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Introduction Introduction The European Commission Concerted Action FAIR CT97-3233 was set up in 1997 following the publication of several scientific papers showing a possible role of lycopene in the prevention of diseases such as cancer and cardiovascular diseases. Epidemiological studies showed that these risks increased with low tomato consumption, the main source of lycopene. A consortium of 15 tomato research institutes and tomato processors from all around Europe was formed, with funding from the European Commission within the framework of the FAIR RTD programme, to review the scientific literature available in order to produce a "white book" on the antioxidants in tomatoes and tomato products. This data would help the industry improve the nutritional quality of the tomato-based products and the scientists to understand better the relationship between nutrition and health. The researchers were organised into 4 working group to study different aspects: • Working Group 1 : Composition of tomatoes and tomato products in antioxidants (methods of analysis; breeding and biotechnology to increase the antioxidant content; evolution of the composition during ripening and storage), chaired by Dr. Pascal Grolier (INRA, Clermont-Ferrand, France) • Working Group 2 : Processing and bioavailability (effects of mechanical and thermal treatments and storage conditions on the antioxidants content and their bioavailability), chaired by Dr. Carlo Leoni (SSICA, Italy) • Working Group 3 : Epidemiological observational studies (protection offered by antioxidants on cardiovascular diseases, cancer and ageing), chaired by Dr. Mariette Gerber (INSERM-CRLC, Montpellier, France) • Working Group 4 : Mechanisms and biomarkers (mechanisms involved in the protection and biomarkers), chaired by Prof. Rod Bilton (Liverpool John Moores University, UK). Project management and co-ordination were provided by Prof. Gérard Bartholin (CTCPA, Avignon, France), Mr. Bernard Bièche & Mrs Sophie Colvine (AMITOM, Avignon, France). Composition of tomatoes and tomato products in antioxidants Working group 1 - Composition of tomatoes and tomato products in antioxidants Chairman: P. Grolier, INRA, UMMM, Vitamin group, 63122 St Genes Champanelle, France. Authors: P. Grolier 1, G. Bartholin2, L. Broers3, C. Caris-Veyrat4, M. Dadomo5, G. Di Lucca6, Y. Dumas7, F. Meddens3, L. Sandei8, W. Schuch9. 1) INRA, UMMM, Vitamin group, 63122 St Genes Champanelle, France. 2) CTCPA, Station Pierre Mainguy, ZA de l’aéroport, BP 1203, 84911 Avignon Cedex 09, France. 3) Nunhems Zaden BV, PO Box 4005, 6080 AA Haalen, Netherlands. 4) UMR INRA-UAPV "Sécurité et Qualité des Produits d'origine Végétale", Site Agroparc, Domaine Saint Paul, 84914 Avignon Cedex 09, France. 5) Azienda Agraria Sperimentale Stuard, Str. Madonna dell’Aiuto, 7/A, 43016 S. Pancrazio, Italy. 6) Sviluppo Produzioni Agricole, Tenuta La Fagianeria, 81015 Piana di Monte Verna, Italy. 7) INRA, Station d’Ecophysiologie et Horticulture, Domaine Saint Paul, Site Agroparc, 84914 Avignon Cedex 9, France. 8) Stazione Sperimentale per l’Indusria delle Conserve Alimentari in Parma, Via Tanara 31/A, 43100 Parma, Italy. 9) Zeneca Plant Science, 3 Marston Lane, Eaton, NR4 6LZ, Norwich, UK. Composition of tomatoes and tomato products in antioxidants (WG1) page 2 __________________________________________________________________________________________ Acknowledgements The authors wish to thank Peter Bramley from the University of London (UK) for it’s participation at the proceedings of the tomato and health seminar in Pamplona (1998). We also wish to thank Wilhelm Stahl from the University of Dusseldorf (Germany) and Marie- Josèphe Amiot from the INRA center of Avignon (France) for the reviewing of our manuscript. Composition of tomatoes and tomato products in antioxidants (WG1) page 3 __________________________________________________________________________________________ Contents p2 Acknowlegdments p3 Contents p9 1. Introduction p10 2. Objectives p11 3. Composition of tomatoes in antioxydants p15 4. Breeding and biotechnology methods for high lycopene content p17 4.1. Breeding p18 4.2. Genetic modification approach p19 5. Influence of maturity stages and storage on the antioxidant composition of tomatoes p20 6. Influence of cultural practices and agronomic aspects on antioxidants content of tomato fruit p20 6.1. Lycopene, β-carotene and total carotene p20 6.1.1. Introduction p21 6.1.2. Influence of temperature p24 6.1.3. Influence of light p25 6.1.4. Influence of cultural techniques Composition of tomatoes and tomato products in antioxidants (WG1) page 4 __________________________________________________________________________________________ p25 6.1.4.1. Variety p26 6.1.4.2. Water p27 6.1.4.3. Mineral nutrition p31 6.1.5. Ripening stage p35 6.1.6. Growth and development regulators p39 6.2. Vitamin C p39 6.2.1. Introduction p40 6.2.2. Influence of temperature p40 6.2.3. Influence of light p42 6.2.4. Influence of cultural techniques p42 6.2.4.1. Variety p42 6.2.4.2. Water p43 6.2.4.3. Mineral nutrition p48 6.2..4.4. Ripening stage p49 6.2.4.5. Growth and development regulators p50 6.3. Vitamin E p50 6.3.1. Introduction Composition of tomatoes and tomato products in antioxidants (WG1) page 5 __________________________________________________________________________________________ p50 6.3.2. Influence of cultural techniques p50 6.3.2.1. Variety p50 6.3.2.2. Ripening stage p51 6.4. Phenolic compounds p51 6.4.1. Introduction p51 6.4.2. Influence of light p52 6.4.3. Influence of cultural techniques p52 6.4.3.1. Variety p52 6.4.3.2. Mineral nutrition p53 6.4.3.3. Fruit development and ripening stage p54 6.4.3.4. Influence of season p54 6.4.4. Conclusions p55 6.5. Conclusions on the influence of cultural practices and agronomic aspects p57 7. Physico-chemical characteristics and properties. p57 7.1. Lycopene p57 7.1.1. Chemical structure Composition of tomatoes and tomato products in antioxidants (WG1) page 6 __________________________________________________________________________________________ p57 7.1.2. Physico-chemical characteristics and properties p57 7.1.2.1. Melting point p57 7.1.2.2. UV-vis spectrophotometry p59 7.1.2.3. IR Spectroscopy p59 7.1.2.4. Solubility p59 7.1.2.5. Sensitivity p61 7.1.2.6. Influence of the environment on physico-chemical properties. p61 7.1.3. Lycopene cis isomers. p61 7.1.3.1. Structures p62 7.1.3.2. Occurrence p62 7.1.2.3. Physico-chemical properties p64 7.1.4. Antioxidant properties p64 7.1.4.1. Quenching of singlet oxygen p65 7.1.4.2. Quenching of free radicals p67 7.2. Vitamin C p67 7.2.1. Chemical structure p67 7.2.2. Physico-chemical characteristics Composition of tomatoes and tomato products in antioxidants (WG1) page 7 __________________________________________________________________________________________ p68 7.2.3. Antioxidant properties p69 7.3. Vitamin E p71 7.3.1. Physico-chemical characteristics p71 7.3.2. Occurrence p71 7.3.3. Antioxidant properties p72 8. Antioxidant analysis in foods and biological tissues p72 8.1. Samples p73 8.2. Storage of the samples p75 8.3. Standards p77 8.4. Extraction p81 8.5. Analysis p87 9. Description of specific methods for tomato antioxidant analyses p87 9.1. Lycopene analysis on tomato matrix p87 9.1.1. Fresh tomatoes p87 9.1.2. Tomato Puree (light) (Passata di pomodoro) and tomato paste p87 9.1.3. Peeled tomatoes and crushed tomatoes p88 9.1.4. Peels from peeled tomatoes. Composition of tomatoes and tomato products in antioxidants (WG1) page 8 __________________________________________________________________________________________ p88 9.1.5. Extractions p88 9.1.6. Recommendations for working with lycopène p89 9.1.7. Differences verified with Hart and Scott method p89 9.2. Vitamin C in tomatoes and in tomato products. p89 9.2.1. Processing effects on Vitamin C p90 9.2.2. Determination of ascorbic acid in tomato products p90 9.2.2.1. Volumetric titration p91 9.2.2.2. Determination of ascorbic acid by HPLC p93 9.3. Polyphenols p93 9.3.1. Colorimetric method p93 9.3.2. HPLC determination p95 10. Conclusions about analysis data p96 References Composition of tomatoes and tomato products in antioxidants (WG1) page 9 __________________________________________________________________________________________ 1. Introduction Several epidemiological studies suggest that the consumption of fruit and vegetables would be beneficial to human health by reducing the risk of developing cancer and cardio- vascular diseases (Koo 1997, Mayne 1996). However, this protective aspect is more difficult to estimate when one particular group of vegetables or one particular family of nutrients or micro-nutrients is considered (WCRF/AICR 1997). Fruit and vegetables are rich in constituents likely to contribute to the prevention mechanisms and the use of only one group of the constituents cannot totally demonstrate the potential interaction amongst them. Recently a great deal of attention has been focused on the tomato and its products. Studies from Giovannucci et al.(1995) and Franceschi et al.(1994) reported that the consumption of tomatoes, tomato sauce and pizza was associated with a reduced risk of developing digestive tract and prostate cancers. Tomatoes are also one of the main part of the Mediterranean diet which has been associated with a low mortality from cardio-vascular troubles. Because tomatoes constitute the almost exclusive source of lycopene, this pigment could be one of the active agents of this protection. Experimental studies reported that lycopene exhibited antioxidant
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