE poultry processing to biodiesel. It was found that high conversions were possible when the reaction was performed applying a residence time between 20 and 30 minutes and a reaction temperature between 300 and 340 °C. These conditions were slightly more severe than required for rapeseed oil.

As compared to the uncatalysed process, a much lower methanol excess was needed. Furthermore, a lower reaction temperature could be applied. The process was also found to be able to cope with a free fatty acid contamination up to 10 wt. %. The presence of free fatty acids was even found to be beneficial for the process. It was also found that methanol contaminated with water was not prohibitive for the process, although the formation of free fatty acids and possible interaction with the catalyst was further to be monitored.

Harsher conditions were found to be needed to convert the commercial rendered fat. In addition, the conversion efficiency was at most around 80 wt. % and indication of catalyst inactivation was observed. In order to account for the slightly more severe reaction conditions necessary for the poultry fats, further research was performed to improve the system. The effect of inefficient premixing was investigated by using a static mixer. Furthermore, the catalyst specific surface and loading was altered, in order to be able to determine the requirements needed. For the improved system, the optimal conversion was obtained by applying 300oC and residence times around 20 minutes. Thermal degradation was noticed under some circumstances for the poly-unsaturated methyl linolenate (C18:3). However, no thermal degradation was observed for the remaining fatty acids, including C18:2. The latter is an improvement as compared to the uncatalysed process and is mainly attributed to the fact that the HTPM-process operates at milder conditions. For poultry fats, the impact of the thermal degradation was limited, because of the small amounts of methyl linolenate (and other polyunsaturated fatty acid methyl esters). Nevertheless, the reaction temperature was limited to 300 °C (at a residence time of around 15 minutes).

In the third year, the need for a pre- and post treatment was also investigated for both the fat from PROSPARE poultry processing as well as for commercial rendered fat. As a basis for the pre-treatment, the procedure of chemical refining was taken, whereby the necessity of the steps of degumming, neutralisation and bleaching was evaluated. As the main goal was to valorise poultry fat to biodiesel, the steps of deodourisation and winterisation were not taken into account. The optimisation of the pre-treatment method was mainly performed on the rendered fat, as this fat was much more contaminated. From the results it was concluded that the fat obtained from PROSPARE was already very pure, containing low concentrations of contaminants, not needing an additional pre-treatment step. The situation was different for the rendered fat, containing soaps and significant amounts of suspended solids which may be responsible for the less efficient process observed.

The biodiesel layer obtained contained approximately 92 wt.% methyl esters, 5 wt.% monoglycerides, 2 wt.% diglycerides and 1 wt.% monoglycerides. As the biodiesel norm EN14214 specifies a methyl ester content of at least 96.5 wt.%, post treatment with vacuum distillation was found to be necessary.

As a final step to complete this task, the full HTPM process as well as the full conventional biodiesel process were applied on the fat obtained from PROSPARE. The resulting samples were then analysed against the EN14214-norm. The results demonstrated the compliance of the biodiesel produced with the HTPM-process for most parameters. Two parameters were not compliant, the water content and the oxidation stability of the biodiesel produced. Furthermore, the cold filter plugging point was rather high and amounted -1 oC in line with the expectations of poultry fat. The water content was in line with expectations as biodiesel is hygroscopic and no special precautions were taken to limit water uptake after vacuum distillation. Also the oxidation stability was too low, but still much higher than in the case of the conventional biodiesel process, another beneficial property of the HTPM-process. It has to be mentioned that no additional measures were taken to enhance oxidative stability by adding anti-oxidants.

Finally, a global processing scheme was defined, incorporating the necessary pre and post-treatment purification steps. A full process scheme was developed in Chemcad 6.3.2. The simulation was based on the optimal conditions for the HTPM production process (i.e. temperature = 300 oC, pressure = 150 bar, residence time = 17 minutes, methanol excess = 0.6 g/g fat) and performed for a 10 kT/yr capacity plant. In the simulation, the fat was mimicked by triolein containing 10 wt.% of FFA to account for a less pure fat stream. The purification unit was designed to allow maximal recovery of the unreacted methanol with purity sufficient to be reused in the process. The recovery system was designed to be able to handle the presence of water in the reaction mixture and to operate at minimal energy duty. Furthermore, a study was performed to incorporate heat integration in the process scheme as well as to allow methanol recovering. This detailed process design was used to determine all necessary utilities and equipment parts, to determine their dimensions and to make a detailed economical evaluation of the process.

In the third year, research was also performed to allow a scaling up of the catalyst, tests were performed on a larger scale to assess possible limitations in the operation of the process and the process economy was determined for a production scale relevant to rendering, which is smaller than biodiesel production from vegetable oil.

The catalyst preparation procedure necessary for scale-up was investigated. The main goal was to obtain a catalyst powder with appropriate particle sizes and with a specific surface area higher than 100 m2/g. Very good results were obtained with the catalyst calcined at 500 °C providing in this way a catalyst preparation procedure compatible with scaling up to pilot.

Initial tests were performed on a pilot-scale installation, allowing to perform the reaction with a heterogeneous catalyst at the specified conditions and to treat up to 100 l/hr fat. Due to its availability, the scaling up tests were performed with rapeseed oil, as this type of oil was expected to be a good model for performing the scaling up. Initial tests performed on the installation indicated two major problems with the installation, i.e. one related to heating of the reaction mixture and one related to the hydrodynamic behaviour. Firstly, the heating system of the reactor was found to be insufficient, resulting in large temperature differences between the heating medium and the temperature of the reaction mixture. This resulted in large temperature gradients and hot spots with danger for product degradation. Secondly, a problem in the hydrodynamic behaviour was observed. This was revealed by testing the hydrodynamic performance of the reactor by spiking with toluene. The toluene was added to the heated reacting mixture at the entrance of the reactor in a short spike of 2 minutes. Then samples were taken at the exit of the reactor as a function of time and were analysed on toluene content. The broadening of the toluene spike was taken indicative of the hydrodynamic performance and the presence of back mixing in the reactor. The toluene peak broadening observed implied that some part of the feed has much larger residence times in the reactor and the reactor has not the real 'plug-flow' behaviour needed.

To address these problems with scaling up, adaptations were made to the pilot installation. The reactor concept for the fixed bed was changed and additional heating capacity was implemented. This resulted in an adapted pilot installation which was used to perform new tests with rapeseed oil, showing the improved heating performance of the reactor. Very similarly, the hydrodynamic performance was significantly improved by adjusting the reactor concept obtaining a reactor with similar plug flow performance as observed for laboratory-scale HTPM-equipment.

In order to evaluate the process at pilot scale, the modified pilot was operated at several conditions and samples were taken at the exit of the reactor. The samples were post treated in the laboratory to remove the glycerol by settling and the remaining methanol by evaporation. The samples were analysed by according to the methods specified in the EN14214:2009-norm. The study showed an optimal process window around 300oC, very similarly as the laboratory results. Similar conversion efficiencies could be attained on pilot scale as on laboratory scale, provided that care was taken on a uniform heating system and a correct operation as a plug flow reactor.

Finally, the economy of the HTPM-process was determined as further input to the scaling-up and commercialisation. The main difficulties encountered in the economical analysis were the strongly fluctuating raw material costs. From the economical analysis it was determined, that the production of biodiesel with the HTPM-process from animal fat with a value of 400 ?/T results in an additional cost price of roughly 110 ?/T for a production unit with a capacity of 10 kT/ yr . The total production cost consists for 89% of raw material costs and only 7% for power and utilities and 4% for the annual costs related to the depreciation and the maintenance of the equipment. When the value of the animal fat increases up to 800 ?/T, the share of the raw material costs further increases up to 94% . Under these circumstances, the production of biodiesel with the HTPM-process results in an additional cost of 140 ?/T . Comparison with the classical biodiesel process indicated that the HTPM process is cost competitive, especially taken into account the smaller scale considered in this study. In addition, this study indicated that the production of biodiesel from animal fat using the HTPM-process is economically cost competitive with the production of biodiesel from vegetable oil using the classical technology. The process remains cost-competitive in time, despite the large fluctuations in raw material cost, as both animal fat as well as vegetable oil prices fluctuate very similarly.

Analytical and functional characterisation of intermediates and end products

The amount of total and free amino acids of FAP samples was detrmined by applying both a commercial LC/fluorescence method and a new LC/MS/MS method newly developed. All FAP samples, prepared on laboratory and pilot scale, both ultrafiltered and not ultrafiltered, were very rich in total and free amino acids. The free amino acid composition of FAP showed a consistent presence of Asp, Glu, Arg, Lys and Leu. The total amino acid composition showed a predominance of Glu and Asp and a quite low content of sulphorated amino acids.

FFP prepared at pilot scale, either by hydrothermal technology alone or by hydrothermal technology followed by enzymatic hydrolysis, were also analysed for their total amino acid content, which again was very high. FFP samples at pilot scale obtained by hydrothermal technology were totally devoid of free amino acids, which were also very low in FFP prepared by hydrothermal technology followed by enzymatic hydrolysis. The total aminoacidic fraction of FFP pilot scale showed a prevelence of Glu, as in lab scale, but in the latter samples cysteine was more consistently present, probably due to the different batches used for lab and pilot scale preparation. In all cases the essential amino acid lysine was present in relatively limited amount.

Molecular weight distribution of FAP and FFP samples was assessed SE-HPLC, indicating that most of the compounds in FAP were in the fraction less than 3 kDa, whereas for FFP the fraction between 3-10 kDa was the most abundant.

The D-amino acid content was checked by chiral GC/MS, confirming the nearly absence of racemisation in FAP, with only Asp, the amino acid most sensitive to racemisation, with a percentage of D-enantiomer less than 4%. This indicate that the process for FAP production is very mild, keeping intact the biological value of FAP hydrolysates. The absence of consistent percentages of D-Ala and D-Glu also indicates that the material by which FAP has been obtained did not underwent extensive fermentation processes by environment bacteria. The D-amino acid content of FFP indicated more consistent racemisation percentages were observed. The most racemised was again Asp, with percentage of D-enantiomer on agerage around 20% (meaning 40% racemisation), but percentage of D-enantiomers, always less than 5%, were also observed for Ala, Ser, Glu, Met, Lys, Val, Ile, Leu, Tyr, Phe. Anyway, in general racemisation only very marginally affected essential amino acids, indicating that the nutritional quality of the FFP were not seriously affected by the hydrothermal treatment.

The volatile fraction of hydrolysates was also characterised. 75 characteristic volatile constituents were identified in FAP including aldehydes (C6-C18), ketones (C7-C19), aliphatic acids (C14-C18), alchohols (C6-C18), hydrocarbons (C11-C13), furan derivates. The composition of volatile fraction of FAP samples obtained at pilot scale was found to be quite stable testifying high reproducibility of FAP production technology.

The composition of volatile fraction of FFP obtained by hydrothermal and enzymatic treatment was also assayed. 41 different constituents were identified. The main volatile constituents were alcohols (C6-C14), aldehydes (C7-C18), hydrocarbons (C11-C12), fatty acids (C10-C18), derivatives of furan and sulfur-containing compounds (benzothiazole, bensenemethanthiol).

The oligopeptide identification was performed by advanced LC/MS/MS methodologies and protein databases including chicken proteins. 419 unique sequences, after redundancy exclusion, have been identified in FAP samples, having MW included between 893 and 4019 Da, derived from 104 precursor proteins. Most of the peptides of FAP were shown to originate from sarcoplasmatic (creatine kynase, enolase, enzymes of glycolysis) and miofibrillar proteins tissue (actin, myosin, troponin, tropomyosin). FAP samples obtained under different scales and hydrolysis conditions are characterised by different number of peptides identified. Significant (less than 0.05) correlation between total number of peptides identified and relative content of medium molecular weight constituents (M.w. 3-10 kDa) in FAP was observed.

Peptide profile of FFP obtained by hydrothermal and enzymatic treatment was also studied by the same methodologies Most of peptides in FFP were shown to originate from feather keratin and ?-keratin related protein. Peptide profile of fermented FFP included 119 peptides with length ranging from 5 to 20 amino acid residues. 49 peptides were found to originate from feather keratin. Only the peptides corresponding to the middle part of the feather keratin sequence, that do not contain disulfide bonds and thus more susceptible to hydrolysis, were established. A database covering the peptides identified by INBI in all samples present in the highest amount was developed. The results clearly demonstrated the potential of High Resolution Mass Spectrometry techniques for the identification of many peptides in complex hydrolyzate mixtures such as FAP and FFP. The 'peptide fingerprint' of FAP and FFP samples was obtained, which can be linked to the proteolytic conditions applied and to the functional activities studied. Comparison of the peptide profiles can be traced to the different proteolytic conditions.

For a semiquantitative approach in peptide analysis, the Proteome Discoverer (PD) tool in the Sequest algorithm has been used to automatically calculate the areas of peptides during sample processing. These areas have been used in order to calculate the area of proteins. PD calculates the area of each protein as the average of the three most abundant peptides with distinct sequences.

Technological properties of FAP samples produces at lab and pilot scales, including water and fat retention capacities, water binding and emulsifying activities and critical concentration of gel formation, were also assessed. FAP samples produced both at lab and pilot scales were characterised by the same values of water-binding and emulsifying activities.Water-binding activity of FAP was shown to be extremely high (94-99%), indicating that it is highly hygroscopic. Water and fat retention capacities of FAPs obtained at pilot scale were 1.5 and 2-fold lower comparably to the corresponding values of FAP samples produced under the same conditions at lab scale. Also, critical concentration of gel formation of pilot FAP samples was shown to be nearly 3.5-fold higher with respect to the lab ones. Those differences in technological characteristics of FAP preparations obtained at lab and pilot plant could be attributed to distinctness in their molecular weight distribution.

Nutritional properties and biological value of FAP could be evaluated based on the data on amino acid score (AAS). AAS of essential amino acids were calculated based on the FAO/WHO (1991) recommended protein standard and reference pattern of essential amino acids in egg protein. Chemical scoring was performed assuming 100% digestibility of FAP. The chemical score of FAP calculated based on the FAO/WHO reference pattern was found to be very high for Lys, Thr, Val, Leu and Ile close or even higher than 100%, whereas it was fairly good for aromatic amino acids (Tyr. Phe, Trp 80-90%) and much lower for the sulphorated amino acids Met+Cys, which comprised on average 70%. Therefore, biological value of FAP1 is limited by the content of Met+Cys. Biological value of FAP1 was also evaluated using thee reference pattern of essential amino acids in egg protein. Again very high chemical scores were observed for Thr, Trp and Lys (close or even higher than 100%), followed by branch chained and aromatic amino acids (Leu, Ile, Val, Phe, Tyr). Sulphorated amino acids Met+Cys were found to be the first amino acids limiting biological value of FAP1.

FFP samples produced by hydrothermal treatment only were also characterised according to their digestibility, using Association of Analytical Communities (AOAC) and International Organisation for Standardisation (ISO) methods. All the samples, produced in the pilot plant, under optimal HTST treatment conditions were characterised by digestibility value exceeding 84%. Thus, the data of the lab scale were confirmed by the data of pilot plant. Enzymatic hydrolysis of FFP increased digestibility up to 99%. Commercial feather meals, sold on European market, were also collected, analysed and compared to FFP. The collected commercial feather meals showed a digestibility that range from 44% to 68%. Only the digestibility of a commercial feather with low protein content reached 80% but its protein content was only 75%. All FAP samples were characterised by digestibility value of about 99%.

Both FAP samples produced at lab and pilot scales showed an antioxidant activity, measured by ABTS and ORAC-HYDRO tests and expressed in Trolox equivalents, comprising on average 650 and 300 µmol Trolox /g respectively. Most of the antioxidant activity of FAP was found to be associated to antioxidant free amino acids (Trp, His, Met) and small non proteolytic peptides (carnosine, anserine) , thus ultrafiltration of FAP did not the antioxidant power. FFP obtained by hydrothermal and enzymatic treatment exhibited antioxidant capacity values comparable to that of FAP. In all cases antioxidant capacity of protein hydrolysates obtained from poultry processing leftovers was predominantly provided by hydrophilic antioxidants.

All FAP also showed a mild antihypertensive ability, with IC50 against Angiotensin Converting Enzyme (ACE) around 1-12 mg FAP / ml solution, with the best values (lowest IC50, around 1mg/ml) shown by the samples richer in peptides and poorer in free amino acids. Ultrefiltered FAP showed an IC50 of about 6 mg/ml, as expected for a sample in which some of the peptides have been lost due to ultrafiltration.

FAP and FFP obtained by hydrothermal and enzymatic treatment were studied in order to determine their ability for the production of probiotic Bifidobacterium and Lactobacillus biomass with improved viability.

The growth promoting activity of the eight FAP and one feremented FFP samples was tested on 18 Bifidobacterium strains and 28 Lactobacillus strains with turbidimetric technique. The growth of Bifidobacteria in medium supplemented with FAPs and FFP was compared with that in basal (B12) and control (B12-Nag) media. The growth of Lactobacilli in media supplemented with FAPs and FFP was compared with that in basal (MRSN) and control (MRSN-YE) media.

FFP obtained by hydrothermal and enzymatic treatment and most proteolysed FAP samples could be consider the optimal growth promoters for Bifidobacterium and Lactobacillus respectively. For this reason they were chosen to evaluate their effect on cells viability. The viable cells number was evaluated by fluorescence microscopic technique, showing that most of the cells were viable. Thus, Lactobacillus growth in the presence of FAPs was very similar, or even better, than in standard media supplemented with yeast extracts. Lactobacillus growth stimulation by FAP could be related to their high content of soluble nitrogen and free amino acids and they consistently showed a poor growth in the FFP sample, rich in proteinaceous material but poor in free amino acids. On the other side, FFP appeared to be the most stimulating supplementation for Bifidobacterium, both in terms of growth rate and viability. This suggests that bifidobacteria are able to hydrolyse the FFP peptides, present in the sample, in a better way as compared to Lactobacillus, or that they find, among the peptides, some specific growth promoting factors. This also suggests that FAPs for Lactobacillus and FFP for Bifidobacterium provide a good alternative to substitute expensive nutrient supplements with cheaper renewable low-cost products for growing Lactobacillus and Bifidobacterium.

FAP were also able to inhibit microbial growth at different temperatures and for several days when mixed with meat. They were demonstrated to inhibit bacterial proliferation in minced meat products, such as hamburgers, through a likely mechanism of aw reduction, allowing for salt subsititution in these products. Although the addition of FAPs to hamburgers cannot substitute totally the salt, still they can integrate the saline function, reducing the amount needed for the control of bacterial contamination without impairing the flavour characteristics and possibly paving the way for producing healthier food for persons subject to hypertension.

For the sensory evaluation of hydrolysates obtained under different technological processes, twenty individual attributes were established to characterise the sensory quality of protein hydrolysates; these attributes were classified into three categories: appearance, odour and taste. The results analysis of sensory evaluation of FAP have shown that the reconstituted product (bouillons) have satisfactory sensory properties.

Finally, all the data on quality and safety aspects of both raw materials and protein hydrolysates, as well as on their composition, functional and sensory properties generated throughout the PROSPARE project were summarised in a database, usable under 'Access' program. The obtained database is a useful tool for exploring the interrelations and correlations of the properties of raw materials, parameters of technological processes and chemical composition, technological and functional characteristics of the intermediate products (chicken fat and protein hydrolysates). The database provides the opportunity to work separately with the data concerning the raw materials and the protein hydrolysates obtained. Moreover, it has corresponding feedbacks that allow retracing the composition and properties of protein hydrolysates using both bottom-up and top-down approaches.

Analysis of the database revealed that sensory characteristics (e.g. odour and taste) of FAP could be mainly attributed to composition of its lipohilic and free amino acids' fractions. However, some peptides, present in FAP could also be responsible for slightly bitter and salty notes in its taste. The influence of variability of raw materials' composition, the technological conditions and production scale on functional properties of protein hydrolysates was examined. Under optimal technological conditions both physical-chemical, microbiological, functional properties of FAP and feather hydrolysates were shown to be perfectly stable. The latter indicates high reproducibility of both biocatalytic and HTST technologies developed in frames of the PROSPARE project.

The database has been made available on the public part of PROSPARE website:

Safety tracing tools

The given WP was focused on the creation and characterisation a set of controlled parameters and analytical procedures to ensure safety of products for the proposed technological process. Specific features of raw material and its processing were taken into consideration, as well as differences in safety regulations between EU and RF. Additional controlled parameters for raw materials were proposed for the developed novel technological process. Besides, levels of lead, cadmium, arsenium, mercury, hexachlorocyclohexane, DDT and its metabolites were selected as chemical indicators of safety; cesium-137 and strontium-90 - as radioactive safety indicators. Basing on analysis of existing regulations, the demands to sampling procedures were revised and sets of samples were collected for the raw materials employed. A special attention was made to ingredient authenticity. The possibility of cross contamination of FAB and FPH preparations by other different animal species will be analysed by molecular methods, by using established procedures according to EU legislation for specific DNA analysis. It was demonstrated that the QIAamp DNA Stool kit (Qiagen) for the DNA extraction, together with Bovine or Swine 'VETfinder MBM Assay' (Generon) for the real time polymerase chain reaction (PCR), is the best solution to control contamination by mammalian ingredients for the sensitivity, specificity and cost.

An integrated raw of chemical and microbiological parameters to be controlled was proposed. Most efficient standard analytical methods were selected taking into consideration priority risks and regulation differences. A specific attention was made to adoption of standard methods to provide safety of the proposed technology, taking into consideration priority risks and regulation differences. For this purpose different analytical techniques were compared, basing on their rapidity, productivity, sensitivity and accordance to ISO regulations. The selected analytical techniques for detection of chemical and microbiological contaminants were applied to FAP/FFP preparations and characterised as tools for efficient safety monitoring of novel products. Hypoallergenic properties of the poultry hydrolysed extract in the IgE inhibition test.

Different microbiological techniques were compared to choose the methods and procedures to use in routine HACCP controls. The selected set of techniques (three ISO methods and two ISO-equivalent methods) allows controlling aerobic colonies, ß glucuronidase positive E. coli, sulphide reducing Clostridia and Enterobacteriaceae. Applicability and repeatability has been checked for all methods. PCR techniques were selected to control the absence of mammalian residues in poultry products. Besides, procedure of contamination control (a tree of decisions) using real-time PCR of Salmonella was elaborated. On the average, the Real-time PCR resulted 32 times more sensitive than the classical microbiological methods that had showed the same sensitivity between each other. A PCR analytical method was successfully be validated for FAP and FFP matrixes against the reference method for the detection of Salmonella spp. from foodstuff and feedstuff as requested from EU legislation. Direct application of the selected PCR analytical method to manufactured FAP and FFP samples demonstrated that all samples were PCR negative thus confirming safety of the proposed technology.

Additional researches in the frame of work package five (WP5) were connected with development and characterisation of novel analytical techniques to improve productivity and informational output for characterisation of chemical and microbiological contamination. Approaches to reduce duration of immunoassays were realised on the example of mycotoxins monitoring and approaches to differentiate alive microorganisms from total biomass content - on the example of pathogens immunosensoric detection.

The need to develop a new analytical method to control microbial contamination of technological samples was stipulated by limitations of existing methods. Traditional microbiological tests include prolonged stages of additional growing for microorganisms from the collected samples before visual identification of colonies or possibility to control their specific enzymatic activity. Immunological methods allow to reduce the analysis time significantly. However, their traditional formats register the total content of specific antigens irrespective of whether they are in living and therefore potentially dangerous cells (rather on their surfaces), in dead or metabolically inactive cells, or in cellular fragments. In this connection, it is desirable to develop and validate a new method that combines the immune selective separation of priority hazardous pathogens from the sample volume, accompanied by their concentration (thickening) and rapid direct detection of metabolic activity, not related to the necessity in additional growing of the target cells or prolonged metabolic changeover to activate enzymatic processes to be registered.

The proposed methodical solution is based on a combination of specific identification of microorganisms via their interaction with antibodies and electrochemical registration of their metabolic processes. The use of magnetic carriers allows to concentrate microbial cells from the volume of the tested sample and to exclude influence of samples compounds on metabolic processes and electrochemical registration.

The registration of electrochemical responses was carried out in a special cell with working glass-carbon electrode, Cl/Ag reference electrode, subsidiary (dammy) electrode. This scheme was in contact with membrane containing immobilised microorganisms. The cell is filled by mediator-containing solution and located in a hermetising holder. The measurements were carried out in a thermostated chamber, signals were transformed by potentiostat IPC 2000 and processed by a computer. Special software has been elaborated to carry out measuring cycles in the electrochemical cell.

Different registering modes were compared for the system including changes of electrode potential, changes of electric current and changes of the rate of potential decrease. The criteria for selection were reproducible signal, the possibility of accurate registration of quantitative parameters at a given time or averaged over a certain period of time, as well as a clear concentration dependence of the signal from the number of immobilised cells. The comparison showed the optimal use for this purpose of the electric current measurements after a certain time after completion of the cycle of potential changes in the cell.

For the developed system values of the recorded electrochemical response for different concentrations of cells in the tested samples were compared. Direct proportional relationship between these parameters was demonstrated that enables accurate registration of the cells content. The indicated above approaches allow to detect E.coli cells in concentrations down to 104 cell/ml. Reproducibility of signals in repeated measurements was no more than +20% being acceptable for reliable control of contamination.

Specific studied were carried out in the frame of the project for more efficient control of mycotoxins. Mycotoxins residues, when present in animal products pose a threat to human health and their control is considered as one of priority tasks in food quality monitoring. At the present time chromatography is the most widely used approach for the control of mycotoxins. However, dependence on complex and expensive equipment limits practical application of chromatographic assays significantly. Immunochemical methods, such as ELISA, allow to reduce assay cost and to increase its productivity. As well as conventional ELISA formats require long incubations with duration of stages of an hour or more, our task was to develop a test system for the rapid (no more than 0.5 h) immunoenzyme control of the most common mycotoxins.

The target analytes considered for this study have been the following: aflatoxin B1, B2, G1 and G2, ochratoxin A, searalenone, fumonisin B1, B2 and B3 and type B trichothecenes (deoxynivalenol, 3- and 15-acetyldeoxynivalenol and nivalenol). Chromatography was considered as reference technique for these studies. Determination was carried out by single target analyses by using LC/fluorescence and LC/MS methods. Two LC/fluorescence methods were applied for the determination of ochratoxin A and aflatoxins, whereas for the remaining mycotoxins LC/ESI-MS methods were applied. In order to get a faster and effective determination of target mycotoxins, a multiresidual method by UPLC-ESI-MS was also developed. Since the detection limits of the applied methods are below the lower EU legal limits for the target mycotoxins in food, the FAP/FFP samples should be considered negative for these contaminants. In consideration of these results, the FAP/FFP preparations were used as blank samples to be spiked for the characterisation of matrix effect for the enzyme-linked immunosorbent assay (ELISA) method.

To prevent influence of samples matrixes of the activity of the label used in the developed immunotechnique for mycotoxins detection, the use of indirect antibody labelling by means of biotin-streptavidin interaction was proposed. Possibility to reduce stages duration up to 5-10 min and total assay duration from around 2 hours to 25 min without lost of sensitivity as compared with common equilibrium assay formats was demonstrated.

Applicability of this ELISA for technological substances (FAP, FFP) and final products was confirmed. The obtained results confirm the robustness of the developed methods for the quantitative determination of mycotoxins in technological samples.

It should be noted that the both developed systems, namely electrochemical immunosensor for specific detection of microorganisms and rapid ELISA for mycotoxins, can be considered as pilot studies of common approaches that may be applied to improve sensitivity and productivity for wide row of analytical techniques in controlling chemical and microbiological contamination.

Additional automation approaches were proposed to increase productivity of recommended and molecular and immune analytical techniques. The proposed robotic system to automate the operations provided by traditional quantitative methods will provide a significant added value not only in terms of reducing the cost of the analysis but also the traceability of the data, risk control for operators and guarantee the performance of tests without manual errors.

The evaluation of the operational characteristics of the proposed robotic system for microbiological control, as the correct execution of the serial dilutions, the accuracy and repeatability made possible to assess the proper functioning of the machine that gave superior results to the corresponding manual tasks; the low values of uncertainty obtained with the test methods developed for the project confirm that. The proposed system complies with the requirements of the ISO 17025:2005. This automation leads to a saving of about 50% of the time of the trial and, consequently, the costs of analysis, relieving the operator of many manual operations.

Food and Feed end-products development

FAP1 and FAP2 obtained at pilot scale complied with all Russian as well as EU requirements in terms of chemical and microbiological safety.

Two FAP1 containing end products namely dry food bouillon and dry food puree soup and FAP-2 based dietetic dry food bouillon were designed using mathematical modeling of protein modules balanced in essential amino acids. Recovery time of instant food bouillon and instant dry food puree soup was optimised by variation of soluble dietary fibres (inuline) content and of the ratio between potato flakes and potato starch respectively. The formula of dry food bouillons was based on protein module, containing 80 mass % of FAP1/2 and 20% of MWPC/MWPH. The bouillons were fortified by addition of ascorbic acid. Dry food puree soup was additionally fortified in microelements by addition of mineral calcium fortifier and iodinated milk proteins. The latter contains bioavailable iodine incorporated into organic matrix.

Dry food bouillons and dry food puree soup contained on average 21.5% and 18.8% of protein respectively. All products contained 9-10% of soluble dietary fibres and 5.0-5.8% of fat. Dry food puree soup additionally contained 10% of insoluble dietary fibres and 4.2 mg% of iron, 1320 mg% of calcium and 210±25 µg% of iodine.

The end products developed complied with all Russian and European safety requirements. Safety and hygienic parameters of FAP1 based end products were verified by the Institute of Nutrition of Russian Academy of Medical Sciences and RF Federal Service for Supervision of Consumer Protection and Welfare. Following normative and technical documentation (technical specifications - TS, technological instruction - TI) was developed: TS # 9197-002-73475455-09 'Fortified instant dry chicken puree soup'; TI on the production of Fortified instant dry chicken puree soup; TS # 9216-00173475455-09 'Fortified instant dry chicken bouillon'; TI on the production of Fortified instant dry chicken bouillon. Sensory evaluation of FAP1-based bouillons and puree soup revealed that their odour and taste were dominated by tones relevant to species, herbs and chicken. FAP1-based puree soup was characterised by pronounced taste of high molecular weight poultry hydrolysate (FAP1) and starchy savour that resulted in reduction of the taste value of the product. Also less pronounced umami and chicken taste of FAP1 based puree soup could be attributed to interactions between protein and carbohydrate ingredients.

The formula of FAP2 based dietetic dry food bouillon had been developed. FAP2-based dietetic dry food bouillon had the highest content of free amino acids (62.42±16.50 mg/g) among all the end products under the study. Free amino acidic fraction of FAP2-based dietetic dry food bouillon is dominated by Glu, Leu, Arg, Tyr and Lys. Content of total amino acids in FAP2-based dietetic dry food bouillon comprised 233.19±8.72 mg/g. Gly, Ala, Asp, Glu and Lys are the most abundant total amino acids in FAP2-based dietetic dry food bouillon. Leu was shown to be the first limiting amino acid in FAP2-based dietetic dry food bouillon. Nevertheless, the product developed has high biological value due to perfectly balanced amino acid composition.

Comparison of composition of volatile fractions in FAP1 and FAP2 based bouillons revealed that they are quite similar. The main volatile constituents of FAP2 based dietetic dry food bouillon are terpenoids (herbs and species) and products of lipids' oxidation (carbonyl substances, furan derivatives). FAP2 based dietetic dry food bouillon is a good source of ascorbic acid and animal protein with balanced amino acid composition.

For certification of FAP2 based dietetic dry food bouillon technical specifications (TS) TS 9216-01835305730-10 'Instant dry Chicken bouillon 'Genuine' were developed. Based on the results of the expert evaluation TS 9216-01835305730-10 and FAP2 based dietetic dry food bouillon was included in the RF Federal catalogue of products. The product was further certified by the authorities of RF Federal Service for Supervision of Consumer Protection and Welfare. As a result the Declaration of compliance was obtained for FAP2 based dietetic dry food bouillon. The declaration of compliance states that FAP2 based dietetic dry food bouillon completely comply with the RF safety requirement for that type of the product, namely Hygienic requirements on safety and nutrition value of food products SanPiN (indices 1.9.10 and GOST R 51074-2003. Sensory evaluation of FAP2-based dietetic dry food bouillon revealed that it has the highest consumer preference among all the end products under the study.

The study of FAP1 sample application as antioxidant in meat matrix has been carried out using poultry Baader and hamburgers (turkey minced meat). Poultry Baader was stored in the presence of various natural antioxidants (HERBALOX, ORIGANOX, NATUREX AND FORTIUM PLUS) and 1.5% of hydrolysates (FAP1). The concentrations of volatile carbonyl compounds (products of lipid hydroperoxides decomposition) in all phenolic natural antioxidant-containing samples have been considerably lower than that in the control indicating high protection of meat against lipid oxidation. Also among volatile carbonyl compounds hexanal was always found to be the most abundant one. In case of tukey minced meat differences in volatile aldehydes content were shown to be evident only between the sample mixed with HERBALOX and the control. The concentrations of volatiles in HERBALOX samples have been lower than the control during the whole storage time (12 months). Regarding the addition of 1.5% of hydrolysates (FAP1) no significant antioxidant effects has been evident during the storage at -20°C, in fact the samples with 1.5% FAP show the same development of aldehydes as the control. Sensory tests showed that FAP1 acts more likely as a flavour enhancer rather than antioxidant in meat products after 8 and 12 months of storage.

FAP1 and FAP2 are novel proteinaceous food ingredients developed in frames of the PROSPARE project. Prior to wide application their functionality and safety should be verified in vivo. Acute toxicity of FAPs was examined according to GOST 12.1.007-76. Both FAP1 and FAP2 were shown to be non-toxic compounds. The study of FAP1 and FAP2 allergenicity using a panel of 104 sera obtained during routine serological analysis from children with different forms of food intolerance revealed that both FAP1 and FAP2 could be considered as low-allergenic ingredients. In vivo experimental model of ovalbumin-induced systemic anaphylaxis was used for analysis of both allergenicity and antiallergic properties of FAP1 and FAP2. The ingestion FAP1 has no effect on the severity of systemic anaphylaxis and serum levels of circulating ovalbumin-specific antibodies and FAP1 could be considered as hypoallergenic ingredient. Ingestion of FAP2 also has no effect on the severity of systemic anaphylaxis but slightly increases serum levels of circulating ovalbumin-specific antibodies and could be considered as low allergenic ingredient. Both FAP1 and FAP2 didn't exhibit antiallergic activity.

Bioavailability of protein and biological value of FAP1 and FAP1 was determined using rat nitrogen balance method. The amount of protein, consumed by rats in different experimental groups is nearly the same. However, protein efficiency ratios (PER) are different for all protein sources under the study. The highest PER was observed for FAP1 (0.58). It was 15% higher than that of casein (0.49). For FAP2 PER (0.29) is 41% lower as compared to casein. True fecal digestibility of FAP1 (89.6%) was found to be comparable to that of bovine casein (90.8%). However, true fecal digestibility of FAP2 was higher (94.7%) comparably to casein. Both FAP1 (63.5%) and FAP2 (77.9%) were shown to have lower biological value (% of absorbed nitrogen that is retained for maintenance and/or growth) as compared to bovine casein (86.9%) used as a standard in nitrogen-balance method. The latter could be attributed to less balanced amino acid composition of FAP1 and FAP2 comparably to casein. Also lower biological value of FAPs with respect to casein could be assigned to inefficiency of utilisation of free amino acidic fractions of FAP1 and FAP2 by rats. The highest excretion of nitrogen in urine was found in group 2, fed with FAP1. FAP2 exhibits higher biological value as compared to FAP1. Nitrogen balance in case of FAP2 and casein is nearly the same. Therefore, FAP2 has higher biological value comparably to FAP1 and lower as compared to casein.

Analysis of the antioxidant status of rats fed with rations, containing different protein ingredients (casein, FAP1, FAP2, protein-free diet), included determination of serum AOC and TBARS concentration values, as well as TBARS content in liver and brain homogenates. No significant difference in serum AOC values was observed among all experimental groups of animals under the study. Comparison of TBARS levels in organs of rats, fed by rations with different protein sources, revealed that no significant differences in liver. However, significantly (less than 0.02) lower concentration of TBARS was observed in brain homogenates of rats fed with FAP1 and FAP2 rations as compared to those fed with casein ration. Therefore FAP1 and FAP2 exhibit mild antioxidant effect in central nervous system.

Analysis of FFP viability for production of pet food was analysed. The standard feather meal due the poor digestibility can be used only in the production of economic pet food, till a maximum percentage of less than 2 % of the total protein rate. On the other hand FFP, for its characteristics can be used more than 6 % of the total protein rate, for both economic products and premium and\or super-premium products. Most dried pet foods would have a low palatability without the use of flavourings or palatability enhancers which areoften sprayed onto the kibble in small amounts to make the food more appetising. FPP being tasteless and odourless has no interference with appetiser normally used in pet food industry, proving also in this case a good source of protein.

Potential impact:

Dissemination & Demonstration (D&D) has been one of the important activities within the project, thanks to the high-potential and value that the innovative results of the project have been generating -from the scientific to the exploitation contexts- in EU and RF, but also on global scale. The dissemination impact of the project's investment have been maximised, for assuring further developments in terms of exploitation and of enhanced research.

Thanks to the D&D activities, a very positive progression has been measured by generating awareness more than interest more than appreciation more than exploitation for the PROSPARE project which, in its inherent scope, intends fostering the introduction on the markets of enhanced innovation with concrete technology and products.

The scientific community (in EU, RF and international) has been quite interested in the novel methods and technology developed and assessed by PROSPARE, which indeed open the way to a new generation of production systems and of unprecedented premium FAP and FFP protein lines.

The industry community has reacted extremely well; contacts in the food chain with selected key industry operators enabled to positively verify the attractiveness and solidity of the project, with confirmation of a clear interest for the innovation exploitable from the research work. Two pilot sites have already been established, with full production in place at the end project and some prospects waiting for feasibility study for new plants. Thus there is now a very clear confirmation of the potential opportunities in view of the post-research exploitation (technology, production systems, R&D enhancements) on the specific industry markets.

In conclusion, while in the first part of the project a clear appreciation was measured for the enhanced R&D objectives of the project, the consumptive final measure of the events and have well responded to the project's D&D objective to optimise cost/value effectiveness to introduce, develop, demonstrate and pre-exploit the PROSPARE project potentiality.

To underline that these direct D&D activities have confirmed the significant scientific but industrial added value being introduced by PROSPARE.

Therefore, thanks to the early pilot installations (at Symbol in Russia and at Proliver in Belgium), PROSPARE has clearly achieved the maturity to fostering a relevant post-project exploitation on the EU and the RF markets, but also internationally. The initial contacts established by the D&D actions have involved Argentina, Belgium, Brazil, France, Egypt, Germany, Greece, Korea, India, Italy, Netherland, Norway, Spain, Russia, Sweden and United States.

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