Identification of New Biomarkers of Banana and Tomato Intake
Phase: NA
Conditions: Biomarkers, Food Intake
Sponsor: Claudine MANACH

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Research project submitted to the local ethics committee « CPP South-East 
VI » 
 
 
 
 
 
 
Identification of new biomarkers of banana and tomato intake 
 
 
 
 
IDRCB Number  
Acronym 
2016-A00153-48 
Project BioBanaTom 
 
Sponsor 
Institut National de la Recherche Agronomique 
147 rue de l'université 
75338 Paris Cedex 07 
Email : alimh@clermont.inra.fr 
 
Principal Investigator 
Pr Ruddy Richard 
Centre de Recherche en Nutrition Humaine (CRNH) d’Auvergne 
58 rue Montalembert, BP371 
63009 Clermont Ferrand Cedex  
Tel : 04 73 60 82 50 
Email : ruddy.richard@udamail.fr 
 
Contact for Ethical committee and Health Agency (ANSM)  
Nathalie Meunier, Attachée de Recherche Clinique 
Centre de Recherche en Nutrition Humaine d’Auvergne 
58 rue Montalembert, BP 321 
63009 Clermont-Fd Cedex 1 
Tel: 04 73 60 82 53  
Email : nathalie.meunier@clermont.inra.fr 
 
 

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SUMMARY  
 
Promotor 
INRA 
Main investigator  Pr Ruddy Richard 
Co-Investigators 
Amandine Prulière, Françoise Laporte, Nicolas Farigon 
Title 
Identification of new biomarkers of banana and tomato intake 
Rational 
In epidemiological studies, dietary intakes are assessed by questionnaires, an 
appraoch with inherent limitations associated with self-reporting : memory bias, 
difficulty to estimate portion size, unsufficient coverage of the dietary 
diversity… The use of biomarkers of food intake should markedly improve the 
accuracy of dietary assessment, However, only few validated biomarkers are 
available. A large European project (FoodBAll) was funded by the JPI HDHL to 
identify a large range of new biomarkers of intakes, using untargeted 
metabolomics on urine ana plasma samples collected in a series of controlled 
nutritional intervention studies.  
Objective 
The BioBanaTom study aims, in the framework of the FoodBAll project, at 
identifying new biomarkers of intake for banana and tomato, two important 
foods of the European diet. 
Evaluation 
criteria 
Main Criteria: Identification using metabolomics of plasma and urine 
metabolites specifically associated to banana and tomato intake, for validation 
as biomarkers.  
Secondary criteria : Obtention of reference metabolomics profiles which may 
be related to metabolic impact of an acute intake of tomato or banana.   
 
Methodology 
Cross-over randomized, open, controlled 
Number 
of 
subjects 
12 
Undesirable 
effects  
No indesirable effect expected beyond those link to blood sampling (small local 
hematoma, slight local inflammation)  
Recruitment 
modalities 
Volunteer file of UEN declared to the CNIL, mailing CHRU and INRA 
Clermont-Ferrand-Theix, local medias. 
The selection of the volunteers will be performed during medical inclusion 
visits.  
Main 
selection 
criteria 
Inclusion criteria 
Men and women, healthy (normal medical and biological exam), non-smoking, 
aged 18 to 40, BMI>18,5 and <30 kg/m2, with no aversion for banana or 
tomato. 
Exclusion criteria 
Smokers, diagnosed with infectious or chronic disease, under medication 
(except contraception pill), having followed an antibiotic treament in the last 3 
months, consuming dietary supplements several times a week, pregnant and 
lactating women, vegetarian as the protocol implies consumption of 
standardized meal containing meat. 
Protocol 
description 
Written consent will be obtained after complete information of the subjects 
about the objectives, and the nature and possible risks of the study (informed 
consent form in duplicate, one copy given to the volunteer). The protocol will 
be explained in details, with objectives and contraints, according to the 
information form, by the scientist in charge. A clinical exam and a check-up of 
all selection criteria will be performed by the main investigator of another 
medical doctor. 
 
At the inclusion visit, subjects will have a medical exam including questionnaire 

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regarding past medical history, family background, medical treatments and 
standard clinical exam (weight, height, blood pressure) and a blood sampling. A 
food frequency questionnaire will also be completed. The conformity to the 
selection criteria will be checked during this medical exam. The selected 
subjects will be definitely included after validation of the selection criteria by 
the investigator. 
 
The volunteers will consume in 3 distinct experimental periods either a control 
drink (oral nutritional complement Fresubin, 250 mL), or 240g banana (and 150 
ml Fresubin), or 300g tomato (with 12 g sunflower oil and 150 mL Fresubin). 
Every volunteer will consume the 3 test foods, in a randomized order. After test 
food intake, urine will be collected over 24h in 7 fractions. Blood samplings 
will be performed before the food consumption (t0) and in post-prandial period 
(4 samplings at 1h, 2h, 4h et 6h), as well as on the following day in fasting state 
(t24h). the volunteers will follow a tomato and banana-free diet during the study 
days, as well as a more restrictive diet, without plant-based food, on the 
sampling days and on the day before. A 24h recall will be filled for the dietary 
intakes on the day before the ssampling days (D2). 
Samples will be analyzed using an untargeted metabolomics approach (LC-MS, 
GC-MS et RMN) with the aim to identify new biomarkers of tomato and banana 
intake.  
Tha total volume of sampled blood will be 154 ml per subject. 
Duration 
Duration of the study for every volunteer : 8 weeks 
Total duration of the study: 6 months for the subject selection and realization of 
all experimental periods for all the volunteers 
Total duration of the project : 3 years  
Indemnities 
350€ per subject 
Duration 
of 
exclusion 
The exclusion duration for the participation of the volunteer to another study is 
fixed at 1 month. 
Location of the 
study realization 
Centre de Recherche en Nutrition Humaine d’Auvergne 
Unité d’Exploration Nutritionnelle 
58 rue Montalembert, BP321, 63009 Clermont-Ferrand Cedex 
Agrément pour la Recherche Biomédicale n°2012 - 476 
Funding 
This study is part of an international research project: Project FoodBAll, JPI 
HDHL 2015-2017. 
 

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TABLE OF CONTENTS 
 
1 – GENERAL INFORMATION  
 
 
 
 
 
 
 
7 
 
1-1 STUDY TITTLE 
 
 
 
 
 
 
 
 
 
7 
 
1-2 STUDY IDENTIFYING NUMBER AND DATE 
 
 
 
 
 
7 
 
1.3 NAME AND ADDRESS OF THE SPONSOR  
 
 
 
 
 
7 
1-4 INVESTIGATOR(S) 
 
 
 
 
 
 
 
 
7 
1-5 PARTNERS 
 
 
 
 
 
 
 
 
 
7 
1-6 ETHIC COMMITTEE 
 
 
 
 
 
 
 
 
8 
1-7 LABORATORY SITE 
 
 
 
 
 
 
 
 
8 
 
2 – INTRODUCTION AND RATIONALE 
 
 
 
 
 
 
9 
 
3 – OBJECTIVES  
 
 
 
 
 
 
 
 
 
10 
 
3-1 PRIMARY OBJECTIVE OF THE CLINICAL STUDY  
 
 
 
 
10 
 
3-2 SECONDARY OBJECTIVES OF THE CLINICAL STUDY 
 
 
 
 
10 
 
4 – PROTOCOL DESCRIPTION  
 
 
 
 
 
 
10 
 
5 – DEFINITION OF STUDIED POPULATION 
 
 
 
 
11 
5-1 POPULATION 
 
 
 
 
 
 
 
 
 
11 
 
5-2 SAMPLE SIZE CALCULATION 
 
 
 
 
 
 
 
11 
 
5-3 SELECTION CRITERIA 
 
 
 
 
 
 
 
 
11 
5-3-1 Inclusion criteria  
 
 
 
 
 
 
11 
5-3-2 Exclusion criteria  
 
 
 
 
 
 
12 
 
5-4 SUBJECT WITHDRAWAL CRITERIA 
 
 
 
 
 
 
12 
 
5-5 EXCLUSION PERIOD AND PARTICIPATION TO THE OTHER CLINICAL TRIAL 
 
13 
 
5-6 COMPENSATION FOR VOLUNTEERS 
 
 
 
 
 
 
13 
 
5-7 SUBJECT RECRUITMENT  
 
 
 
 
 
 
 
13 
 
6 – DESIGN OF TRIAL TO BE CONDUCTED 
 
 
 
 
13 
6-1 STUDY PROCEDURES 
 
 
 
 
 
 
 
 
13 
 
6-2 SCHEME OF STUDY AND FLOW CHART  
 
 
 
 
 
17 
 
6-3 BIOLOGICAL SAMPLES  
 
 
 
 
 
 
 
18 
6-3-1 Blood samples 
 
 
 
 
 
 
 
18 
6-3-2 Urine sampling 
 
 
 
 
 
 
 
18 
6-4 DURATION OF SUBJECT PARTICIPATION AND DURATION OF ALL TRIAL PERIODS AND 
FOLLOW UP IF ANY 
 
 
 
 
 
 
 
 
 
 
18 
 
7 – TRIAL DESIGN 
 
 
 
 
 
 
 
 
 
19 
 
7-1 STUDY PARAMETERS/ENDPOINTS 
 
 
 
 
 
 
19 
7-1-1 Primary study parameter   
 
 
 
 
 
19 
7-1-2 Secondary study parameter 
 
 
 
 
 
19 
 
7-2 DESCRIPTION OF THE RESEARCH METHODOLOGY 
 
 
 
 
19 
 
8 – TRIAL TREATMENT, DOSAGE FORM, AND DOSAGE REGIMEN OF 
THE INVESTIGATIONAL PRODUCTS 
 
 
 
 
 
20 
8-1 INVESTIGATIONAL PRODUCT/TREATMENT 
 
 
 
 
 
20 
 
9 – STATISTICAL ANALYSIS 
 
 
 
 
 
 
 
21 

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10 - BALANCE BENEFITS/RISKS 
 
 
 
 
 
 
21 
 
11 – ETHICAL CONSIDERATIONS 
 
 
 
 
 
 
21 
 
11-1 INDEPENDENT ETHIC COMMITTEE 
 
 
 
 
 
 
21 
11-2 REGULATION STATEMENT 
 
 
 
 
 
 
 
21 
11-3 INSURANCE 
 
 
 
 
 
 
 
 
 
22 
11-4 DOCUMENTATIONS OF THE RESEARCH 
 
 
 
 
 
22 
11-5 INFORMED CONSENT 
 
 
 
 
 
 
 
 
22 
11-6 ASSESSMENT OF SAFETY 
 
 
 
 
 
 
 
22 
11-6-1 Definitions 
 
 
 
 
 
 
 
22 
11-6-2 (Serious) Adverse events handling 
 
 
 
 
23 
11-6-3 New relevant and safety information 
 
 
 
 
23 
11-7 DATE HANDLING  
 
 
 
 
 
 
 
 
24 
11-8 REGISTRATION ON THE NATIONAL REGISTER OF VOLUNTEERS 
 
 
25 
11-9 AMENDMENTS TO THE PROTOCOL 
 
 
 
 
 
 
25 
11-10 MONITORING   
 
 
 
 
 
 
 
 
25 
11-11 BIOLOGICAL COLLECTION 
 
 
 
 
 
 
 
25 
11-12 DATA RETENTION 
 
 
 
 
 
 
 
 
25 
11-13 PUBLICATIONS POLICY 
 
 
 
 
 
 
 
26 
 
12 – REFERENCES 
 
 
 
 
 
 
 
 
 
27 
 
10 – APPENDIX  
 
 
 
 
 
 
 
 
 
28 
 

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LIST OF ABBREVIATIONS 
 
ANSM : Agence Nationale de Sécurité du Médicament 
CPP : Comité de Protection des Personnes 
CRNH : Centre de Recherche en Nutrition Humaine d’Auvergne 
INRA: Institut National de Recherche Agronomique 
JPI HDHL: Joint Programing Initiative Healthy Diet for Healthy Life 
FoodBAll : Food Biomarker Alliance project 
LC-MS = Liquid  Chromatography- Mass spectrometry 
GC-MS = Gas chromatography-Mass spectrometry 
RMN or NMR : Nuclear Magnetic Resonance 

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1 – GENERAL INFORMATION 
 
 
 
1-1 STUDY TITTLE 
 
Identification of new biomarkers of banana and tomato intake 
 
 
 
1-2 STUDY IDENTIFYING NUMBER AND DATE 
 
Study identifying number 
Type 
JPI HDHL 2015-2017 
2016-A00153-48 
 
BioBanaTom 
European Number 
IDRCB 
CPP 
Acronym 
 
 
 
1-3 NAME AND ADDRESS OF THE SPONSOR 
 
Institut National de la Recherche Agronomique 
Represented by Jean Dallongeville, Head of Human Nutrition Department 
INRA, Centre de Paris 
147 rue de l'université 
75338 Paris Cedex 07 
Email : alimh@clermont.inra.fr 
 
1-4 INVESTIGATOR(S) 
 
Principal investigator 
 
Pr Ruddy Richard, PU-PH, Directeur adjoint CRNH 
Centre de Recherche en Nutrition Humaine (CRNH) d’Auvergne 
Unité d’Exploration en Nutrition 
58 rue Montalembert, BP371 
63009 Clermont Ferrand Cedex  
Tel : 04 73 60 82 50 
Email : ruddy.richard@udamail.fr 
 
Co-investigators  
 
Amandine Prulière, Medical Doctor 
Françoise Laporte, Medical Doctor 
Nicolas Farigon, Medical Doctor 
Unité d’Exploration Nutritionnelle, CRNH d’Auvergne 
58 rue Montalembert, BP 321, 63009 Clermont-Ferrand Cedex 1 
Tel 04 73 60 82 51 
 
1-5 PARTNERS 
 
Scientific partners  
 
 
Dr C. Manach, INRA 
Natalia Vazquez Manjarrez, PhD student INRA 
Unité de Nutrition Humaine 
INRA Centre Auvergne-Rhone-Alpes- Site deTheix 
63122 Saint Genès Champannelle 
Coordinator of the Project   
Pr E. Feskens, 
Univ Wageningen,   
Bomenweg 4 

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6703HD Wageningen 
Netherlands 
 
 
Clinical research assistants  
Nathalie Meunier, Adeline Blot 
Nurses : 
 
 
 
Hélène Parrot, Dominique Dumas 
Technician 
 
 
 
Véronique Pidou 
Dieticians :   
 
 
Aurélie Caille, Noëlle Lyon 
Cooker :  
 
 
 
Guy Manlhiot 
 
Unité d’Exploration Nutritionnelle 
Centre de Recherche en Nutrition Humaine d’Auvergne 
58, rue Montalembert – BP 321 
63009 Clermont-Ferrand Cedex 1 
 
1-6 ETHIC COMMITTEE 
 
CPP Sud-Est VI 
Direction générale - CHU de Clermont-Ferrand 
58, rue Montalembert - 63000 Clermont-Ferrand 
 
1-7 LABORATORY SITE 
 
The centre is located in the Human Nutrition Research Centre (Centre de Recherche en Nutrition 
Humaine d’Auvergne) Clermont-Ferrand. 
The study will take place in the Unité d’Exploration Nutritionnelle (UEN) – Centre de Recherche en 
Nutrition Humaine d’Auvergne, 58 rue Montalembert, 63009 Clermont Ferrand France (Autorisation 
de lieu n°2012 - 476). 
 
 

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2 – INTRODUCTION AND RATIONALE 
 
Determining dietary exposure is a challenging endeavor in nutritional research. The most 
common tools used in dietary assessment are based on self reporting methodology such as 24-hour 
recall questionnaires, food frequency questionnaires and weighted dietary records. However, the 
information obtained through these tools may not be entirely reliable since they depend on the 
individual’s memory and honesty which compromises the objectivity of the dietary assessment. 
Through the identification of dietary biomarkers in biological fluids, nutritional researchers have 
found a more objective way to determine dietary exposure1. A dietary biomarker may be defined as a 
specific measurement in a biological sample that accurately reflects the intake of a specific food or 
food constituent. By knowing how foods are metabolized the possibility of identifying unique 
biomarkers for each food or food groups is possible.   
 
There are many factors that influence biomarkers of intake discovery; individual’s 
characteristics such as genetic variants, environmental factors, food matrix and the lifespan of the 
compound may change the metabolites identified in biofluids. Another key factor for discovery of 
biomarkers of intake is the nature of the biological sample, where accessibility plays an important 
role. Urine and plasma are easy access biofluids and therefore, they are the main biological sources 
used to assess metabolites of intake in different studies2. 
 
The rise of metabolomics along with different platforms such as liquid chromatography mass 
spectrometers (LC-MS), have allowed the assessment of thousands of metabolites simultaneously in 
biological samples and the recognition of patterns that may constitute a fingerprint of the intake of 
different foods. Recent studies demonstrated the great potential of metabolomics to discover new 
biomarkers of intake in intervention and cohort studies 3,4 
The diversity of compounds found in food metabolomics represents a major challenge and so in an 
international effort to improve dietary biomarkers identification and validation, the Food Biomarkers 
Alliance (FOODBALL) has been created. In this project, 22 institutions from 11 different countries 
will collaborate in three main tasks: 1) Discovery of new dietary biomarker using a metabolomic 
approach, 2) systemic validation of existing and newly discovered biomarker to achieve a good 
coverage of food intake in different European populations and 3) exploring biological effects using 
biomarkers of intake (http://foodmetabolome.org/). With the latter, the necessity of building a 
chemical library that allows the use of standards for further identification arises. Along with 
FOODBALL, The Food Compound Exchange (FoodComEx) aims to improve availability of 
analytical standards of biological compounds to achieve a better and easier biomarker identification 
(http://foodcomex.org/).  
 
As part of INRA collaboration to FOODBALL and FoodComEx, the present project attempts 
to identify biomarkers of banana and tomato intake, through the exploration of the serum and urine 
metabolome of subjects who consumed these foods in a controlled intervention. The same study 
design will be applied in six different centers within Europe to assess biomarkers of the intake of 12 
public health related foods. 
 
Tomatoes were introduced to European countries in the 16th century. The origin of tomatoes 
may be tracked back to the Aztec civilization in Mexico. There are fifteen leading countries in the 
production of tomato China, US, India, Turkey, Egypt, Italy, Spain, Brazil, Iran, Mexico, Greece, 
Russian Federation, Ukraine, Chile and Uzbekistan which supports the fact that tomatoes are between 
the five most eaten vegetables in the world 5. Tomatoes contain a large variety of micronutrients (-
carotene as pro-vitamin A, vitamin C, folate, and potassium) and other chemical compounds, 
including lycopene and polyphenols that are considered beneficial for health 6. Most of the published 
work on tomato is mainly focused on the association between the intake of lycopene and flavonoids, 
such as naringenin and hesperetin, and a lower risk for chronic diseases and cancer 7, however 

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lycopene and flavonoids are not robust exposure biomarkers of tomato intake for various reasons (low 
concentrations, interindividual variation in intestinal absorption and metabolism). Tomato contains 
many other phytochemicals that could be revealed as exposure biomarkers or bioactive compounds 
contributing to the health effects of tomato. 
 
On the other hand, bananas (musa spp) are among the world’s leading food crops as a source 
of energy, mainly in tropical regions. The use of pulp and peel of the banana as medicine remedies has 
been documented in traditional medicine in different continents such as Africa and America. The 
dietary recommendations for banana intake are based mainly in their content of pro-Vitamin A 
compounds such as trans-α and trans-β carotenes and in their energy contribution8. Like tomatoes, 
there are no validated exposure biomarkers on the literature.  
 
Determining biomarkers of intake of banana, tomato and different other public health related 
foods will allow a more precise dietary assessment in nutritional epidemiology, opening the 
possibility of better investigating the health effects of foods and dietary patterns. Biomarkers will also 
be very useful to monitor compliance in controlled intervention studies. 
 
 
3 – OBJECTIVES  
 
The overall aim of the project is to identify potential novel biomarkers of intake of tomato and banana 
in urine and blood of volunteers after acute consumption of these foods. 
 
 
 
 
3-1 PRIMARY OBJECTIVE OF THE CLINICAL STUDY 
 
The primary objective, within the FOODBALL project, of the present study is to identify biomarkers 
of food intake after an acute dietary intervention with banana, tomato and a control drink on 12 
healthy subjects, by comparing the metabolomic profiles analyzed for urine and serum samples 
collected over 24h. 
 
 
 
3-2 SECONDARY OBJECTIVES OF THE CLINICAL STUDY 
The secondary objectives of the BioBanaTom project are: 
 
1. Collect reference urine and serum samples and associated metabolomic profiles after acute intake 
of banana and tomato to be shared in the FoodComEx library for analytical purposes. 
2. Explore the modifications in metabolomics profiles that may be associated with metabolic effects 
of acute consumption of tomato and banana. 
 
 
4 – PROTOCOL DESCRIPTION 
 
The present project is a randomized, controlled, crossover study with 12 subjects. A cross-over design 
has been selected as each subject can serve as his/her own control thereby minimizing variations. 
Limitations attributed to cross-over designs are possible order or carry-over effects. These effects will 
however be minimized due to a randomized intervention order. The study design does not allow 
blinding as intervention is the ingestion of different foods. 
In this study, we will assess the metabolomic profiles of human biofluids after consumption of two 
different foods: banana (240g peeled fruit) and tomato (300g fresh fruit) in order to identify novel 
biomarkers for each food. As a control diet, a high energy high protein drink, Fresubin ® 2kcal fiber 
(Fresinius kabi) will be used. As shown in the general study plan, the intervention comprises three 
dietary intervention periods of 4 days each for each food of interest and the control drink. Each 

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intervention period will be separated by a washout period of minimum 3 days (and maximum 10 
days). Volunteers will be randomly assigned to a different food at each time point. 
Volunteers will be asked to maintain their normal physical activity and follow the dietary 
recommendations between intervention periods; they will receive an information form containing the 
description and objectives of the project, and detailed instructions. A volunteer’s booklet will also 
include simple information about nutritional metabolomics and a calendar marked with their visits to 
our facilities.  
 
 
General study plan 
 
 
 
 
 
5 – DEFINITION OF STUDIED POPULATION 
 
5-1 POPULATION 
 
In total 12 subjects (with the aim of 10 subjects completing the study) will be recruited for the present 
project of which 50% (n=6) will be men and 50% will be women (n=6) between 18 and 40 years of 
age. Subjects will have a BMI above 18.5 and under 30 kg/m2, will be nonsmokers and with no 
disease diagnosis.  
 
 
 
5-2 SAMPLE SIZE CALCULATION 
 
As untargeted metabolomics is a data driven approach, which consists in detecting thousands of 
metabolites in biological samples with various analytical and inter-individual variability, it is not 
relevant to calculate a unique statistical power to determine the number of subjects. Nevertheless, 
previous studies showed that metabolomics allows identification of food intake biomarkers in 
controlled intervention studies with 4 to 20 volunteers3,9  
 
5-3 SELECTION CRITERIA 
 
5-3-1 Inclusion criteria  
 Healthy males and females  
 Aged 18 - 40 years 
 BMI >18.5 and < 30 kg/m2 
 Willing/able to consume all test foods (tomato, banana, Fresubin drink) and the standardized 
meals (rice and chicken) 
 
 
5-3-2 Exclusion criteria 
 Smokers 
 Diagnosed health condition (chronic or infectious disease) 
 Taking nutritional supplements (e.g. vitamins, minerals) several times a week 
 Taking medication (oral contraceptive pill is allowed). 
 Pregnant, lactating 

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 Antibiotics treatment within 3 months prior to intervention 
 Vegetarians, as standardized meals will contain meat 
 Not willing to follow nutritional restrictions, including drinking alcohol during study days 
 Not willing/able to give informed consent or to sign informed consent. 
 Not affiliated to National Health Insurance  
 Being in exclusion on the National Volunteers Data file or refusing to be registered on the 
National Volunteers Data file 
 Currently participating or who having got 4500€ in this year to have participated in another 
clinical trial. 
 Subjects deprived of their liberty by judicial or administrative decision.  
 
 
 
5-4 SUBJECT WITHDRAWAL CRITERIA 
 
Subjects may discontinue their participation in the study at any time.  
The clinical Investigator may remove a subject, if, in his opinion, it is in the best interests of the 
subject.  
A subject may be withdrawn from the study for any of the following reasons: 
 Withdrawal of consent - any subject may withdraw from the study at any time 
 Significant deviation from the protocol 
 Lost of follow up 
 Death 
 Incidental illness 
 Occurrence of a serious or considered serious adverse event or significant alteration in clinical 
and/or laboratory parameters. In these cases, appropriate actions will be taken. The sponsor 
will be notified immediately. 
 
When the investigator had no news of the participant, he must have made every effort to contact 
him, to establish the reason for the discontinuation of treatment, and to suggest the participant to come 
to an end-of-study visit. If all these attempts to contact the participant failed, the investigator could 
then declare the participant “lost to follow-up”. The investigator should document all these attempts in 
the corresponding medical file. If a volunteer discontinues the study before the end of recruitment 
period, a new volunteer will be included with the same treatment. The investigator will record the 
reason(s) and the exact time of premature withdrawal of the study in the case report form. If more than 
one reason will be given, the investigator will indicate the main reason. The investigator will notify 
the sponsor of study withdrawals immediately using the page provided for this purpose in case report 
form by faxing to the study monitor. 
 
In the case of withdrawal from the study due to a serious or non-serious adverse event, the 
investigator will make every effort to collect the information relating to the outcome of the event. If 
necessary, the information will be collected afterwards. This information will be recorded in that part 
of the case report form which concern adverse events. If the investigator cannot collect the 
information from a visit, he will collect it from the doctor ensuring the follow-up of the participant. 
 
 
 
5-5 EXCLUSION PERIOD AND PARTICIPATION TO THE OTHER CLINICAL TRIAL 
 
The subject is not allowed to participate in another clinical trial for the duration of this protocol. In 
addition, the exclusion period defined in this study is 1 month, timeframe during which the subject 
cannot participate in another clinical research protocol. 
 
 
 
5-6 COMPENSATION FOR VOLUNTEERS 
 

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The subjects will receive 350 € of compensation for the entire participation.  
In the case of a premature withdrawal, a calculation of the compensation will be made in proportion to 
protocol completion, as follows: 50€/kinetics. 
Withdraw after the baseline visit: 0 € 
 
Very exceptionally, the investigators may exclude an obviously non-compliant subject. In that case, 
the volunteer will receive no compensation. 
 
 
 
5-7 SUBJECT RECRUITMENT 
 
Subjects may be recruited in the community through advertisements in local newspapers, CRNH web 
site, emailing at INRA and CHRU, flyers at public places, and from existing UEN volunteer database 
registered at the CNIL. 
 
 
6 – DESIGN OF TRIAL TO BE CONDUCTED 
 
6-1 STUDY PROCEDURES 
 
All the participants will give freely their written informed consent before their selection in the study. 
The investigator or co-investigator will collect written consent from each participant before, or at the 
latest on, the day of the first study visit. Prior to this, the investigator or his delegate will inform each 
participant of the objectives, benefits, risks and requirements imposed by the study, as well as the 
nature of the study products. 
 
The participant will receive an information and consent form in clear, simple language. He will be 
allowed ample time to inquire about details of the study and to decide whether or not to participate in 
the study.  
 
Subjects will come to the Investigational Site for screening to determine whether they are eligible to 
participate in the study. Information on demographics, anthropometrics, vital signs, relevant medical 
history and concomitant medication and nutritional supplements (including vitamins) will be 
collected. A blood sample will be collected to measure: full blood count, platelets, CRP (C Reactive 
Protein), glycemia, ionogramme (Na+, K+, Cl-), hepatic, lipid and renal profile. A total volume of 
10ml will be collected to perform this biological test. All inclusion and exclusion criteria will be 
checked by the investigator or co-investigator. Subjects will also complete a food frequency 
questionnaire (Appendix 2).  
 
Selected subjects will be definitively included after validation of their biological test by the 
investigators. Subjects will be personally informed by the medical doctor in case of non inclusion if 
their biological test presents anomalies. Results will be delivered to them confidentially. Their 
medical doctor will be informed with a letter from the investigator. The results of biological tests will 
be kept in the envelope dedicated to each volunteer for this study and only people involved in the 
research will have access to documents (Nurses, co-investigators, dieticians, Clinical Research 
Assistant) in accordance with Good Clinical Practices. 
 
The volunteers recruited for this project will be asked to eat three different foods, banana, tomatoes 
and a control drink (Fresubin ® 2kcal fiber) in order to identify novel biomarkers for each test food. 
Subjects will be randomly assigned to each test food at different time points of the study. As shown in 
the general study plan, there will be three dietary interventions: 
 
 Intervention 1: Volunteers will consume the control beverage Fresubin ® 2kcal fiber (250 
mL). 

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 Intervention 2:  Volunteers will consume 240g of peeled banana plus Fresubin ® 2kcal 
fiber (150 mL). 
 Intervention 3: Volunteers will consume 300g of raw tomato with sunflower oil (12g) and 
Fresubin ® 2kcal fiber (150 mL). 
 
 
Fresubin is a beverage composed mainly of milk protein, vegetable oils (rapeseed oil, sunflower oil) 
carbohydrates, vitamins minerals and trace elements (Detailed composition in Appendix 3). For the 
purpose of this study volunteers will drink the neutral flavored beverage. 
 
Each intervention period will be separated by a washout period of at least 3 days (maximum 10 days). 
Volunteers will be asked to maintain their normal physical activity and diet between intervention 
periods; they will receive an information form containing the description and objectives of the project. 
Volunteers will be randomly assigned to a different food at each intervention period.  
 
Following the study design shown in the general study diagram, the run-in phase (Day1 and Day2) 
participants will follow a restricted diet (described in Appendix 4) and will avoid the intake of banana 
or tomato in any form. On Day 2 volunteers will also avoid the intake of banana and tomato in any 
form and will exclude phytochemical-rich foods such as tea, coffee, wine and other plant foods to 
avoid noise in the metabolomic profile (recommendations described in Appendix 5). For dinner in this 
day, volunteers will prepare a standardized diner which composition will be given by the investigators 
(rice and chicken meal, composition in Appendix 6). After dinner subjects will begin the overnight 
fasting period. All foods consumed on day 2 will be reported in the 24-hour recall questionnaire 
applied by a nutritionist the day of the dietary intervention. 
 
In the morning of Day 3, subjects will arrive fasted at Investigational Site at around 7.30 am. As stated 
previously volunteers will be assigned randomly to one of the three interventions, Fresubin ® 2kcal 
fiber, 240g of banana plus control drink, or 300g of tomato plus control drink. Throughout the 
intervention, subjects will have free access to water, maximum 250ml of water per hour until 6 hours 
after intervention (1.5L in total). 
A trained phlebotomist will place a catheter on the subject’s arm before the intake of the test foods to 
collect the baseline sample. Then four other samples will be collected postprandially after 1h, 2h, 4h, 
and 6h.  
 
A total of 7 urine samples will be collected. The first void of urine will be collected by the subjects at 
home upon the morning of Day 3 and the rest of the samples after the intake of the test foods as 
follows: 0-1h, 1h-2h, 2h-4h, 4h-6h. The urine samples corresponding to 6h-12h and 12-24h interval 
will be collected by volunteers at home until the morning of Day 4. Urine samples will be kept at 4ºC 
between collections; the use of preservatives will not be needed. 
 
After the 6h collection of blood, the peripheral catheter will be removed and subjects will have lunch 
composed of rice and chicken meal, then subjects will be allowed to go home. Before leaving the 
Investigation center, participants will be instructed to prepare a standarized dinner based on chicken 
and rice. Volunteers will not be allowed to eat or drink anything except water and the standardized 
dinner. 
On the morning of Day 4, subjects will arrive fasted at the investigation center at around 7.30 am in 
order to collect the 24h blood sample and will deliver the 6h-12h and 12-24h urine collection. 
Afterwards participants will receive a breakfast (free composition) on site and be allowed to follow 
their normal diet until the run in phase of the following intervention period initiates. This procedure 
will be repeated for each intervention (1,2,3) separated by a minimum 3 day period (=7 days between 
visits). 
Table 1.  Summary of trial procedures 

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Who 
Where 
Day 1: Run in 
 
 
Restricted diet: no banana or 
tomato intake 
Volunteer 
At home 
Day 2: Run in 
 
 
Restricted diet: no intake of 
tomato, banana, tea, coffee, 
wine, fruit juices, plant foods. 
Dinner based on chicken and 
rice,   
Volunteer 
At home 
After dinner, beginning of 
fasting period (8-12 hr) 
Volunteer  
At home 
Day 3: Intervention day 
 
 
Urine collection 1 (first void 
of the morning) 
Volunteer 
At home 
Placement 
of 
peripheral 
catheter 
Phlebotomist 
UEN 
Blood sampling 1 at baseline 
(0h) 
Phlebotomist 
UEN 
Intake of test food (banana, 
tomato or control drink) 
Volunteer 
UEN 
Blood sampling 2 (1h) 
Phlebotomist 
UEN 
Blood sampling 3 (2h) 
Phlebotomist 
UEN 
Blood sampling 4 (4h) 
Phlebotomist 
UEN 
Blood sampling 5 (6h) 
Phlebotomist 
UEN 
Urine collection 2 (0-1h) 
Volunteer 
UEN 
Urine collection 3 (1-2h) 
Volunteer 
UEN 
Urine collection 4 (2-4h) 
Volunteer 
UEN 
Urine collection 5 (4-6h) 
Volunteer 
UEN 
Urine collection 6 (6-12h) 
Volunteer 
At home 
Urine collection 7 (12-24h) 
Volunteer 
At home 
At T6h, Lunch intake (rice 
and chicken meal) 
Volunteer 
UEN 
Restricted diet: Dinner based 
on chicken and rice. No intake 
of  tomato, banana, tea, 
coffee, wine, fruit juices, plant 
foods intake 
Volunteer 
At home 
After the standardized dinner, 
beginning 
of 
the 
fasting 
period 
Volunteer 
At home 
Day 4: Day after intervention 
 
 
Blood sample collection 6 
(24h) 
Phlebotomist 
UEN 
Urine Collection (first void of 
urine) 
Volunteer 
At  home 
Breakfast 
Volunteer 
UEN 
 
 
 

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6-2 SCHEME OF STUDY AND FLOW CHART 
 
Table 2: Measurement calendar 
 
 
 
Visit 
 
 
 
 
 
 
 
 
 
 
 
 
 
Day of visit 
 
Screening 
visit 
Day 
1 
Run 
in 
Day 2 
Run in 
Day3 
Acute 
intervention 
Day 4 
 
Day 1 
Run in 
Day 2 
Run in 
Day3 
Acute 
intervention 
Day 4 
 
Day 1 
Run in 
Day 2 
Run in 
Day3 
Acute 
intervention 
Day 4 
 
Signature of subject 
information form and 
consent form 
Checking of eligibility 
criteria 
Clinical examination 
Biological test 
X 
 
 
X 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Restricted diet 
 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
X 
 
Biological collection 
Blood  
Urine 
 
 
 
 
 
 
X 
X 
 
 
X 
X 
 
 
 
 
X 
X 
 
 
X 
X 
 
 
 
 
X 
X 
 
 
X 
X 
 
Body assessment 
Height, weight, waist, hip 
circumference, body mass 
index. 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dietary assessment 
24-hour recall questionnaire 
Food frequency 
questionnaire 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
Test Food  A 
Test Food B 
Test Food C 

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6-3 BIOLOGICAL SAMPLES 
6-3-1 Blood samples 
We will collect 6 blood samples (8 ml), one at baseline and 5 others after the acute intervention  (t= 
0hr, 1hr, 2hr, 4hr, 6hr, 24hr) at each of the 3 experimental interventions (Banana, Tomato, Control 
drink Fresubin). These samples will be collect at the Unité d’Exploration Nutritionnelle and will be 
stored at the Human Nutrition Unit, INRA Centre of Theix. Volunteers will arrive at the 
Investigation Center in an 8-12 hour fasting state and after two days of a restricted diet. All blood 
samples will be processed immediately after they are drawn allowing clotting at room temperature 
for 30 minutes. After clotting serum will be collected in aliquots and then stored at -80ºC until 
analysis. The whole volume of blood collected during the experimental periods is 8mL x 6 time 
points x3 experimental periods = 144 mL. In addition, 10 mL blood will have been collected at the 
screening visit. The sample collected at the screening visit will be analysis the same day of the 
collect by the Biochemistry Unit of Clermont Ferrand Hospital. 
In total, the volume of blood collected for the whole BioBanaTom study is 154 mL.  
 
6-3-2 Urine sampling 
Participants will be instructed on collecting urine according a standardized protocol and they will be 
provided with containers. Subjects will be asked to collect all urine samples in marked containers at 
the following schedule: first void of urine before intervention, 0-1h, 1-2h, 2-4h, 4-6 h, 6-12h and 
12-24h (Day 4). Urine samples will be kept at 4ºC until processing.  
After complete collection, urine samples volumes will be recorded, urine will be centrifuged at 
1800xg for 10 minutes at 4ºC and separated in aliquots and stored at -80ºC at the Unité de Nutrition 
Humaine, INRA Centre of Theix. until analysis. 
 
 
Table 3: Analyses performed  
 
ANALYSES 
Partner 
Measurements 
UNH, INRA Theix, France 
Serum LC-MS metabolomics 
Urine LC-MS metabolomics 
Max Rubner-Institut (MRI) Karlsruhe, Germany 
Serum GC-MS and NMR metabolomics 
Urine GC-MS and NMR metabolomics 
 
 
6-4 DURATION OF SUBJECT PARTICIPATION AND DURATION OF ALL TRIAL PERIODS 
AND FOLLOW UP IF ANY 
 
The duration of the subject’s participation is of 18 days distributed as follows: 
 
 3x run-in phase of 2 days (Day1 and Day2) = 6 days 
 3x intervention periods (Day3) = 3 days 
 3x collection of 24 h blood samples (Day 4) = 3 days 
 2x 3 days washout periods between interventions= 6 days 
 
The duration of the study may be extended to 5 weeks if the wash-out periods are extended (up to 
10 days). 
 
The participation of the subjects enters within the framework of articles L1121-1 and following 
ones of the Public health code. 
 
 
 

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7 – TRIAL DESIGN 
 
 
 
7-1 STUDY PARAMETERS/ENDPOINTS 
 
 
 
7-1-1 Primary study parameter  
The primary parameter in this study is the assessment of metabolites present in blood serum and 
urine before the dietary intervention (t=0h) and post-prandially at different time points over 24h. 
Metabolites will be analyzed using a non-targeted metabolomics approach (GC-MS, LC-MS and 
NMR). The endpoint is the identification of biomarkers of acute intake of the foods of interest 
through the comparison of metabolomes after either single dose of tomato, banana or control drink. 
 
 
 
 
7-1-2 Secondary study parameter 
A secondary endpoint is to obtain pools of urine and serum samples collected after acute intake of 
tomato or banana that can be shared with the international scientific community through the 
FoodComEx library, to be used as analytical standard or for the identification of specific 
metabolites of banana or tomato components. 
 
Other secondary parameters in this study are the possible variations in endogenous metabolites 
detected by the metabolomics profiles that may reflect a metabolic effect of acute consumption of 
the tested foods. The secondary endpoint is the identification of new hypotheses on possible 
metabolic effects of banana or tomato that may be investigated further in others studies. 
 
 
 
7-2 DESCRIPTION OF THE RESEARCH METHODOLOGY 
 
 
 
7-2-1 Anthropometry 
In order to minimize variation, all anthropometric measurements will be taken by the same person 
at the screening visit. These measurements will be conducted on the subject while they are wearing 
light, indoor clothing and while they are still fasting. Shoes and belts will be removed and pockets 
emptied. 
 
Height measurement 
The subject is placed standing at a stadiometer while wearing socks or bare feet. Hats, hair slides or 
highly placed hair-dos must be removed or undone. The subject is asked to stand with their back to 
the stadiometer’s backboard ensuring their feet, buttocks and occiput (back of skull) touch the 
board. The measuring arm is brought down to touch the top of the subject’s head, pushing hair 
down if necessary. This is done in duplicate, obtaining measurements to the nearest 0.1cm which 
will be averaged. 
 
Weighting method 
The scale is turned on (if previously off) and allowed to equilibrate. The subject removes their 
socks and stands on the scale. They are instructed to hold still for about 5 seconds until the scale has 
obtained its reading. Body weight is taken to the nearest 0.1kg. Antibacterial cleansing wipes are to 
be used on the scales in between uses. 
 
Method for measuring waist circumference  
For both measurements a non-stretch tape should be used. The subject should be standing with the 
abdomen relaxed, arms at the sides, feet together and breathing normally. If agreed by the subject, 
the measurement will be made directly on the skin. It should not be taken over heavy clothing.  
The tape is placed around the waist, which is defined as the mid-point between the lowest rib and 
the supra-iliac crest. The measurement should be taken at the end of a normal expiration ensuring 
the tape is not compressing the skin. If the waist cannot be easily found, the investigator should ask 
the subject to bend over to the side - the fold is where the waist is. The measurement is read while 
standing at the subject’s left side and it should be recorded to the nearest 0.1cm. This should be 
done in duplicate. 
 

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Method for measuring hip circumference 
The subject should wear non-restrictive briefs or thin bottoms. They should stand erect with hands 
held slightly away from the sides. The investigator should squat to the side of the subject and pass 
the measuring tape around the greater trochanter, without compressing the skin. The measurement 
is recorded to the nearest millimeter. 
 
 
 
 
7-2-2 Health status and dietary assessment 
Dietary and nutritional status evaluation 
In order to assess the dietary habits of the volunteers two instruments will be applied, 24-hour recall 
questionnaire and a food frequency questionnaire. The 24-hour recall will be applied in the day on 
intervention (Day 3) to ensure compliance to the restricted diet on day 2. During the 24r-hour recall 
questionnaire volunteers will be asked about the amount and type of foods and beverages they 
consumed the day before.  
The food frequency questionnaire (FFQ) is a self-administered questionnaire where the dietary 
habits of individuals for the past year are assessed using a food list and beverage items categorized 
in 22 categories. Usual frequency of consumption will be assessed for the 22 categories of foods. 
The objective is to get an overall estimation of the dietary pattern of the volunteer to identify dietary 
patterns far outside the standard dietary patterns observed in the French population. 
 
 
 
 
7-2-3. Metabolomics analysis 
Urine and Blood samples will be analyzed using a UPLC-QTOF-MS (Waters Corporation, 
Manchester, United Kingdom) platform for the detection of metabolites, equipped with an 
electrospray source and a lock mass sprayer to ensure accuracy. Later on, a small number of urine 
samples will be analyzed using a high resolution Thermo Scientific LTQ Orbitrap Velos hybrid 
mass spectrometer to obtain accurate masses and molecular formulas of potential biomarkers of 
intake.  Several online chemical databases will be queried using the accurate formula or mass to 
search for possible hits of chemical structures and compounds.  
GC-MS and NMR analyses of the samples will also be conducted in a collaborative research unit of 
the FoodBAll project, at the Max-Rübner Institute in Karlsruhe. 
 
 
8 – TRIAL TREATMENT, DOSAGE FORM, AND DOSAGE REGIMEN OF 
THE INVESTIGATIONAL PRODUCTS 
 
8-1 INVESTIGATIONAL PRODUCT/TREATMENT 
Bananas and tomatoes will be bought in a local grocery. A unique dose of 240g banana and 300g 
tomatoes will be given to the volunteers.  
The control drink will be neutral flavored Fresubin ® 2kcal fiber. Volunteers will drink 250 mL 
on the Intervention  1 (volunteers only drink Fresubin ® 2kcal fiber as a control) and 150 ml of 
Fresubin plus the test food of interest on Intervention 2 (Fresubin ® 2kcal fiber plus 240g of 
banana) and Intervention 3 (Fresubin ® 2kcal fiber plus 300g of tomatoes).  
 
Nutrimental composition of Fresubin ® 2kcal fiber is as follows:  
 
Nutritional Composition 
  
Per 100ml 
Per 
200ml 
Energy 
kcal 
200 
400 
Protein 
g 
10 
20 
Nitrogen 
g 
1.6 
3.2 
Carbohydrate 
g 
22.5 
45 
Of which sugars 
g 
3.2-5.8* 
6.4-11.6 

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Of which lactose 
g 
≤0.3 
≤0.6 
Fat 
g 
7.8 
15.6 
Of which saturated fatty acids 
g 
0.6 
1.2 
Of which polyunsaturated fatty acids 
g 
1.4 
2.8 
Of which monounsaturated fatty acids 
g 
5.8 
11.6 
Fibre 
g 
0, 1.5-1.61 
0, 3.21 
Water 
ml 
69-70* 
138-140* 
Osmolarity 
mosmol/l 
495-650 
  
Osmolality 
mosmol/kg 
H2O 
720-950 
  
Information obtained from: http://www.fresenius-kabi.co.uk/4727.htm 
 
 
9 – STATISTICAL ANALYSIS 
 
Data will be collected in ad hoc developed databases and analyzed according to the classical 
workflow used for metabolomics data. Data will be cleaned in a standardized way. Univariate 
(ANOVA with Benjamini Hochberg correction) and multivariate analyses (Principal Component 
Analysis, Partial-Least-Square-Discriminant Analyses, and Hierarchical Clustering analysis) will be 
applied to pinpoint the detected metabolites which significantly vary along time after the intake of 
banana or tomato.  
 
The statistical analysis will be centralized and performed by the INRA investigators, in close 
collaboration with the statistician of the Metabolomics platform (Mélanie Pétéra, PFEM: Plateforme 
d’Exploration du Métabolisme). 
 
 
10 - BALANCE BENEFITS/RISKS 
 
The risks involved in participating in this experiment are minimal. A venous punction is 
comparable to a normal blood drawn and the only risk is that of a small local hematoma. 
The inconveniences which can result of blood collection are possible infectious or inflammatory 
problems. These complications are exceptional and all the necessary measures of asepsis are taken 
to avoid them. 
The food products that will be provided are commercially available products and for this reason do 
not bring any health risks.  
 
 
11 – ETHICAL CONSIDERATIONS 
 
 
 
11-1 INDEPENDENT ETHIC COMMITTEE 
 
The protocol and the Subject informed form and consent will be submitted to The Independent 
Ethics Committee (CPP Sud-Est VI) and written approval from the Chair of the Ethics Committee 
is required before the initiation of the study. 
The notification of the approval will be forwarded to the French authority ANSM. A request for 
authorization will be sent by the sponsor to ANSM before the start of the study. 
 
11-2 REGULATION STATEMENT 
 
The study will be performed in full accordance with the ethical principles stated in the Declaration 
of Helsinki 1964, as revised in 2008 (59th WMA General Assembly, Seoul, October 2008). The 

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participation of the subjects will be performed in accordance with the Code de la Santé Publique 
(National Health Codes), Law n°2004-806 of august 9th, 2004. 
 
The clinical study will be conducted in compliance with the protocol, International Conference on 
Harmonisation (ICH), guidelines for Good Clinical Practice (GCP, as mentioned in the National 
Health Codes, article L.1121-3) and in the decree of the November 24th, 2006. 
 
11-3 INSURANCE 
 
The sponsor, INRA has an insurance (N° 4623359604) which is in accordance with the legal 
requirements. This insurance provides cover for damage to research subjects through injury or death 
caused by the study product and/or study procedures. 
 
It is to note that the non compliance with the legal requirements of the research (absence of CPP 
opinion, absence of ANSM’s authorization, non consent of the person, the continuation of a 
suspended or speechless research) is an exclusion clause of the guarantee. 
 
11-4 DOCUMENTATIONS OF THE RESEARCH 
 
Before starting the research, the investigator will supply to the sponsor a copy of his personal 
curriculum vitæ dated and signed and containing his number of registration in the Medical 
association, as well as all the investigators.  
The version of the protocol accepted before submission with its appendices will be jointly signed by 
the investigator and the scientific in charge of the study. 
For every new version of the protocol, (amendments and\or requests of the authorities), a new 
number and the date will be attributed and the same signatures will be collected. 
 
The investigators agrees that the study is conducted in accordance with the Public Health law N° 
2004-806 - 9 August 2004 on biomedical research, the implementing decree N° 2006-477 from 
26/04/2006 amending chapter I of title II of book I of the first part of the code of Public Health 
relating to biomedical research and the applicable orders. 
 
11-5 INFORMED CONSENT 
 
All the participants will give freely their written informed consent before their selection in the 
study. Prior to this, the investigator or his delegate had inform each participant of the objectives, 
benefits, risks and requirements imposed by the study, as well as the nature of the study products. 
 
The participant will provided with an information and consent form in clear, simple language. Two 
original information and consent forms will be completed, signed and dated personally by the 
participant and by the investigator or co-investigator for collecting the informed consent. The 
participant will be given one signed original information and consent form, the second original will 
be kept by the investigator and archived during, at least, 15 years. 
 
11-6 ASSESSMENT OF SAFETY 
11-6-1 Definitions 
-ADVERSE EVENT (AE) 
AE is any untoward medical occurrence in a patient or clinical investigation subject administered a 
study product and which does not necessarily have a causal relationship with the study product 
(derived from the ICH-GCP definition). 
 
-SERIOUS ADVERSE EVENT (SEA) 
SAE is any untoward medical occurrence that at any dose: 
- 
results in death, 

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- 
is life-threatening (at the time of the event), 
- 
requires inpatient hospitalisation or prolongation of existing hospitalisation, 
- 
results in persistent or significant disability or incapacity,  
or 
- 
is a congenital anomaly or birth defect 
 
-SUSPECTED UNEXPECTED SERIOUS ADVERSE REACTION (SUSAR) 
A SUSAR is both: 
- 
an SAE that is judged to be at least possibly related to the study product by the either investigator or 
sponsor, and 
- 
unexpected (not listed in the Product Information Brochure) 
 
11-6-2 (Serious) Adverse events handling 
-(S)AE RECORDING 
Any (S)AE as reported spontaneously by the subject or observed by the investigator or staff, is 
recorded on the AE form during the course of the study. The investigator must ensure that all 
required information is captured on this form, including the nature, onset, duration, and severity of 
the event, the relationship to the study product, and the action taken.  
 
Only untoward medical events occurring after first study product intake are recorded as (S)AE 
(Events occurring during the Baseline period and/or before first study product intake are recorded as 
pre-existing condition on the Relevant Medical History and Pre-Existing Conditions page in the 
CRF). 
When a subject undergoes a medical intervention or hospitalisation in absence of an Adverse Event 
(such as treatment for a pre-existing condition or hospitalisation for elective surgery or diagnosis), 
this intervention/hospitalisation must be reported on the AE page and not as SAE. These procedures 
will be handled like AEs, and timelines for reporting are the same as for reporting AEs. 
Complications or prolongations of hospitalisation that result from such procedures must be reported 
as (S)AEs, according to the applicable reporting timelines and procedures.  
 
The severity of any (S)AE is scored as follows: 
 
- 
Mild: 
transient or mild discomfort; no medical intervention/therapy required 
- 
Moderate: 
mild to moderate limitation in activity; some assistance may be needed; 
and/or minimal medical intervention/therapy required 
- 
Severe: 
marked limitation in activity; some assistance usually required; and/or 
significant medical intervention/therapy/hospitalisation required 
 
The relationship of the (S)AE to the study product is assessed as being not related / unlikely related 
/ possibly related / probably related / definitely related.  
 
-SAE REPORTING BY THE INVESTIGATOR 
The investigator must completed SAE report. The initial report must be sent to the delegated person 
by the sponsor within 48 hours (2 working days) after first notice, the follow-up report as soon as 
relevant new information is available. 
-SAE REVIEW AND REPORTING BY THE SPONSOR 
In case the SAE is a potential SUSAR, the Medical Monitor must decide whether unblinding is 
required. 
All SAEs are reported to the accredited Ethics Committee that approved the protocol. SAEs are 
reported annually as line listings or according to the requirements of the Ethics Committee, SUSARs 

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are reported within 7 days (fatal and life threatening events) or 15 days (other events). 
 
-FOLLOOW UP OF (S)AES 
(S)AEs are followed-up by the investigator until they have abated, or until a stable situation has 
been reached. Depending on the event, follow-up may require additional tests or medical 
procedures as indicated, and/or referral to the general practitioner, a medical specialist, or another 
health care provider. 
 
 
 
 
11-6-3 New relevant and safety information 
The Sponsor will inform the investigator and the reviewing accredited Ethics Committee if anything 
occurs that may negatively affect the burden or risks of participation as foreseen in the research 
proposal. The study may be suspended pending further review by the accredited Ethics Committee, 
provided that suspension does not jeopardise the subjects’ health. The investigator will take care 
that all subjects are kept informed. 
 
11-7 DATE HANDLING  
 
CRFs are records of data on each subject as defined by the study protocol. Entries on CRFs shall be 
made complete, legible and correct using a ball-point pen. Any mistakes shall be corrected by 
drawing a line over the old entry and by initialling and dating next to the correction. The 
investigator should file all data per subject. An explanation for the omission of any required data 
should appear on the appropriate CRF page or other data collection forms.  
The last page of each visit shall be signed and dated by the investigator to indicate the overall 
responsibility. 
Original completed CRFs will be collected by the sponsor for data entry and further data 
management and statistical applications. A copy of each CRF will be archived by the investigator. 
At each visit the clinical monitor will review all completed CRFs (since last visit) for completeness 
including signature, and will compare selected data with the subject’s records.  
 
In accordance with GCP and with the national data protection laws, all information concerning the 
subjects in the study must be treated as strictly confidential by all persons involved in the study 
including the clinical, medical and statistical monitor. 
Such consent shall be deemed to have been given for disclosure to any person for whom the 
investigator is responsible at his centre, but only so far as required for the purposes of the study, 
and, in the case of disclosures to staff, only if such staff are bound by obligations of confidentiality 
no less strict than those set out herein. 
 
The Commission Nationale de l’Informatique et des Libertés (CNIL) is responsible for ensuring 
that information technology remains at the service of citizens, and does not jeopardize human 
identity or breach human rights, privacy or individual or public liberties. The Commission fulfils its 
duties in pursuance of the law of January 6, 1978 as amended on August 6, 2004. 
In accordance with this law, healthy volunteers are informed that their data are processed in an 
automated way and that they can access this file directly through the investigator or through the 
physician of their choice. 
 
INRA, sponsor of the study, signed a commitment to comply with the "Reference Method" of the 
CNIL for the processing of personal data carried out in biomedical research (MR-001) conducted by 
INRA. The investigative team of the project (UEN-CRNH) is committed to the methodology of 
reference MR001. 
 
 

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11-8 REGISTRATION ON THE NATIONAL REGISTER OF VOLUNTEERS 
 
The healthy volunteers will be recorded on the national database of volunteers who participate to 
clinical researches, VRB (Volunteers for Biomedical Research) in accordance with the French 
regulations. 
 
11-9 AMENDMENTS TO THE PROTOCOL 
 
There will be no alterations or changes to the protocol without agreement of all investigators and 
sponsor. 
If such an agreement, the planned changes will constitute an amendment that will be attached to the 
protocol. 
Any amendment must be notified to the ethic committee if the planned changes affect the ethical or 
medical-scientific study (evaluation criteria, addition of a new center ….). Minor modifications do 
not require a review of the ethic committee. 
 
11-10 MONITORING  
 
The investigator must permit study-related monitoring visits, audits, review by the Ethics 
Committee and regulatory inspections, and allow direct access to source data and source documents 
provided that subject confidentiality is protected.  
 
Monitoring includes on-site visits to assure that the investigation is conducted according to the 
protocol and in order to comply with applicable regulations and deadlines. On-site review of Case 
Report Forms (CRFs) includes the review of forms for completeness, clarity, and consistency with 
source documents available for each subject. 
 
 
 
11-11 BIOLOGICAL COLLECTION 
 
The collection of biological samples described in this protocol (blood and urine samples) is 
reported to the competent authority (Article L1123-12). It can be stored at the end of the research 
for the study of certain biological parameters of interest in the same purpose (metabolomic analysis) 
initially planned in the protocol All collected samples will be stored during 15 years after the end of 
the clinical study.  
As mentioned in the protocol and subject information sheet, the collected biological samples 
can be stored at the end of the research, without change of purpose.  
 
In case of a change of purpose, the Ministry of Research, the CPP and the Regional 
Hospitalization (if the facility is a health organization) are notified. Moreover, subjects will receive 
a new information which will permit them to express their opposition if they desire. 
 
At the end of the study, urinary and blood samples collected will be stored at the Human 
Nutrition Unit, INRA Centre of Theix. 
 
11-12 DATA RETENTION 
 
The following documents will be archived in the CRNH of Auvergne to the end of the period of 
practical use (15 years). 
These documents are:  
- 
Protocol and appendix, any amendments, 
- 
Information and consent forms signed (originals) 
- 
Individual data (authenticated copies of raw data) 
- 
Follow-up documents 
- 
Statistical analysis 

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- 
Final report of the study 
 
At the end of the period of practical use, all documents to be archived, will be under the 
responsibility of the INRA for 15 years after the end of the study according to institutional 
practices. 
No destruction can be performed without the consent of the sponsor. At the end of the 15 years, the 
sponsor will be consulted for destruction. All data, all documents and reports may be subject to 
audit or inspection. 
 
11-13 PUBLICATION POLICY 
 
Investigator will have full and unrestricted access to the database with all anonymized data. 
Anonymised data and outcomes of this study will be shared within partners of the FOODBALL 
consortium (Appendix 7). 
 
The outcomes of this study will be published in field relevant peer-reviewed journals thereby 
presenting the results to the scientific community. Other forms of communication include 
presentations at national and international meetings as well as media releases. Outcomes may be 
included in databases for dietary biomarkers created within the project FOODBALL.  
 
Rules for publications have been defined in the FOODBALL consortium agreement. 

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12 – REFERENCES 
 
 
1.  
Andersen MBS, Kristensen M, Manach C, et al. Discovery and validation of urinary 
exposure markers for different plant foods by untargeted metabolomics. Anal Bioanal Chem. 
2014;406(7):1829-1844. doi:10.1007/s00216-013-7498-5. 
2.  
Manach C, Hubert J, Llorach R, Scalbert A. The complex links between dietary 
phytochemicals and human health deciphered by metabolomics. Mol Nutr Food Res. 
2009;53(10):1303-1315. doi:10.1002/mnfr.200800516. 
3.  
Scalbert A, Brennan L, Manach C, et al. The food metabolome: A window over dietary 
exposure. Am J Clin Nutr. 2014;99(6):1286-1308. doi:10.3945/ajcn.113.076133. 
4.  
Manach C., Brennan L, Drasgted L.O. Metabolomics to evaluate food intake and utilization 
in nutritional epidemiology. In: Metabolomics as a Tool in Nutritional Research, Woodhead 
publishing 2015. pp.167-196 
5.  
Peralta I, Spooner DM. Genetic Improvement of Solanaceous Crops Volume 2: Tomato. CRC 
Press; 2006. https://books.google.com/books?hl=en&lr=&id=1m7RBQAAQBAJ&pgis=1. 
Accessed December 18, 2015. 
6.  
Re R, Bramley PM, Rice-Evans C. Effects of food processing on flavonoids and lycopene 
status in a Mediterranean tomato variety. Free Radic Res. 2002;36(7):803-810. 
http://www.ncbi.nlm.nih.gov/pubmed/12180131. Accessed December 18, 2015. 
7.  
Giovannucci E. Tomatoes, Tomato-Based Products, Lycopene, and Cancer: Review of the 
Epidemiologic Literature. JNCI J Natl Cancer Inst. 1999;91(4):317-331. 
doi:10.1093/jnci/91.4.317. 
8.  
Pereira A, Maraschin M. Banana (Musa spp) from peel to pulp: ethnopharmacology, source 
of bioactive compounds and its relevance for human health. J Ethnopharmacol. 
2015;160:149-163. doi:10.1016/j.jep.2014.11.008. 
9.  
Pujos-Guillot E, Hubert J, Martin JF, et al. Mass spectrometry-based metabolomics for the 
discovery of biomarkers of fruit and vegetable intake: Citrus fruit as a case study. J Proteome 
Res. 2013;12(4):1645-1659. doi:10.1021/pr300997c. 
10.  
Kesse-Guyot E, Castetbon K, Touvier M, Hercberg S, Galan P. Relative validity and 
reproducibility of a food frequency questionnaire designed for French adults. Ann Nutr 
Metab. 2010;57(3-4):153-162. doi:10.1159/000321680. 
 
 
 

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13 - APPENDIX 
 
Appendix 1 : Signature page 
Appendix 2 : Food Frequency Questionnaire 
Appendix 3 : Nutritional Composition of Fresubin ® 2kcal fiber 
Appendix 4 : Restricted diet for Day 1 
Appendix 5 : Restricted diet for Day 2 
Appendix 6: Composition of the standardized dinner for Days 2 and 3 
Appendix 7: FOODBALL consortium 
 
 
 
 
 

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APPENDIX 1 
 
PROTOCOL SIGNATURE PAGE 
 
I, the undersigned, acknowledge that I agree :  
 
To 
participate 
in 
the 
study 
described 
in 
the 
present 
document 
of 
the 
…………………………………….…., according to the Clinical Good Practices, in 
accordance with the current regulatory texts and as required by the protocol approved by the  
ethics committee CPP Sud-Est VI and the ANSM and approved by the promotor. 
 
 
Pr Ruddy Richard principal investigator 
………… / ………… /…………  
 
 
……………………… 
 Jour 
mois 
année 
 
 
Dr Amandine Prulière co-investigator 
 
………… / ………… /…………  
 
 
……………………… 
 Jour 
mois 
année 
 
 
Dr Françoise Laporte co-investigator 
 
………… / ………… /…………  
 
 
……………………… 
 Jour 
mois 
année 
 
 
Dr Nicolas Farigon co-investigator  
………… / ………… /…………  
 
 
……………………… 
 Jour 
mois 
année

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Exemple of questions translated from original in French for the volunteers: 
 
Breakfast 
At what time, foods usually consumed with quantities ? 
Standing or sitting? 
Same every day or changing? 
 
Do you eat snacks in the day?  
At what time? At what frequency? 
What food do you eat as snacks? 
 
What do you drink? 
Water: less than 0.5l per day or 0.5 to 1l per day, or 1 to 2l/day, or more? 
Wine: no or 2-3 glasses per day, or 4-7 glasses per day, or more? 
Beer or cider: no or 1 can or more? 
Fruit juice: no or 1 glass per day or 2-3 glasses per day, more (how many)? 
Sweetened beverages: no or 1 glass per day or 2-3 glasses per day, more (how many)? 
 
What do you drink? 
Raw vegetables: every meal or once a day or once a week 
Cooked vegetables: every meal or once a day or once a week 
Meat: every meal or once a day or less (how many?) or never 
Fish: never, once a week, more (how many?) 
Eggs: never, once a week, more (how many?) 
 
 
 
 
 
 
 
Appendix 2 : Dietary questionnaire 

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Appendix 3 : Nutritional Composition of Fresubin ® 2kcal fiber 
 
Fresubin ® 2kcal fibre is a high energy nutritional supplement developed by Fresenius Kabi. It is 
composed mainly by protein milk, vegetables iols (rapeseed oil, sunflower oil), carbohydrates, 
vitamins, minerals and fibre. It is a lactose and gluten free product. Data available at: 
http://www.fresenius-kabi.co.uk/4727.htm 
 
 
Energy and Macronutrient Composition 
 
Nutritional Composition 
  
Per 100ml Per 200ml 
Energy 
kcal 
200 
400 
Protein 
g 
10 
20 
Nitrogen 
g 
1.6 
3.2 
Carbohydrate 
g 
21.8 
43.6 
Of which sugars 
g 
3.3-6* 
6.6-12* 
Of which lactose 
g 
≤0.3 
≤0.6 
Fat 
g 
7.8 
15.6 
Of which saturated fatty acids 
g 
0.6 
1.2 
Of which polyunsaturated fatty acids 
g 
1.4 
2.8 
Of which monounsaturated fatty acids 
g 
5.8 
11.6 
Fibre 
g 
1.5 
3 
Water 
ml 
68 
136 
Osmolarity 
mosmol/l 
590-615 
 590-615 
 
 
 
 
 
 
 
Micronutrient composition 
 
Minerals and Trace Elements 
  
Per 100ml Per 200ml 
Sodium 
mg (mmol) 
60 (2.6) 
120 (5.2) 
Potassium 
mg (mmol) 
160 (4.1) 
320 (8.2) 
Chloride 
mg (mmol) 
80 (2.3) 
160 (4.6) 
Calcium 
mg (mmol) 
205 (5.1) 
410 (10.2) 
Phosphorus 
mg (mmol) 
120 (3.8) 
240 (7.6) 
Magnesium 
mg (mmol) 
16 (0.66) 
32 (1.32) 
Iron 
mg 
2.5 
5 
Zinc 
mg 
1.6 
3.2 
Copper 
μg 
375 
750 
Manganese 
mg 
0.5 
1 
Iodide 
μg 
37.5 
75 
Chromium 
μg 
12.5 
25 
Molybdenum 
μg 
18.8 
37.6 
Fluoride 
mg 
0.25 
0.5 
Selenium 
μg 
13.5 
27 
 
 
 
 
 
 

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Vitamine 
  
Per 100ml 
Per 200ml 
Vitamin A 
μg 
150 
300 
Beta-carotene 
μg 
375 
750 
Vitamin D 
μg 
2.5 
5 
Vitamin E 
mg 
3.75 
75 
Vitamin K 
μg 
21 
42 
Vitamin B1 
mg 
0.3 
0.6 
Vitamin B2 
mg 
0.4 
0.8 
Nicotinamide 
mg 
3.75 
7.5 
Vitamin B6 
mg 
0.43 
0.86 
Vitamin B12 
μg 
0.75 
1.5 
Pantothenic Acid 
mg 
1.5 
3 
Biotin 
μg 
9.4 
18.8 
Folic Acid 
μg 
62.5 
125 
Vitamin C 
mg 
18.8 
37.6 
 
 

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Appendix 4 : Restricted diet for Day 1 
 
No intake of tomato in any form : raw tomato or cooked tomato, tomato products (sauce, paste, 
ketchup) and dishes containing tomato (pizzas, soup, ratatouille, chili con carne...). 
 
No intake of banana in any form. 
 
No alcohol. 
 
 
 
Appendix 5 : Restricted diet for Day 2 
 
No intake of tomato in any form : raw tomato or cooked tomato, tomato products (sauce, paste, 
ketchup) and dishes containing tomato (pizza, soup, ratatouille, chili con carne...). 
 
No intake of banana in any form. 
 
No intake of plant-based beverages : coffee, tea, herbal teas, chocolate, fruit juices, wine, beer.  
 
No intake of fruits and vegetables, legumes, salad, breakfast cereals, wholegrain products. 
 
No intake of soft drinks.  
No alcohol 
 
Animal products (meat, fish, eggs, etc…), dairy products (butter, cream, yogurt, cheese), pasta, white 
rice, potatoes, white bread are allowed. 
Water and milk are the only beverages allowed. 
 
 
 
 
Appendix 6: Composition of the standardized dinner for Days 2 and 3 
 
- 
The dinner will be prepared at home by the volunteers, using food bought in local grocery. 
- 
The dinner will be composed of chicken breast simply pan-fried with butter or margarine, boiled 
white rice with salt, butter or margarine, and white bread. 
- 
The volunteers can eat as much as they want. 

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Appendix 7: FOODBALL consortium 
 
Wageningen UR**, The Netherlands 
University College Dublin*, Ireland 
University Oslo; Norway 
University of Copenhagen*; Denmark 
National Institute of Public Health and the Environment (RIVM), The Netherlands 
Ghent University, Belgium 
University of Alberta, Canada 
Technical University Denmark, National Food Institute, Denmark 
Institut National de la Recherche Agronomique (INRA)*, France 
Technical University München*, Germany 
Research Center on Food and Nutrition (CRA-NUT), Italy 
Fondazione Edmund Mach, Italy 
University of Bologna, Italy 
University Medical Center Groningen, The Netherlands 
Maastricht University, The Netherlands 
University of Barcelona, Spain 
Max-Ruber-Institut*, Germany 
ILSI Europe 
Agroscope*, Switzerland 
Helmholz Centre München, Germany 
TNO, The Netherlands 
Le Centre Hospitalier Universitaire Vaudois (CHUV)*, Switzerland 
ZonMw, The Netherlands 
Vitas AS, Norway 
University of Auckland, New Zealand 
University of Eastern Finland, Finland 
International Agency for Research on Cancer (IARC) 
 
*Partners within Work Package 1 (WP1) – Acute Study 
**coordinator