EUR 22393 EN

PROJECT SYNOPSES

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EUROPEAN COMMISSION Directorate-General for Research Directorate E — Food, Agriculture and Biotechnologies Unit E1 — Horizontal aspects and coordination

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EUROPEAN COMMISSION

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2007

Directorate-General for Research 6th Research Framework Programme Thematic Priority 5 - Food Quality and Safety

EUR 22393 EN

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FOREWORD BY THE COMMISSIONER

Even though these are not times of food scarcity for Europe, people are still affected by food-related anxieties. When the European Commission started to develop the ideas behind the Sixth Research Framework Programme, the role of microbes, vitamins and carbohydrates were already familiar, a new era of discourse on food began. At that time, citizens started to concern themselves with questions of pesticide in fruits, nutritional content, bovine growth hormones in milk, mad cow disease, allergies and obesity. There was only one way to differentiate between the good, the bad, and the inedible and to support the new emerging rights of the “informed choice”; we needed to invest in food safety and quality research across the board, from fork to farm. Food industry is Europe’s largest manufacturing sector. Surprisingly, it remains nonresearch intensive. Less vulnerable than others to economic fluctuations, it is instead affected by consumer habits and perceptions more than anything else. If we want to remain competitive and rise to the challenge in providing our citizens with the high-quality safe food they request, we have to discover and unveil the potential of new technologies and transfer the knowledge to large companies as well as to the many SMEs that represent the economic backbone of Europe. By responding to pressing requests from consumers, market and Member States, the forward-looking thematic priority Food Quality and Safety in the Sixth Framework Programme has been a great success. It effectively supported research to develop an environmentally friendly production and distribution chain able to deliver safer, healthier and more varied food to people. I hope you will look forward to the new Seventh Research Framework Programme with equal enthusiasm.

Janez Potočnik European Commissioner for Science and Research



STEADFAST COMMITMENT TO HIGH-QUALITY AND SAFE FOOD IN EUROPE Over the last decade, food quality and safety has grown into a significant sector, in the public opinion and the media. At the beginning of 2000, related research needed to become a priority as various food scares had caused a serious lack of confidence among EU citizens. Recognising that food safety is a fundamental and ongoing issue, EU policymakers developed Thematic Priority 5 (TP5), Food Quality and Safety. The driving force for setting up TP5 in the Sixth Research Framework Programme (FP6) was the need to improve the health and well-being of European citizens through higher quality food and improved control of food production and related environmental factors. Using the classic ‘farm-to-fork’ approach, the programme gave priority to identifying the major issues for consumers, and then proceeded along the production chain, outlining issues associated with primary production, animal feeds, processing, distribution, consumption and environmental health risks. In addition to combining production, processing, nutritional and analytical expertise, the projects funded through TP5 drew on expertise from such areas as genomics, medicine, information technologies, ethics, environmental, economic and the social sciences, to achieve their aims. In doing so, the strategy provided the fertile ground for the development of a ‘total food chain’ approach to food quality and safety. 

Accordingly, the food sector shifted its focus towards consumer needs, resulting in improved links between production, distribution and consumption. This all-encompassing approach has helped allay concerns and restore consumer confidence across Europe. This catalogue highlights the challenge undertaken by Food Quality and Safety during FP6. A total of EUR 751 million in funding was injected into research activities between 2002 and 2006 to deepen understanding of the links between food production, consumption and health. TP5 supported 181 research projects to help develop an environmentally friendly production and distribution chain able to deliver safer, healthier and varied food to European citizens. The scope of the theme is indicative of the scale of the research issues addressed – from food-related risks and diseases (relying in particular on biotechnology and the results of post-genomic research) to the health risks associated with environmental changes. The research activities funded by FP6 also contributed to the realisation of a European Research Area promoting mobility, cooperation and training of EU scientists through the pooling of know-how and expertise. Contemporary philosophers have already noted the key role played by modern food production in western societies. Furthermore, food safety regulation and the governance of regional and global biotechnology markets present new and more complex challenges for European policymakers. The selected research projects served to reinforce and establish European governance around food production and consumption. Combined, they offer valuable insight into the complexity of research issues regarding one of the most pressing international problems of today.

* Statistical analysis of the 181 projects funded under TP5 in FP6 is available at http://cordis.europa. eu/food/

FP6 portfolio of instruments* The Food Quality and Safety Priority 5 (TP5) of the Sixth Framework Programme (FP6) encompassed many of the research topics and themes funded by the previous FP5 Quality of Life programme. However, new scientific areas had been introduced: total food chain; epidemiology of food-related diseases and allergies; impact of food on health; ‘traceability’ processes along the production chain; methods of analysis, detection and control; safer and more environmentally friendly production methods and technologies; impact of animal feed on health; and environmental health risks. Between 2002 and 2006, 3 130 participants of 181 projects were selected under four main calls incorporated in ten deadlines. FP6-2002-FOOD-1; FP6-2003-FOOD-2-A; FP6-2003FOOD-2-B; FP6-2004-FOOD-3-A; FP6-2004-FOOD-3-B; FP6-2004-FOOD-3-C; FP6-2005FOOD-4-A; FP6-2005-FOOD-4-B; FP6-2005-FOOD-4-C and FP6-2003-ACC-SSA-FOOD. In addition to the traditional funding instruments of the scientific research community such as Specific Targeted Research Projects (STREPs), Coordination Actions (CAs) and Specific Support Actions (SSAs), FP6 moved towards funding larger, more ambitious projects. In particular, two new funding instruments were introduced: Integrated Projects (IPs) and the Networks of Excellence (NoEs).

STREPs The 60 Specific Targeted Research Projects were designed to gain knowledge or demonstrate the feasibility of new technologies. Available to small and emerging players, they played an important function for the scientific community because they financed research on new technologies that do not necessarily have a direct impact on the market. FP6 supported the improvement of innovative non-thermal processing technologies to develop the quality and safety of ready-to-eat meals; determined whether pharmaceutical products were present in food and whether they affected human fertility; and assessed if the composition of flavonoids in various foods had similar effects as antioxidants in decreasing the risk of cardiovascular diseases and enhancing the body’s immune system. STREPs also helped to create recommendations for lifestyles and healthy habits to improve the quality of life of EU citizens.

CAs The 7 Coordinated Actions (CAs) covered the definition, organisation and management of joint initiatives that aimed to avoid duplication of efforts in different Member States and sought to build synergies between existing national and other international initiatives so as to better integrate European research.

SSAs The 71 Specific Support Actions (SSAs) helped to prepare and support new research activities. The SSAs also aided in the preparation of Seventh Framework Programme, encouraging and facilitating participation in European collaborative research efforts. In particular, FP6 Specific Support Actions (SSA) were directed at the following seven objectives: achieving



ERA objectives; promotion of SME participation; stimulating international cooperation; linking with candidate countries; supporting policy development; stimulating exploitation; and contributing to the EU strategy for life sciences and biotechnology.

IPs The 31 Integrated Projects were designed to deliver new knowledge, a competitive advantage to European industry, and respond to SME needs by integrating and mobilising the critical mass of research activities and resources. Some IPs put into service innovative biomarkers to detect the exposure to chemicals and metals in food and set up diagnosis procedures to help food industries to manage allergies.

NoEs The 12 Networks of Excellence were aimed at strengthening excellence by connecting resources and expertise and supporting effective integration and cooperation in the research activities of the network partners, as well as advancing the overall topics of interest.



7

Food Quality and Safety projects in FP6: SSA STREP

31 (17%)

IP CA NOE

60 (33%)

7 (4%)

12 (7%) 71 (39%)

NOCHEMFOOD

PAVING THE WAY FOR VEGETAL-BASED FOOD ADDITIVES

268

LIST OF PARTNERS • Bioma Agro Ecology CO AG (Switzerland) • CNR Avellino -Istituto di Scienze dell’Alimentazione (Italy) • Centro Tecnólogico de la Industria Cárnica de la Rioja (Spain) • CSIC-Institute of Industrial Fermentation-Madrid (Spain) • Università del MoliseDipartimento di Scienze e Tecnologie Agro-Alimentari e Microbiologiche (Italy) • Innova S .p.A (Italy) • Salumificio Spiezia (Italy) • Embutidos Dany SL (Spain) • Evaggelopoulos Apostolos (Greece)

T

he NOCHEMFOOD project endeavours to develop a novel class of food additives based on plant sources. These new vegetalbased extracts are being tested with a view to replacing currently used classes of chemicals. Keeping in mind the economic importance of the food additive industry, attempts will be made to fully exploit the market potential of the innovative technologies that are likely to emerge from NOCHEMFOOD. The first step is to test these new extracts on a specific food group widely consumed in Europe: the sausage. Food Quality and Safety - Call FP6-2004-FOOD-3-B

VEGETABLE ALTERNATIVES TO CHEMICAL ADDITIVES

IMPACTING THE EUROPEAN FOOD MARKET

NOCHEMFOOD employs a host of biotechnological, genomic and biochemical techniques to extract and process vegetal-based substances and study their applicability in the food industry. One of the project’s goals is to develop and scale-up appropriate extraction methods, which are not only friendly to the environment but also capable of meeting industrial demands. Characterising and potentially optimising the extracted substances is an important part of the project’s overall strategy. These efforts include a variety of tests aimed at examining the effect of these substances, not only on food preservation but also on taste and overall quality.

Following EC decisions to reduce authorised levels of nitrates and nitrites in meat products, NOCHEMFOOD can offer alternative additives that result in food products with improved quality and safety. These vegetal-based bioorganic additives could eventually replace chemicals such as nitrites and nitrates throughout the food industry. The first industry to be included in this initiative is meat processing. Sausages rank high among European consumers’ preferences and as such offer a viable market diffusion tool for any new class of food additives developed within the NOCHEMFOOD consortium. The sausage/pork meat market is particularly sensitive to pathogens; therefore the economic benefits of NOCHEMFOOD could be significant indeed.

Acronym: NOCHEMFOOD Full title: novel vegetal-based extracts additives for chemical-free food Contract n°: 023060 Project co-ordinator: BIOMA Agro Ecology CO AG EC Scientific Officer: Ebba Barany [email protected] EU contribution: € 2.3M

Specific Targeted Research or Innovation Project

© European Communities, 2007 - Reproduction is authorised provided the source is acknowledged.

European Commission EUR 22393 — Food Quality and Safety in Europe Luxembourg: Office for Official Publications of the European Communities 2007 — 396 pp. — 14.8 x 21 cm ISBN 92-79-02693-3

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European Commission THE SIXTH FRAMEWORK PROGRAMME

3rd Year Activity Report (Month 36) CONTRACT N° : 023060 ACRONYM : NOCHEMFOOD TITLE : NOvel Vegetal-based Extracts Additives for CHEMical-Free FOOD PROJECT CO-ORDINATOR : Company: Bioma Agro Ecology CO AG E-mail: [email protected] Fax n°: +41 918 40 10 19 PARTNERS : Part. No.

1 2 3 4 5 6 7 8

Participant name

Bioma Agro Ecology CO CNR Avelllino -Istituto di Scienze dell’alimentazione Centro Tecnólogico de la Industria Cárnica de la Rioja CSIC-Institute of Industrial Fermentation-Madrid Università del Molise-Dipartimento di Scienze e Tecnologie Agro-Alimentari e Microbiologiche Innova S.p.A. Salumificio Spiezia Embutidos Dany SL

Participants Short name BIOMA CNR

Country

CTIC

ES

CSIC DISTAAM

ES IT

INNOVA SALS EMB

IT IT ES

CH IT

REPORTING PERIOD: FROM 1st August 2008 to 31th July 2009 PROJECT START DATE : 1st August 2006 DURATION : 36 months Date of issue of this report : September 2009

1 NOCHEMFOOD-Specific Targeted Research Project-Contract no.: 023060

M36 Report

European Commission

Publishable Executive Summary NOVEL VEGETAL-BASED EXTRACTS ADDITIVES FOR CHEMICAL-FREE FOOD The NOCHEMFOOD Project (NOvel Vegetal-based Extracts Additives for CHEMical Free FOOD), aims at developing a new strategy to produce foods containing mainly natural ingredients and from which possibly chemical additives, considered harmful for human health and subjected to regulation restrictions, have been removed. In particular, the project explores the potential use of these natural preserving agents, constituted by mixtures of active molecules extracted from vegetal sources, in the sausage industry, in substitution of commonly used nitrites and nitrates. More specifically, the main scientific and technological objectives of the project are the following:



To develop new vegetal-based extracts as additives for food, with the scope of substituting some currently used chemicals, which are subject to regulation restriction and might be harmful for human health. A full characterization from the chemical, biochemical and microbiological point of view of these products, with the subsequent attempt to understand their mechanism of action




To demonstrate the effectiveness of these novel vegetal-based mixtures of compounds as preserving agents in some largely consumed foods in Europe. Target foods selected for this purposes are sausages and other food classes which will be selected and preliminarily evaluated during the course of the project.




To develop an effective dissemination plan to fully exploit the innovation potential of these products and enhance the commercial potential of some of these typical European productions.

During the final period, WP1 activities were focused on the optimisation of the extraction process, in order to obtain the best extraction yield and the best antioxidant and antimicrobial activities. The influence of different concentration of ethanol on the extract compositions were investigated. Tests on homogeneity of the formulation, which was identified as the main critical parameter in the extraction process, were carried out. The optimized formulation NC was prepared and extensively analysed and tested.

Page 2

European Commission WP2 was focused on the analysis of the optimized formulation NC, its antioxidant activities and polyphenols content. Chemical and functional characterization of all extracts, including subcritical batches, supercritical batches (CO2 + 6%, 3% and 0% ethanol) and formulations (SC, LC and NC).was done. Two new additional analyses were performed to NC formulation to measure the nitrites content and metal contaminant. In WP3, pilot scale and industrial experiments performed by CTIC and Dany revealed that the size of minced meat influence the final color of sausages. Experiments performed by Spiezia revealed the color uniformity in products treated with vegetal extracts is slightly lower with respect to the conventional one. In WP4 the biochemical and microbiological analyses performed on meat products, revealed that the NC formulation, well reproduces the effect of nitrites and nitrates by inhibiting the undesirable microorganisms growth without affecting the useful ones. Results evidenced that the best NC concentration is 0.5%. The same results were obtained in pilot and large scale production. The activities of WP6 were focused on testing the theoretical formulation NC in other food classes. In particular, cooked ham (Spiezia), pathè (CTIC and DANY), and Chorizo fresco (CTIC, DANY) were prepared and preliminarily analysed from the biochemical and microbiological point of view. The preliminary analyses evidenced good results for cooked ham and Chorizo fresco: the products containing vegetal extracts are very similar to the conventional one. In case of pathè, good results from the microbiological and sensorial point of view were observed, nevertheless the substitution of nitrites with vegetal extracts strongly affects the colour of product, from pink with nitrites to brown with NC formulation. During the last year the exploitation and dissemination activities were intensified, through: patent application: a joint European patent application is being filed • participation to scientific conferences and congresses, • publication on scientific journal (Food Chemistry) and on the annual eStrategies publication addressed to wide public (39'000 of the most important delegates in this area in Europe), • presentation of NOCHEMFOOD to CLITRAVI (liaison centre for the meat processing industry in the E.U.) • interview for “SME Success Stories in the area Food Agriculture and Fisheries and Biotechnology” catalogue. •

During the last year the interaction between partners was very strong: the project is structured in a way that WP’s activities strongly depend one each other. This allows a strong collaborative and fruitful work inside of the consortium.

Page 3

European Commission Section 1 - Project objectives and Major achievements during the reporting period 1.1 General Objectives of the project The table below summarise the achievements in the M24-M36 period against the general project objectives formulated in Annex I. Objective Number 1

Objective Description

Comments on Achievement

To develop new vegetal-based extracts as additives for food, with the scope of substituting some currently used chemicals, which are subject to regulation restriction and might be harmful for human health.

2

A full characterization from the chemical, biochemical and microbiological point of view of these products, with the subsequent attempt to understand their mechanism of action.

During the third year of the project, the NOCHEMFOOD formulation was designed on the basis of statistical analysis performed by CSIC and prepared by BIOMA. T-P conditions were defined on the basis of the best yield of extractions obtained. Extractions with 3 different concentrations of Ethanol (0%,3% and 6%) were carried out in order to evaluate the influence of the ethanol on the extracts compositions and on the yield of extraction. T-P parameters for extraction scale up process were defined. Chemical, biochemical and microbiological characterizations of the optimized formulation were done and compared to the results on the other formulations previously obtained. Moreover additional analysis were carried out on the NC formulation for measuring the nitrites content and metal contamination.

3

To demonstrate the effectiveness of these novel vegetal-based mixtures of compounds as preserving agents in some largely consumed foods in Europe. Target foods selected for this purposes are sausages and other food classes which will be selected and preliminarily evaluated during the course of the project.

4

To develop an effective dissemination plan to fully exploit the innovation potential of these products and enhance the commercial potential of some of these typical European productions.

Experiments on pilot and industrial scale production of salami Napoli and Chorizo Vela were performed. The experiments were performed on: a) meat products containing nitrites and nitrates, b) meat products containing NC formulation and c) meat products without chemical or vegetal additives. Biochemical and microbiological analyses were performed on all productions. Sensory panels of the products were carried out. Preparation of 3 other food classes (cooked ham, pathè and Chorizo fresco) containing NC formulation were undertaken and preliminarily analysed from the physicochemical and microbiological point of view. Preliminary panel tests on cooked ham were carried out . The exploitation framework was analysed and the exploitation plan was defined at consortium and partner level. The consortium participated to different conferences and exhibition, such as CLITRAVI (Meat European Association). The consortium was invited to publish an article in the annual eStrategies publication. Finally a patent application is being jointly filed.

1.2 Summary of work performed and Partners involved during the reporting period WP n° 1 2 3 4 5

Work Performed Extraction at 3 different concentrations of ethanol (0%, 3% and 6%). Preparation of the NC formulation. Chemical biochemical and biological characterization of NC formulation. Antioxidant activity and polyphenols content of NC. Nitrites content and metal contamination of the NC formulation Preparation of Salami Napoli and Chorizo Vela containing NC formulation: pilot and large production Microbiological and physico-chemical characterizations of the meat products prepared in pilot and industrial scale Sensory panels of salami Napoli and Chorizo Vela. Acceptability and

Partners Involved BIOMA CSIC, CNR SPIEZIA, CTIC SPIEZIA, CTIC, CNR, DISTAAM CNR

Page 4

European Commission 6

7 8

preference indices Production of 3 other food classes containing NC formulation: Cooked ham, pathè and Chorizo fresco. Preliminary physico chemical and microbiological analyses of the 3 classes. Preliminary panel test of 3 food classes Dissemination activities and patent application Management and coordination of the project

SPIEZIA, CTIC, CNR, DISTAAM All partners BIOMA, INNOVA

1.3 Timeline The table below illustrates the GANNT chart for the 36 months of the project.

1.4 Summary of key objectives for the reporting period Obj. Number 1

2

Objective for the reporting period

Current Status

Evaluation of the ethanol concentration on the extracts composition. Preparation of the designed NC formulation Full chemical characterisation: genotoxicity and proteotoxicity tests

Extractions have been made at 3% of Ethanol as concentration and the extracts have been analysed and compared to the extraction at 0 and 6% of Ethanol. NC formulation has been completely characterized, as previous formulations; additionally nitrites content and metal contamination were measured. Meat products containing NC formulation have been prepared following a production protocol optimised during the project. Salami Napoli (one diameter, 50 mm) was prepared on pilot and industrial scale, as Chorizo Vela. A large production of Salami Napoli (calibre 40) has been also prepared Biochemical and Microbiological analyses have been performed on Salami Napoli and Chorizo Vela produced on pilot and industrial scale.

3

Preparation of Salami Napoli and Chorizio Vela on pilot and industrial scale

4

Biochemical and microbiological analyses on meat products prepared. Investigation of the concentration of NC on undesirable and useful microorganisms Sensory panel and consumer accettability tests of Italian and Spanish salami and sausages Selection of 3 other food classes: cooked ham, pathè and Chorizio fresco, and tests on these of the NC formulation

5

6

7

Produce dissemination material for scientific and public audience

The sensory panels have been performed on meat products containing NC formulation. Comparison with corresponding conventional products have been done The production of 3 other food classes containing NC formulation have been done, Preliminary microbiological and biological analyses were carried out. Sensory panel of cooked ham has been performed 1 publication was submitted to peer-reviewed journal. 1 article was done on Science Technology and

Page 5

European Commission

8

Ensure delivery of the reports and deliverables

Innovation journal. 3 posters were produced. 5 abstract for conferences were sent off. 2 oral presentations were done. Patent application is in progress. All reports and deliverables were submitted with slightly in delays.

Page 6

European Commission Section 2 – Workpackage progress for the period WP1 – Environmentally Friendly Extraction Processes2.1.1 Summary of progresses towards objectives and contractors involved Starting point of work

Objectives

Progress

T-P parameters were defined on the basis of the extraction yields. The homogeneity of the formulations was observed as the most important scale-up parameter

Definition of the formulation NC optimisation of industrial process

new and the

The new formulation was designed on the basis of statistical analysis and prepared by BIOMA. Concerning the scale up experiments, homogeneity tests were performed in order to detect T-P anomalies during the industrial process. Extractions at 3% of ethanol concentration were done and the extracts were characterized and compared to the results obtained at the other 2 ethanol concentrations.

Contractors involved BIOMA

2.1.2 Deliverables and milestones Table a: Deliverable

Deliverable N°

Deliverable Title

M2

D1.1

First extracts to be characterized in WP2

D1.2

M12 Parameters allowing to obtain a given composition Identification of scale-up M24 parameters

D1.3

Delivered Yes/No

Delivery date from Annex I

Actual / Foreseen date

Yes

M2

Yes

M12

Yes

M29

Table b: Milestones

Milestone N°

Milestone Title

Delivered Yes/No

Actual / Foreseen date

M1.1

Exploration of a given temperature and pressure parameter space

Yes

M18

M1.2

Establishment of rules allowing to obtain a given extracts composition starting from a given extraction parameter set.

Yes

M24

M1.3

Critical parameters for the subsequent scale up have been identified

Yes

M24

2.1.2 Activities Performed Task 1.2: Solvent extraction experiments with fluid CO2 and Task 1.3 Variation of the extraction conditions During the second annual meeting and considering the results obtained by the project, all the partners had a discussion on the definition of the final formulation for preserving meat products. The ideal formulation (NC) is described below in table 1. The composition is based on all the

Page 7

European Commission functional properties evidenced in previous reports and it include Raphanus niger as component, considering its antioxidant properties, its effects on colour and its activity versus undesired and useful micro-organisms. This extract, that showed inhibition on useful micro-organisms, substituted Medicago composita, that affects both undesired and useful bacteria. Some minor modifications to a statically obtained formulation were performed in order to round the amount of each component in the final formulation and make easy the preparation. The T-P conditions were selected considering yields of extraction processes.. Table 2.1. Definiton of the new formulation (NC)

Extraction Conc. (g/l)

T (°C)

P (atm)

Formulation Comp. (mg/l)

Formulation Comp. (%)

Spirulina pacifica

100

40

73

-

-

Raphanus niger

50

40

73

1

6.1

Malpighia punicifolia

250

40

73

3.5

21.2

Rosmarinus officinalis

300

40

73

1.5

9.1

Propolis

100

40

73

-

-

Medicago composita

100

40

73

4

24.2

Carica Papaya

50

40

73

3

18.2

Citrus compositum

210

40

73

3.5

21.2

Vegetal Source

This formulation was produced and sent for experiments (see WP3, current report) to CTIC-DANY and SPIEZIA. The comparison between supercritical (SFE) and sub-critical (SBFE) conditions was completed preparing extracts under supercritical conditions using the 3% of ethanol as solvent. This batch was sent to analytical partners for characterization.

Task 1.4 Scale-up experiments The starting point for the optimization of the industrial process is represented by the observations obtained by the pilot scale. The goal is to maximize the yield of the extraction process and to obtain the best antioxidant and antimicrobial properties for each raw material. In report M12 and M24 are described all the experiment carried out during Task 1.1 and Task 1.2, performed on ten different T-P couples and replicating analyses in some of them. Considering all the obtained results, the homogeneity of the formulation produced in an industrial plant was identified as the main critical parameter. Homogeneity tests were carried out in order to detect T-P anomalies during the industrial process, that can affect the final composition of the final product. This test was focused on antioxidant properties that can be estimated by EC50. The antioxidant activity of two batches (extracted at 40°C and 73 atm), produced by two different runs of the industrial plant, respectively was measured. Each batch was divided in 10 samples whose activity was measured twice, which means 20 measurements by batch. The number of samples has been decided in function of the Harmonized Protocol Procedure (M. Thompson and R. Wood, Pure Appl. Chem. 1993, 65, 2123) which gives indications for testing for “sufficient homogeneity”. Samples were sent to CSIC laboratories for the analytical work. For further details see D1.3. The formulation NC prepared by the large plant was sent to partners in order to perform the scheduled activities on meat products and additional food classes (see WP2, 3, 4, 5 and 6).

Deviation No deviation

Page 8

European Commission WP2 – Extracts Characterization 2.2.1 Summary of progresses towards objectives and contractors involved Starting point of work

Objectives

Progress

Full chemical characterization of SC3 and LC3 formulations and evaluation of their antioxidant properties

Chemical and biochemical characterisation of the new formulation NC ,its characterization of antioxidant activity

Genotoxicity and protoetoxicity tests were performed on SC3, LC3 and on the new formulation NC. The chemical,biochemical and functional characterization of all extracts, including subcritical batches, supercritical batches (CO2 + 6%, 3% and 0% ethanol) and formulations (SC, LC and NC) were done. Moreover additional analyses were performed on NC formulation in order to test the possible presence of nitrites and metal contaminations

Contractors involved CSIC, CNR

2.2.2 Deliverables and milestones Table a: Deliverables

Deliverable N°

Delivered Yes/No

Delivery date from Annex I

Deliverable Title

Actual / Foreseen date

D2.1

Mutageniticity, proteotoxicity and genotoxicity reports

M12

Yes

M12

D2.2

Data on antimicrobial properties

M6/M12

Yes

M6/M12

D2.3

Preliminary GC and HPLC data

M6

Yes

M6

D2.4

Optimized analytical protocols M34 Chemical composition of the M34 extracts

Yes

M36

Yes

M36

D2.5

Table b: Milestones

Milestone N°

Milestone Title

Delivered Yes/No

Actual / Foreseen date

M2.1

The mutageniticity, genotoxicity, and proteotoxicity characteristics of the mixtures have been assessed

Yes

M30

M2.2

The antimicrobial properties of the extract have been evaluated

Yes

M18

M2.3

The main organic components and their relative concentrations in the extracts have been identified

Yes

M18

M2.4

The best analytical methodologies for the characterization of the extracts have been identified

Yes

M18

2.2.3 Activities Performed Task 2.1- Mutagenicity, genotoxicity, proteotoxicity and antimicrobial activity tests Page 9

European Commission Genotoxicity Test (CNR) Further experiments on the potential degradation of genetic material deriving from eucariotic cells caused by the SC3 and LC3 vegetal extracts formulations, as well as by the new designed NC formulation, were performed using the Single Cell Gel Electrophoresis (SCGE), a microgel electrophoresis technique of single cells, commonly known as “Comet Assay”. In such assay, cells with enhanced DNA damage display increased migration of the DNA from the nucleus towards the anode under the electrical field, giving the appearance of a “comet tail”. All details regarding this methodology have been widely described in the previous M24 Report. In the following Table 2.1 results are reported, indicating that percentages of damaged cells by the tested vegetal formulations were very similar to those relative to negative controls. Table 2.1 - Effects of SC3, LC3 and NC vegetal extracts formulations on Jurkat cells in the Comet assay

Damaged cells , % Untreated cells (Negative Control)

20.1 ± 3.2

Cells incubated with SC3 (1%)

23.4 ± 6.1

Cells incubated with LC3 (1%)

19.8 ± 4.2

Cells incubated with NC (1%)

21.5 ± 5.1

Cells treated with H2O2 (Positive Control)

81.2 ± 3.5

Jurkat cells, at a concentration of 106 cells/ml, were incubated for 1 hour with each of the above mentioned vegetal extracts formulations at 1% concentration, before SCGE electrophoresis.

Proteotoxicity Test (CNR) The evaluation of possible protein damage caused by vegetal extracts was performed, also in this case, by means of electrophoretic methodologies, investigating the effect of both SC3 and LC3 formulations and the new prepared NC one on a model of carrier protein, the Bovine Serum Albumin (BSA) at physiological conditions. Samples containing 1 ml of a 4% BSA solution were incubated, for 2 hours at 37 °C, in the presence of different amounts of the vegetal extracts, in a total volume of 2 ml. After incubation, all prepared samples were analyzed for potential breakages in fragments of the BSA protein by means of SDS-PAGE electrophoresis. As control test, the BSA protein was treated with 0.25 ml of Dimethylsulphoxide (DMSO) in the same conditions as with the different vegetal extracts. In the Fig. 2.1 the electrophoretic pattern of the BSA samples after incubation is reported.

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European Commission

1

2

3

4

5

6

7

Figure 2.1 - SDS-PAGE electrophoresis of 4% BSA samples, incubated for 2 h at 37°C in the presence of different vegetal formulations.

Before incubation, all samples were normalized to a total volume of 2 ml with distilled water. After incubation, 20 µl aliquots of samples were loaded onto the gel. Lanes: 1. BSA 1 ml + SC2 0.2 ml; 2. BSA 1 ml + LC2 0.2 ml; 3. BSA 1 ml + SC3 0.2 ml; 4. BSA 1 ml + LC3 0.2 ml; 5. BSA 1 ml + NC 0.4 ml; 6. BSA 1 ml + H2O 1 ml (negative control); 7. BSA 1 ml + DMSO 0.2 ml (positive control). Despite the notable breakage in fragments of the BSA protein caused by the highly polar organic solvent DMSO (Fig. 2.1, Lane 7), any changes in the BSA protein structure could be detected in the case of the tested vegetal formulations, even at enhanced concentrations.

Task 2.3.- Optimization of analytical protocols Antioxidant activity The antioxidant activity of supercritical CO2 extracts was measured with the DPPH radical scavenging test, as seen in previous reports. As can be seen in Fig. 2.2, the batch SFE1 showed pretty similar activity, which in general terms is slightly higher (lower EC50) than the mean of subcritical extracts. But SFE2 showed worse antioxidant activity, this low activity could be associated to the dehydration process, because this batch was the only that was dried before sending the samples.

Page 11

European Commission 170 160 150 140 130 120 EC50 (µ g/ml)

110 100 90 80 70 60 50 40 30 20 10 0 Citrus

Batch 1

Batch 2

Papaya

Batch 3

Batch 4

Batch 5

Medicago

Batch 6

Batch 7

Propolis

Batch 8

Batch 9

Raphanus

Batch 10

Batch 2b

Batch 2c

Rosemary

Batch 10b

Batch 10c

Spirulina

Batch 7B

SFE1

*Malpighia extracts precipitated in the reaction media, activity not measured

Figure 2.2- Antioxidant activity of raw extracts expressed as EC50 (µg/ml), the lower EC50, the higher antioxidant activity.

In terms of chemical composition, extracts were analyzed by HPLC-DAD for the quantification of antioxidant compounds (vitamins, polyphenolic and pigments) and GC-MS for the qualitative analysis of volatile antimicrobial compounds.

GC-MS (antimicrobial compounds) Table 2.2 shows the composition of SFE extracts. As can be seen, the composition of SFE1 looks pretty similar to subcritical batches, but SFE2 showed, for all the raw materials tested, an extremely, an anomalous, high amount of 2-furanmethanol and some other compounds. In order to confirm this point, analysis were repeated twice but results were always the same. This compound was only found previously in such high levels in propolis, while it was also present in subcritical extracts of malpighia, papaya and raphanus, but in very low amount. These results depict some kind of contamination which could come from the extraction process, recovery, dehydration, rehydration, etc. The presence of these compounds could also explain the lower antioxidant activity showed by this batch.

RT 3,54 4,84 7,43 8,96 9,09 9,29 12,44 14,56 15,93 16,58 17,30 17,80

Compound Acetic acid, methyl ester 2-Propanone, 1-hydroxy2-furanmethanol 1,3-Cyclopentanedione 2-cyclohexen-1-ol 2(3H)-Furanone, 5-methyl1,8-cineole Linalool Camphor Endo-borneol L-4-Terpineol A-terpineol

SFE1 0,67 0,49 ----7,83 1,75 8,05 1,94 1,74 1,65

SFE2 7,98 -25,04 4,35 1,51 2,09 -10,14 -7,81 -2,07

18,34

2-Furancarboxaldehyde, 5-(hydroxymethyl)-

1,83

13,44

14,07

18,91

Glicerol acetate

1,74

--

14,50

21,64

2,4(3H,5H)-Furandione, 3-propyl-

--

6,06

16,17

24,05

2,1,3-Benzothiadiazole

6,56

--

17,42

Propolis

Rosemary

Table 2.2- Main results of the GC-MS analysis of the SFE extracts, results expressed as % of Area, retention time (RT) in minutes. RT 3,29 3,64 4,93 6,51 7,06 7,97 8,11 8,30 8,99 9,12 9,33 13,34

Compound Acetic acid Acetic acid, methyl ester 2-propyn-1-ol 2-Furanmethanol Acetamide, N-(aminocarbonyl)Cyclopentanone 2-Cyclohexen-1-ol 1,3-Cyclopentanedione 2(3H)-Furanone, 5-methyl2-cyclohexen-1-ol 1,3-Cyclopentanedione Cyclobutanol 2-Furancarboxylic acid, methyl ester 2-Propanamine, N-methyl-Nnitroso4H-Pyran-4-one, 3-hydroxy-2methyl2-Furancarboxaldehyde, 5(hydroxymethyl)-

SFE1 SFE2 10,60 -2,52 -1,32 -33,63 36,26 -1,38 -2,53 2,56 1,27 -1,89 4,02 -1,36 -2,81 --4,97 2,54

--

2,10

--

3,62

3,34

--

24,86

Page 12

SFE2

European Commission 26,19

Ascorbic

52,82

--

29,42 30,15 31,39 34,05

Proline, 3-hydroxy-4-methylHexadecanoic acid Isomenthol Betulin Phenol, 4,4'-methylenebis[2-(1,1-dimethylethyl)6-methylTotarol-7-one Retinoic acid, 5,6-epoxy-5,6-dihydro-, methyl ester 4H-1-Benzopyran-4-one, 5-hydroxy-7-methoxy-2(4-methoxyphenyl)-

-1,29 2,35 --

12,14 --7,37

1,03 4,50

---

2,02

--

1,74

--

35,34 35,62 36,40 37,13

24,32 27,70 30,16 30,46

Ascorbic Quinoline, 3-hidroxi-2,4-dimethylHexadecanoic acid 5,5'-oxy-dimethylene-bis(2-furaldehyde)

-6,34 1,64 6,98

21,07 ----

27,27 27,97 28,38

RT 3,45 3,86 5,11 6,41 7,90 8,04 8,07

Compound Formic acid Acetic 2-Propanone, 1-hydroxy2-furanmethanol Cyclopentanone 2-cyclohexen-1-ol 2(3H)-Furanone, dihydro-

SFE1 9,81 1,73 1,25 1,97 --2,76

SFE2 ---34,03 2,22 1,27 --

RT 3,49 4,00 6,64 8,10 8,21 8,40 13,42

8,24

1,3-Cyclopentanedione

0,82

1,59

15,89

9,30 10,92 13,32 13,94 15,52

Trisulfide, dimethyl Sugar Cyclobutanol Methyl cinnamate Trimethyl urea 4H-Pyran-4-one, 2,3-dihydro-3,5dihydroxy-6-methyl2-Pyrrolidinone 2-Furancarboxaldehyde, 5(hydroxymethyl)Glicerol acetate 2,4(3H,5H)-Furandione, 3-propylAscorbic N-acetil cytidine Proline, 3-hydroxy-4-methyl-

0,77 1,11 ----

--3,63 3,88 8,17

9,47 --

-25,65

15,96 17,11

Raphanus

17,44

15,98 17,41 18,37 18,93 20,74 25,63 27,49 28,41

SFE1 SFE2 1,70 -3,48 --19,84 18,75 -2,18 -6,82 --6,74 2,32 -9,40 --

-6,40

RT 3,26 3,68 5,11 8,05 8,46 13,39 13,99 15,39 Papaya

20,22 21,91

Compound Acetic 2-furancarboxyaldehide 2-furanmethanol Furan 2,5-Furandione, 5-methyl 2,5-Furandione, dihydro-3-methylene4H-Pyran-4-one, 3-hydroxy-2-methylFurancarboxylic acid, methyl ester 4H-Pyran-4-one, 2,3-dihydro-3,5dihydroxy-6-methyl2(3H)-Furanone, dihydro-5-propyl2-Furancarboxaldehyde, 5(hydroxymethyl)Propanoic acid, 2-methyl-, 2methylpropyl ester Ethyl-2-Furancarboxylic acid

15,96 17,12

16,79 45,95

17,34

19,41 4,18

---

18,80 20,09

5,27 -9,30 --13,71 26,57 -29,18 --5,84

Medicago

Citrus

RT 3,24 6,36 6,44 9,72 10,27 12,56 13,93 14,05

18,61 2(3H)-Furanone, dihydro-5-propyl- 17,53 -Pentanoic acid, 3-hydroxy-, methyl 20,83 ester -14,71 28,66 Proline, 3-hydroxy-4-methyl-1,00 29,50 Furaltadone 15,40 7,79

Compound Acetic acid Acetic acid, methyl ester 2-furanmethanol N,n-bis(2-methoxyethyl)-methylamine 1,3-cyclopentanedione Cyclobutanol Methilcinnamate 2-Propanamine, N-methyl-N-nitroso4H-Pyran-4-one, 2,3-dihydro-3,5dihydroxy-6-methyl2(3H)-Furanone, dihydro-5-propyl2-Furancarboxaldehyde, 5(hydroxymethyl)-

SFE1 SFE2 24,53 6,87 3,12 4,66 1,92 39,58 3,53 5,52 -2,31 -6,61 -2,22 2,04 5,32

Glicerol acetate 2,4(3H,5H)-Furandione, 3-propyl1,2-Benzenedicarboxylic acid, bis(2methylpropyl) ester Furaltadone Proline, 3-hydroxy-4-methyl-

17,34 20,62 27,99 28,38 28,41

Compound Acetic acid Acetic acid, methyl ester 2-Furanmethanol Cyclopentanone 3,6-dihydro-2h-pyran-2-one 1,3-Cyclopentanedione Cyclobutanol 4H-Pyran-4-one, 2,3-dihydro-3,5dihydroxy-6-methyl2-Furancarboxaldehyde, 5(hydroxymethyl)2,4(3H,5H)-Furandione, 3-propylFuraltadone Sugars Proline, 3-hydroxy-4-methyl-

30,16 30,40

Hexadecanoic acid Hexadecanoic ethyl ester

31,24 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl31,41 Ethyl linoleate 31,59 Linoleic acid

18,24 --

-6,01

--

4,54

16,48 12,98

-4,82

17,16 ---

-2,95 8,58

SFE1 SFE2 11,17 2,98 1,48 --40,88 -4,38 -1,78 1,30 1,99 5,55 9,99 3,60

--

-16,58 -8,43 -2,81 58,65 --10,17 2,40 1,45

---

2,50 7,26 4,66

----

Page 13

Spirulina

European Commission Compound Acetic acid, methyl ester 1,2-propanediol 2-Furanmethanol N,N-bis(2-methoxyethyl)-methylamine 1,3-Cyclopentanedione Methilcinnamate

15,59

2-Propanamine, N-methyl-N-nitroso2-Furancarboxaldehyde, 5(hydroxymethyl)Phenol, 5-methyl-2-(1-methylethyl)-

17,37 20,13 20,40 27,99 28,43 29,17 31,25 31,33 31,41 31,55

SFE1 SFE2 0,38 3,37 2,06 --34,95 -4,67 -4,11 -2,31 --

Malpighia

RT 3,45 4,80 6,62 8,09 8,21 13,99

7,29

-0,85

13,39 -7,08Phenol, 2-methyl-5-(1-methylethyl)0,86 Hexadecanoic acid -3,91 Hexadecanoic acid ethyl ester -11,50 C18 acid isomers 49,33 7,42 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl 12,16 -5,8,11-Heptadecatrienoic acid, methyl ester 9,72 -Linoleic acid 17,12 -Ethyl linoleate 7,52 --

RT 6,45 8,17 9,10 11,32 12,26 13,23

SFE1 SFE2 1,32 --0,80 0,59 0,79 0,88 0,59 -0,80 0,97 --

17,39

Compound 2-Furancarboxaldehyde N,Nbis(2-methoxyethyl)-methylamine 2-Furancarboxilic acid 2-hexenol Furan, 2,5-dihydro2-Furancarboxilic acid methyl ester 2-Furancarboxaldehyde, 5(hydroxymethyl)-

19,81 27,25

Methyl 4-methoxy-3-pentenoate Ascorbic

-6,06 78,75 78,97

27,78

Sugars

8,07

9,41

3,07

8,92

HPLC-DAD (antioxidant compounds) Results of chemical characterization of supercritical extracts can be seen in Table 2.3. As could be seen in the GC-MS analysis, it seemed to be a contamination, in this case the “rare” compound is a cinnamic acid (cinnamic 25). This compound appeared previously only in Propolis. As demonstrated in the statistical analysis, cinnamic acids were the main compounds responsible of lowering the antioxidant activity, therefore this data can explain the lower antioxidant activity found in SFE2 respect SFE1. The rest of compounds seem to be in accordance with previous results. Globally, these results clearly show that the tested supercritical conditions do not improve largely the results obtained in previous batches, when subcritical conditions were used. Table 2.3- Main results of the HPLC-DAD analysis of the SFE extracts, results expressed as % of Area, retention time (RT) in minutes. SFE1

Medicago

Papaya

Citrus

Name

RT (min)

SFE2

mg comp/g extract

Ascorbic

2,6

1,22

--

Thiamine

3,5

27,55

26,09

cinnamic 01

8,6

5,11

3,43

Flavanone 01

9,5

4,79

3,26 1,91

Flavonol 02

10,7

4,46

Flavanone 09

11,1

10,93

7,74

Flavanone 12

11,5

11,91

10,56

Cinnamic 25

12,5

--

77,26

cinnamic 28

12,6

2,43

2,41

Thiamine phenolic 04 Cinnamic 25 Anthocianin 01

3,5 9,5 12,5 11,0

53,50 5,07 -3,47

52,72 4,97 44,69 0,85

Ascorbic

2,4

9,51

9,39

Thiamine

3,0

142,07

141,59

Isoflavone 01

9,3

5,65

4,05

cinnamic 11

10,4

3,77

--

cinnamic 14

10,8

4,98

4,60

cinnamic 17

11,1

4,06

2,88

Page 14

Rosmarinus

Raphanus

Propolis

Malpighia

European Commission Flavonol 04

11,4

12,45

9,34

Flavonol 06

11,6

2,63

--

Flavonol 10

11,9

15,01

12,26

Flavonol 12

12,1

12,73

12,60

Isoflavone 02

12,3

3,31

--

Cinnamic 25

12,5

--

54,87

Isoflavone 03

12,7

4,44

2,34

Flavonol 18

13,2

8,38

7,20

Flavanone 24

14,5

5,90

3,95

Ascorbic

3,0

127,03

126,37

Thiamine

3,1

23,45

20,70

Phenolic 01

8,6

2,50

--

Pheno diol 01

9,3

5,04

3,79

Flavanone 05

10,2

0,73

--

Cinnamic 25

12,5

--

31,69

n.i. "isoflavone" 2

13,1

4,24

3,68

n.i. "isoflavone" 3

13,5

3,06

2,51

Ascorbic Thiamine n.i. 1 n.i. 2 Cinnamic 15 n.i. 3 Flavanone 13 Cinnamic 22 Cinnamic 24 Cinnamic 25 Flavanone 16 Flavonol 16 Cinnamic 26 Flavanone 18

2,4 3,4 9,5 10,6 11,0 11,3 11,5 11,7 12,1 12,5 12,8 13,0 13,2 13,4

4,20 30,13 19,63 20,67 30,53 4,75 16,81 41,60 69,29 41,61 52,52 9,66 92,45 35,08

2,20 27,79 16,69 18,86 28,64 -14,96 38,47 66,05 79,46 52,04 7,41 90,96 33,21

Flavanone 19

13,6

33,20

31,58

Ascorbic

3,0

3,19

3,08

Thiamine

3,4

90,10

89,70

Nicotinamide

5,9

11,81

9,93

cinnamic 05

9,3

2,19

1,15

cinnamic 06

9,5

1,27

--

cinnamic 09

10,2

3,99

3,00

cinnamic 12

10,4

1,80

--

cinnamic 20

11,4

5,50

3,63

Cinnamic 25

12,5

--

64,69

Thiamine

3,2

31,36

30,56

Phenolic 05

9,5

2,39

--

Flavanone 07

10,6

3,91

2,88

Flavanone 08

10,9

5,89

3,55

Flavonol03

11,3

20,55

19,72

Flavonol07

11,6

8,39

6,45

Flavonol09

11,8

13,83

12,69

Rosmarinic 01

12,0

1,25

--

Rosmarinic 02

12,2

37,93

36,45

Cinnamic 25

12,5

--

64,24

Flavonol 13

12,5

10,27

--

Flavanone 17

12,8

63,64

60,84

Page 15

European Commission Flavonol 15

12,9

18,37

15,97

Flavonol 17

13,1

13,23

10,53

Flavonol 20

13,3

4,43

2,65

Flavonol 21

13,7

19,86

18,24

Flavanone 20

13,8

10,24

10,24

Flavanone 21

14,1

6,18

5,07

Flavonol22

14,7

14,38

14,22

Flavonol23

14,7

14,84

12,82

Phenolic 09

15,6

14,19

12,96

Phenolic 10

16,3

10,11

8,35

Antioxidant activity of formulation

EC50 mg/L

In the Logroño meeting, a formulation was agreed based on the statistical analysis of previous data (bioactivities of pure extracts and formulations) and “commercial interests” (colour, yield, smell…). Its composition resulted as follows: Spirulina 0%, Citrus 21,2%, Rosemary 9,1%, Propolis 0%, Medicago 24,2%, Malpighia 21,2%, Raphanus 6,1%, Citrus 18,2%. The antioxidant activity of The Formulation was measured by DPPH antioxidant activity test, as previously described for extracts. On a first set of analysis, the antioxidant activity of one formulation produced at pilot plant scale was measured giving an average value of antioxidant activity equal to 71,50 µg/ml, which mean a better value compared with the activity found for previous formulations SC and LC, as can be seen in Fig 2.3. It also provide a similar but slightly better value than the predicted by the Multiple Linear Regression Model prediction, which was 74,94 µg/ml. 110

Experimental value

100

MLR Prediction

90 80 70 60 SC1

SC2

SC3

LC1

LC2

LC3

Pilot Scale

Figure 2.3- Antioxidant activity of The Formulation produced at Pilot Scale compared to SC and LC formulations

The analytical protocols followed during the last 6 months have benne the same used until now and it is well described on the D2.4. Nevertheless, the new analysis were performed only to NC formulation to measure the nitritis and metal contaminat contents. The protocols followed are here reported.

Nitrites content: Griess method Griess first described a colorimetric assay to measure the levels of nitrite (NO2–) in aqueous solutions 130 years ago1. When sulphanilic acid is added, the nitrites form a diazonium salt. When the azo dye agent (alpha-naphthylamine) is added a pink colour develops. The Griess (nitrite) test is a simple, inexpensive, and rapid method for detecting nitrites. Despite some other fluorimetric or ionic chromatographic methods can be found with higher sensibilities, none of them is as rapid and used as Griess. A typical commercial Griess reagent contains 0.2% naphthylenediamine dihydrochloride, and 2% sulphanilamide in 5% phosphoric acid. 1

Ivanov, V.M., The 125th Anniversary of the Griess Reagent Journal of Analytical Chemistry, 2004. 59(10): p. 11091112.

Page 16

European Commission Procedure: 1. Mix equal volumes of Griess Reagent (Fluka, Madrid, Spain) and sample. The calibration curve is done within working range: 0.15-500 nmolar NaNO2 (Sigma, Madrid, Spain). Two blanks are included Griess+water and FormulationNC+water. 2. Read the absorbance at 540 nm after 15 minutes.

Heavy metal content (ICP-MS) The inorganic impurities generally originate from various sources and phases, i.e., raw materials, reagents, solvents, electrodes, catalysts, reaction vessels, plumbing and other equipments. Monitoring of heavy metals in process intermediates and final drug substances is an important activity in food industry. It is not only because of their ability to catalyze decomposition but also potential for toxicity. Heavy metals like lead and cadmium in food pose the risk of serious health hazards even at very low doses2. Atomic spectrometric techniques, AAS (Atomic Absorption Spectroscopy), ICP-AES (Inductively Coupled Plasma-Atomic Emission Spectroscopy) and ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) are widely used for analysis of trace elementals. AAS is probably the most extensively used technique for determination of metals in different sample matrices. Generally flame-AAS (FAAS) is used when concentration of the analyte is high enough or graphite furnace AAS (GF-AAS) when it is low. Its application to the analysis of impurities is limited due to relatively high detection limits. Highly specific hollow cathode lamps are used for determination of each metal. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) plays a significant role in the analysis of food and pharmaceuticals. It is a multi elemental technique but suffers from complex spectral interferences and accuracy at ultra trace levels. Mass spectrometer coupled with inductively coupled plasma ionization (ICP-MS) is one of the most sensitive analytical techniques for fast multi element determination of heavy metals in trace and ultra trace concentrations in different sample matrices. Recently, it has emerged as a powerful technique and at present, it is the most suitable technique for the analysis of trace elements in bulk drugs and pharmaceuticals. It provides a major service to the pharmaceutical industry in the analysis of heavy metals in drugs2. The Elan 6000 ICP-MS (Perkin-Elmer, Sciex, USA) fitted with a peristaltic pump was used for the determination of metals and metaloids in sample digests. The instrument was calibrated using multi-element standards solutions prepared in 3% nitric acid (Merck) by mixing and diluting ICP stock solutions of individual elements (Merck). Formulation NC was dried at 60ºC overnight in a polycarbonate plate to avoid metal contamination. Dried samples were resuspended in 3% nitric acid (200 mg of NC formulation in 50 ml) and 1 ml was injected in the ICP. Rhodium (Merck) was used as internal standard. Due to the lack of this kind of equipment in the dependences of CSIC-UAM group, this analysis was subcontracted to the Servicio Interdepartamental de Investigación of Universidad Autónoma de Madrid (SIDI-UAM) and performed by their personnel.

Task.2.4: Characterization directed to scale-up experiments In order to test the formulation homogeneity, the antioxidant activity of two batches, 1010 and 1011 or NC prod 1 and 2, respectively was measured. Each batch was divided in 10 samples whose activity was measured twice, which means 20 measurements by batch. The values of the antioxidant activity obtained looked pretty similar to the antioxidant activity found for the pilot scale experiment (Fig. 2.4)

2

Nageswara Rao, R. and M.V.N. Kumar Talluri, An overview of recent applications of inductively coupled plasma-mass spectrometry (ICP-MS) in determination of inorganic impurities in drugs and pharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis, 2007. 43(1): p. 1-13.

Page 17

European Commission 90 80 70

EC50 (µg/ml)

60 50 40 30 20 10 0 Batch 1010 Sample 1

Sample 2

Sample 3

Batch 1011 Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Sample 9

Sample 10

Pilot

Figure 2.4- Antioxidant activity of Large Scale extraction experiments, antioxidant activity of pilot scale experiment is shown in yellow for comparison.

In order to confirm how similar were both batches in terms of antioxidant activity, their values were treated using a T-Student test. This test confirmed certain difference among both samples, but this difference was lower than the differences due to the analytical process itself; Fig. 2.5 clearly shows the high reproducibility of the extraction process. In the T-Student test, the differences among formulations were not statistically equal to cero (p