<figure><img src="image_1.png" /></figure>

HAL
open science

# Bioactives in Fruit and Vegetables and their Extraction Processes: State of the Art and Perspectives

Catherine M.G.C. Renard

<figure><img src="image_2.png" /></figure>

## To cite this version:

Catherine M.G.C. Renard. Bioactives in Fruit and Vegetables and their Extraction Processes: State of the Art and Perspectives. Bio2actives 2017, Jul 2017, Quimper, France. 2017. hal-01605299

HAL Id: hal-01605299

https://hal.science/hal-01605299

Submitted on 2 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_3.png" /></figure>

Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License
<figure><img src="image_1.png" /></figure>

# Bioactives in fruit and vegetables and their extraction: state of the art and perspectives

Catherine M.G.C. Renard

UMR408 Sécurité et Qualité des Produits d'Origine Végétale,
INRA, Université d'Avignon et des Pays du Vaucluse

<figure><img src="image_2.png" /></figure>

## Outline

- Introduction

- Bioactive compounds in fruit and vegetables

- Fruit and vegetable co-products and their stabilisation

- Extraction technologies

- Traditional methods

- New extraction methods

- Alternative solvents

- Research ... and organisation needs

<figure><img src="image_3.png" /></figure>
# Bioactives in Fruit and Vegetables and their Extraction Processes : State of the Art and Perspectives

## Catherine M.G.C. Renard

UMR408 SQPOV Sécurité et Qualité des Produits d'Origine Végétale, INRA,
Université d'Avignon, F-84000 Avignon, France
catherine.renard@inra.fr

### Abstract

Fruit and vegetables are rich in bioactive compounds that contribute to prevention of a number of degenerative diseases. These bioactive compounds are also present, often in even higher concentrations, in the co-products from fruit and vegetable processing. This makes these co-products an attractive source for extraction of bioactives, or extraction bioactives an attractive valorisation of the co-products. There has been recently renewed interest in extraction methods, notably with process intensification using physical phenomena and the search for alternative solvents. This paper will review the main bioactives in fruit, vegetables and their co-products, the precautions to preserve these bioactives in the food processing chain, and the new developments in the extraction methods.

Keywords : micronutrients, functional properties, stability, process intensification, solvent

### Introduction

The increase in demand for naturalness from consumers and the trends towards plant-based foods has sparkled a renewed interest in fruit and vegetables and their co-products as sources of bioactive and functional components. Traditionally, most hydrocolloids (polysaccharides) as well as a number of colorants (anthocyanins, carotenoids, betains) are extracted from plants. A high level ("five-a-day") of consumption of varied fruit and vegetables is associated with better health, and there has been much interest in identifying the molecules behind this effect. In recent years, this has progressed beyond the oversimplifying "antioxidant" hypothesis towards identification of mechanisms by which these molecules, or rather their metabolites, interact with specific targets in the Human organism (Dangles, 2012).

Concurrently there has been renewed interest in methods to obtain these bioactive components either for food uses, which are mostly relevant for molecules with functional properties (texture, colour, antioxidant, even aroma or taste), as food supplements, though their use as isolated molecules has proven disappointing, or as cosmetics. For economical reasons, preferred sources are the co-products from fruit and vegetable processing, as these are cheap and often highly concentrated in some bioactives. The ideal situation would be a complete use of the fruit and vegetable biomass in a biorefinery that would take into account food uses, high value bioactives as well as use of the remaining bulk material. A challenge is to conciliate the different aims and qualities of the products, as well as deal with availability issues.

Classical extraction techniques such as maceration or supercritical fluid have been supplemented with many "assisted" extraction techniques where an additional
<figure><img src="image_4.png" /></figure>

# Why a renewed interest in bioactives from plants?

## Socio-cultural factors

- Consumers' attraction for « naturality »

- Good image of plant products

## But also a long history

- Many colorants and hydrocolloids

- Antioxydants

- Presumed health effects - nutraceuticals

## Recent innovations in extraction

- Process intensification : « XX-assisted extraction »

- REACH regulation (2006)

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

# « Bioactive compounds »

## They are

- Secondary metabolites - but not all secondary metabolites

- Present among others in fruit and vegetables

- Demonstrated or supposed preventive impact on chronic degenerative pathologies

- OR with interesting functionalities: color, antioxidant...

## They are not

- Nutrients sensu stricto (vitamins, minerals)

- With pharmalogical (or toxic) activities

- Used in traditional medecine

<figure><img src="image_7.png" /></figure>
physical phenomenon is used to intensify the process. There has been an explosive growth of publications on these topics since the year 2000. The physical phenomena range from ultrahigh pressure to void (DIC instantaneous controlled depressurization), from microwave to ultrasound or electric fields. Many of these techniques were first developed for analytical purposes in order to have true quantification of the bioactive molecules, and some have proven to be profitably upscalable, at least to pilot level. Newer challenges concern the solvents used in the extraction, as REACH comes into application, and costs of solvent recovery become more and more significant. Biosourced solvents, subcritical liquids or ionic liquids thus appear now more frequently in the scientific literature, and also pose specific challenges.

# 1 Bioactives in fruit and vegetables: chemical classes and activities

The very notion of “bioactive” or bioactive compounds can have different definitions in the scientific literature. It is generally associated with the existence of a positive effect on chronic pathologies, with different levels of proof (Amiot et al., 2012). In the most common meaning of the term, it does not cover vitamins, although proven to be indispensable for human health, dietary fibers, or compounds with demonstrated acute pharmacological or negative activities (notably alkaloids). Indeed fruit and vegetables by definition have no detectable acute effect in a normal dietary pattern, at least in the Western world, while many drugs were originally identified in plants, giving rise to the large field of phytochemistry. Some chemical classes such as saponins are “borderline”. The level of proof or activity needed for a health claim appropriate to a functional food or nutraceutical is therefore a tight balance.

In terms of plant physiology, most of these compounds are secondary metabolites, i.e. they are not necessary to the basic metabolism (energy production, protein synthesis...) of the plants. It is generally believed that their role in planta concerns interactions with the outer environment, where they may act as protection against UV radiation (flavonols), as deterrents against predators (glucosinolates, some polyphenols), as attractants for pollinisation (anthocyans). The underlying chemical complexity explains that relatively few of these molecules have been studied for their action in preventing life-style diseases, and the relatively low levels of proof for many of them. This also may explain why most developments have been concentrated on a few classes that are either remarkably abundant (like the polyphenols or carotenoids) or remarkably active (glucosinolates, isoflavones).

Table 1 summarizes the classes of compounds that are most commonly called “bioactives” and which can be found in fruits or vegetables sensu largo, i.e. including herbs and spices, but not medicinal plants. Some of the classes are very specific of a given botanical family, for example the capsaicinoids (Capsicum) or glucosinolates (Brassica) while others are very widespread, like the phenolic acids or flavonols.

<table><caption>Table 1: The main chemical classes of bioactive compounds in fruit and vegetables (from Renard et al., 2012)</caption><thead><tr><td>Chemical classes</td><td>Main Fruit & vegetable sources</td><td>Bioactivity</td></tr></thead><tbody><tr><td>Terpenoids</td><td></td><td></td></tr></tbody></table>
Abundant recent scientific
publications

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

TrAc 71 (2015) 100

Two most studied technologies:

- Microwaves and ultrasounds

- For extraction or for analytical purposes

Most of the literature is from Eastern countries

- Polyphenols: China >> India - Spain

- Essential oils: China > Iran >> France

<figure><img src="image_11.png" /></figure>

Outline

Introduction

Bioactive compounds in fruit and vegetables

Fruit and vegetable co-products and their stabilisation

Extraction technologies

- Traditional methods

- New extraction methods

- Alternative solvents

Research ... and organisation needs

<figure><img src="image_12.png" /></figure>
<table><tr><td>Monoterpenes</td><td>"herbs", citrus</td><td>Aroma, digestion, antiseptic...</td></tr><tr><td>Diterpenes: carnosic acid</td><td>"herbs" (rosemary)</td><td></td></tr><tr><td>Triterpenes: Phytosterols,</td><td>Plant oils</td><td>Cardiovascular health</td></tr><tr><td>Saponins</td><td>Soy, chestnut</td><td></td></tr><tr><td>Tetraterpenes: Carotenoids:</td><td></td><td></td></tr><tr><td>Carotens: β-caroten</td><td>Orange fruit & vegetables, carrot</td><td>Provitamin A</td></tr><tr><td>lycopene</td><td>Tomato, watermelon</td><td>Prostate cancer</td></tr><tr><td>Xanthophylls: lutein</td><td>Green vegetables</td><td>Age-related macular degeneration</td></tr></table>

<table><thead><tr><th>Chemical classes</th><th>Main Fruit & vegetable sources</th><th>Bioactivity</th></tr></thead><tbody><tr><td colspan="3"><strong>Phenolic compounds</strong></td></tr><tr><td>Flavonoids</td><td></td><td></td></tr><tr><td>Flavonols</td><td>Onions, fruit, vegetables</td><td></td></tr><tr><td>Anthocyans</td><td>Red fruits, berries</td><td></td></tr><tr><td>Flavanols and proanthocyanidins</td><td>Tea, wine, chocolate, cider, fruits</td><td></td></tr><tr><td>Flavanones</td><td>Citrus</td><td></td></tr><tr><td>Isoflavones</td><td>Soy, legumes</td><td>Phyto-oestrogens</td></tr><tr><td>Non flavonoids</td><td></td><td></td></tr><tr><td>Phenolic acids</td><td>Coffee, fruits & vegetables</td><td></td></tr><tr><td>Lignans</td><td>cereals</td><td>Phyto-oestrogens</td></tr><tr><td>Hydrolysable tannins</td><td>Strawberry, fruits</td><td></td></tr><tr><td>Stilbens: resveratrol</td><td>Wine, peanut (stressed)</td><td>Heart disease?</td></tr><tr><td>Tyrosol & derivatives</td><td>Olive</td><td></td></tr><tr><td>Coumarins:</td><td>Apiaceae</td><td>photosensibilisation</td></tr><tr><td colspan="3"><strong>Sulphur-containing compounds</strong></td></tr><tr><td>Thiosulfinates</td><td>Allium</td><td></td></tr><tr><td>Glucosinolates</td><td>Brassicaceae</td><td>Thyroid hypertrophy, inhibition of H. pilori...</td></tr><tr><td>Capsaicinoides</td><td>Capsicum</td><td>Analgesic</td></tr></tbody></table>

Many of these compounds have antioxidant properties. It is now generally recognized that the health effects of bioactives are mediated by specific interactions of their circulating metabolites with cellular targets (Dangles, 2012). However the antioxidant properties may be relevant when focussing on stabilisation of food or cosmetics, for replacement of synthetic antioxidants such as butylhydroxytoluene or prevention of oxidation of polyunsaturated fatty acids in the gut (Brewer, 2011).
<figure><img src="image_13.png" /></figure>

# Main classes of bioactives in F&V

<figure><img src="image_14.png" /></figure>

## Terpenes

- Tetraterpenes: carotenoids

- Monoterpenes (essential oils)

- Saponins, phytosterols...

<figure><img src="image_15.png" /></figure>

## Phenolic compounds

- Non flavonoids: phenolic acids, hydrolysable tannins, lignans, stilbenes (resveratrol), coumarins...

- Flavonoids: anthocyanins, condensed tannins, flavonols, flavanones...

<figure><img src="image_16.png" /></figure>

## Organosulfur compounds

- Thiosulfinates and glucosinolates

<figure><img src="image_17.png" /></figure>

## Nitrogen-containing compounds

- Capsaicinoids

- Betalains

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

# Carotenoids

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

Red to yellow pigments - hydrophobic

Include provitamins A, lutein and lycopene

Two subclasses: carotens (hydrocarbons) and xanthophylls

Colorants and Antioxydants

<figure><img src="image_22.png" /></figure>
## 2 Co-products from fruit & vegetable processing and their stabilization

Many of the secondary metabolites are concentrated in the outer, least palatable, parts of fruit and vegetables and therefore are present in high concentrations in co-products from fruit and vegetable processing (Martins & Ferreira, 2017; Banerjee et al., 2017; Baiano et al., 2016; Strati & Oreopoulou, 2014). However these high concentrations are relative: polyphenols may reach from 10 to 50 g/kg dry weight in pomaces from fruit juice extraction, while carotenoids stay under 10 g/kg even in tomato by-products (Lavelli & Torresani, 2011) dietary fibers represent over 500 g/kg (Kolodziejczyk et al., 2009; Garcia et al., 2009).

A major limiting factor is that of an eventual co-product resource, as the question of bioactives can not be addressed from the target property but from the availability. We do not ask ourselves "from what can I extract this molecule of interest" but "what can I do with the co-product I have". Another point to be considered is the high variability of the resource: concentrations of the target compounds are likely to vary, sometimes over a factor 10, depending on the exact variety (and its maturity stage) (Garcia et al., 2009).

The alternative is establishment of a specific production chain, which demands a demonstrated and sufficient final value of the bioactive. Indeed, there are often alternatives using specific plants or other parts of plants that make more sense economically than relying on fruit and vegetables. For example lutein is extracted from Tagetes flowers and alfalfa, β-caroten from specific varieties of carrot with high concentrations, phloridzin is much more abundant in apple leaves than apple fruit (Gaucher et al., 2013), ...

In practical terms, this means that research on bioactives from fruit and vegetables is still very much "opportunity-driven" i.e. focussed on products that meet conditions of availability and potential interest. Availability of a co-product is in turn linked to the existence of a production chain. The most studied sources are thus pomace from grapes (wine) (Barba et al., 2016), tomato (pulp & concentrate), apples (juice), berries (juice), peels from Citrus (juice) or mango (puree or dried mangoes), sugar-beet pulp from sugar refining, waste water from olive processing, peels from onion or salad. Dietary fibers and polyphenols are the main chemical classes of interest, only tomato co-products being abundant and rich in carotenoids.

The treatments that take place for the initial product preparation (for example preparation of cloudy or clear juice, Kolodziejczyk et al., 2009) as well as those used for stabilisation of the co-product will also impact its composition and potential as source of bioactive. Two phenomena must be taken into account when envisioning extraction of polyphenols from e.g. apple pomace: one is that the molecules present in the pomace may not be not those of the original fruit, as there can be extensive enzymic oxidation during the juice extraction and pomace processing (Herrera-Ramirez et al., 2012) as well as chemical degradation, and the other is that some polyphenols, notably the condensed tannins, form strong adducts with the cell wall material (Renard et al., 2017) that may become un-extractable. Whether the pomace was initially treated to inhibit enzymes, the mode and temperature of drying, its final water content and storage temperature all impact the final composition (Herrera-
## Polyphenols

### 🏠 Ubiquitous

- Presents in F&V but mostly in tea, coffee, cocoa, wine, cider...

### 🏠 Many have interesting properties

- Color: anthocyanins and flavonols

- Antioxydants

- Nutraceutical effects, often mediated by microbiota

### 🏠 But also a number of drawbacks

- Bitter and/or astringent

- Brown color after oxidation (enzymic browning)

- Enzyme inhibition (technological but also digestive enzymes

- antinutrients)

<figure><img src="image_23.png" /></figure>

## Classification and a few formulae

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>
Ramirez et al., 2012; Lavelli & Corti, 2011; Lavelli & Torresani, 2011; Rahja et al., 2014; Yu et al., 2013). Carotenoids, in contrast to polyphenols, appear less stable at the lower aw values (Lavelli & Torresani, 2011).

A major difficulty in going from a laboratory demonstration to actual industrial production lies in the seasonal availability of fruit and vegetable and also of their co-products. For example, industrial tomatoes are harvested and processed over a period of about 2 months. Therefore their pomace, although remarkably high in lycopene, is only available fresh during this short period. The implementation of co-products, which are prone to microbial waste, may require immediate processing or a stabilization step, most often by drying, which may lead to some loss of bioactive by thermal degradation of the more fragile molecules or interfere with the extraction procedure itself (Rahja et al., 2014).

## 3 Recent developments in extraction methods

### 3.1 Conventional methods

The more classical methods for extraction of bioactives rely on maceration (with more or less intense stirring) in a solvent of appropriate polarity (Galanakis, 2012). A first improvement is increasing the extraction temperature for better dissolution and lower solvent viscosity, as is done e.g. in heated reflux extraction (which also takes advantage of concentration equilibria) or in a Soxhlet apparatus (which further has the advantage of separating the soluble from the insoluble fractions).

Supercritical fluids, and among them mostly carbon dioxide, have been used industrially in the food and cosmetics industry. Supercritical carbon dioxide has a low critical temperature and pressure (31°C, 7.39 MPa), which are of interest for labile molecules, and its polarity can be increased by adding co-solvents (e.g. ethanol). It is GRAS and non explosive, relatively cheap, and the extracted compounds can be recovered by evaporation of the gas. It is commonly used for extraction of essential oils and has a good potential for carotenoids.

Enzymic pretreatment have also been used in improving extraction yields, because it has long been recognised that plant cell walls are one of the limiting factors. However pretreatments with cellulases or pectinases demand long incubations (typically a few hours) in aqueous media.

<table><caption>Table 2: Conventional and emerging technologies for extraction of plant bioactives</caption><thead><tr><th>Method</th><th>Advantage</th><th>Disadvantage</th></tr></thead><tbody><tr><td colspan="3">Extraction methods</td></tr><tr><td>Maceration</td><td>Low investment cost; modulation of selectivity by solvent choice</td><td>Long; low recovery</td></tr><tr><td>Heating reflux, Soxhlet</td><td>Low investment cost; increased yields</td><td>High temperature; solvent</td></tr><tr><td>Supercritical fluid extraction</td><td>Low temperature; high yields; mostly for molecules of low polarity but can be modulated</td><td>High investment costs</td></tr></tbody></table>
## A few common flavonoids

<figure><img src="image_26.png" /><figcaption>### Anthocyanins</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>### Flavan-3-ols</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>### Catechins</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>### Flavanones</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>### Proanthocyanidins</figcaption></figure>

<figure><img src="image_31.png" /></figure>

## Organo-sulfur compounds

### Thiosulfinates

- Allicin - alliin

- In Alliaceae

- Degradation by alliinase to give typical odor

<figure><img src="image_32.png" /></figure>

Aliphatic
Aromatique
Indolique

### Glucosinolates

- R: aliphatic, aromatic or indolic

- Degradation by myrosinase to active thiocyanates, isothiocyanates...

- In Brassicaceae

- Odor and pungency...

<figure><img src="image_33.png" /><figcaption>sulforaphane</figcaption></figure>
<table><tr><td>Microwave-assisted extraction</td><td>Reduction of processing time and solvent use</td><td>Locally high temperatures; polar solvents.</td></tr><tr><td>Ultrasound-assisted extraction</td><td>Reduction of processing time, low temperature</td><td>Swelling of the plant material</td></tr><tr><td>Pressurized solvent extraction</td><td>Reduction of processing time and solvent use</td><td>Investment costs; temperature; low throughput</td></tr><tr><td>Pulsed electric fields</td><td>Reduction of processing time and solvent use</td><td>Requires conductivity; activity of enzymes</td></tr><tr><td colspan="3">Tissue destructuration methods</td></tr><tr><td>Enzyme-assisted extraction</td><td>Facilitated extraction from a plant tissue</td><td>Additional long operation in wet conditions</td></tr><tr><td>Negative pressure cavitation</td><td>Moderate temperature, possibility of anaerobic conditions</td><td>Lack of background data</td></tr><tr><td>High pressure homogeneisation</td><td>Increased yields by tissue disintegration</td><td>No selectivity, requires separation</td></tr><tr><td>Instantaneous pressure drop</td><td>Increased yields by tissue disintegration</td><td>Pretreatment only; requires pre-drying.</td></tr></table>

## 3.2 Emerging technologies

More recent developments concern the use of non-thermal concepts to facilitate extraction without risking overheating of the matrix, and decreasing energy use. Recent reviews describe application of conventional and emerging technologies to extraction of various classes of bioactives (Ameer et al., 2017; Lu et al., 2017; Poojary et al., 2016; Gil-Chavez et al., 2013; Wijngaard et al., 2012). All aim to destroy cell integrity by elimination of the cell membranes or cell walls. The most studied applications since 2005, as identified from scientific publication trends, concern microwave and ultrasound-assisted extraction (Mandal & Tandey, 2016).

Microwaves are electromagnetic waves, generally used at 2.45 GHz, which interact with polar molecules (typically water, ethanol...) and generate heat (Zhang et al., 2011). In microwave-assisted extraction the moisture inside the cell is heated and its evaporation increases the porosity of the biological matrix, which in turns allows better penetration of a solvent. The elevated temperature also generally increases solubility and improves yield. Its main advantage is reduction of the extraction time and solvent use. Elevated temperatures may still result in some degradation of the more labile molecules.

Ultrasound (> 20 KHz) improves extraction through acoustic cavitation: above certain energy levels the acoustic waves interact with the solvent and dissolved gas by creating free bubbles that can expend to a maximum size and violently collapse, generating locally extreme heat and pressures (Tiwari, 2015). Due to this cavitation phenomenon the cell walls can be ruptured, providing channels for solvent access, and mass transfer is improved. The small size of the bubbles means that the heat generated upon collapse can be dissipated rapidly and bulk temperature increase can stay limited. However liquid-solid separation may be hindered as ultrasound can lead to swelling and disintegration of the plant material.
## Nitrogen-containing compounds

<figure><img src="image_34.png" /></figure>

### Capsaicinoids

- capsaicine, dihydrocapasaicine

- Alcaloids present in Capsicum

- Responsible for their « burning » sensation

- Pain relief

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

### Betalaines

- Betacyanines (red to purple) or betaxanthines (yellow to orange)

- In Caryophylaceae

- Beet, prickly pear, pitaya, ...

<figure><img src="image_37.png" /></figure>

13

## Different compounds distribution in different species

<figure><img src="image_38.png" /></figure>

Flavan-3-ols (incl. procyanidins), hydroxycinnamic acids, flavonols & dihydrochalcones

<figure><img src="image_39.png" /></figure>

Anthocyanins, flavonols, phenolic acids, flavan-3-ols (procyanidins)

<figure><img src="image_40.png" /></figure>

Flavan-3-ols (incl. procyanidins), flavonols, hydroxycinnamic acids & anthocyanins

<figure><img src="image_41.png" /></figure>

Flavan-3-ols (incl. procyanidins), anthocyanins, flavonols & hydroxycinnamic acids

14
Among electrotechnologies, different pulse protocols and intensities of electric fields have been used to generate cell disintegration and thus enhance extraction of intracellular compounds. The most studied is Pulsed Electric Fields, in which the sample is submitted to very short periods (several nanoseconds to several milliseconds) of an intense electric field. This causes the formation of pores in the cell membranes (electroporation) which helps in solvent diffusion and facilitates mass transfer (Rastogi, 2003; Puertolas et al., 2012)). PEF is also used to increase juice yields, i.e. non selective extraction of intracellular fluids.

Pressurized liquid extraction (or accelerated solvent extraction) relies on increased temperature and pressure in a solvent that is kept below its boiling point to enhance mass transfer and modify surface equilibria in solid – liquid extraction (Mustafa & Turner, 2011). Pressure is used primarily to maintain the solvent n the liquid phase, though it may also facilitate solvent entrance in the pores of the matrix. It is commonly used in analytical quantification of bioactives but also pesticides. The elevated temperature also modulates the polarity of the solvent and thus its extraction selectivity.

Negative pressure cavitation is a patented technology that aims to produce cavitation by depression to generate an intense erosion of solid particles, and increase turbulence and mass transfer from the solid matrix to the solvent (Roohinejat et al., 2016).

Another potential pretreatment is high pressure homogeneization (Xi, 2017; Corrales et al., 2009), a wet milling process where plant particles are disintegrated by high intensity mechanical stresses as a consequence of the liquid flow through an homogeneization chamber at high pressures (50-500 MPa). It can be used on wet samples.

Another method which has been proposed for sample disintegration prior to extraction is instant controlled pressure drop (DIC) (Mounir et al., 2014). In DIC, the sample is subjected for a short time to saturated steam then to a sudden pressure drop at low pressures. This causes instantaneous vaporisation of water, resulting in cell wall expansion and a rapid cooling. The instantaneous vaporization allows recovery of essential oils, while polyphenols or carotenoids are more easily extracted from the remaining fine powder. Similar results may be obtained by intense grinding, provided care is taken to prevent temperature increase during grinding.

### 3.3 Alternative solvents

The solvent choice is a concern as it impacts selectivity, removal and disposal method, costs and safety. The REACH directive has notably been an incentive for "green" solvents in replacement to *n*-hexane. For example, among alcohols ethanol is often proposed due to its low boiling point and GRAS status, but may not be efficient for less polar molecules such as carotenoids. Water is also often tested, alone or in mixture with ethanol or acetone, in spite of its high energy requirements for evaporation. More apolar solvents, mostly terpenes (limonene, alpha-pinene) have also been proposed (Filly et al., 2015). Another option is the use of subcritical solvents such as liquefied gases (e.g. *n*-butane), which may ally low polarity and
<figure><img src="image_42.png" /></figure>

Contents are also very variable

<figure><img src="image_43.png" /></figure>

Sanoner et al., JAFC 47 (1999) 4847

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

They can be concentrated in
some fractions of the F&V

Often in higher concentrations in tough, outer parts

- So that they are more concentrated in co-products

- Epidermis: anthocyanins, flavonols

- Sometimes higher concentrations in pips or stones

<figure><img src="image_46.png" /></figure>

Markovic et al., 2010

<figure><img src="image_47.png" /></figure>
facile solvent elimination by decompression at or close to room temperature (Rapinel et al., in press). However safety issues must be addressed for these new solvents.

Recently ionic liquids have been proposed for extraction of bioactives. Ionic liquids *senso strictu* are organic salts in a liquid state. Although they are viscous, their polarity can be adjusted across a wide range of hydrophobicity / hydrophilicity, and some of them are distillable in conditions compatible with recovery of bioactives (Almeida et al., 2014; Lu et al., 2013)). The most recent trend in this field is the use of "natural deep eutectic solvents" (NADES), which are combinations of natural components such as sugars, organic acids or aminoacids, with tailored solvent properties (Dai et al., 2013; Radosevic et al., 2016). However solvent removal becomes more complex.

## Conclusion

A lot of experimental work has been devoted in the last 10 – 20 years to development of “alternative” technologies for extraction of bioactives from plants, including fruit and vegetables. It is time that this body of experimental data, which mostly relies on statistical optimisation of yields, gives place to a more mechanistic oriented research. In addition, post-extraction treatments (elimination of the solvent, purification...) are the poor relatives in this research.

A number of technical issues have to be addressed before and after extraction: will the raw material be stabilised or only extracted fresh, i.e. over a short period? If it is stabilized, how do we do it with the least loss of bioactive? Is a purification step needed? Most of these molecules are only present in low concentrations, therefore their extraction will still leave large amounts of waste: can that waste in turn become a co-product? This requires anticipation and conceiving the whole chain in an integrated manner for valorisation of all the biomass.

Two main conditions must be met for the extraction of bioactives from fruit and vegetables to be economically interesting:

- The activity or functionality of the molecules must have a market potential;

- It must be present in an amount compatible with its market in a cheap co-product – or the market potential must be sufficient for specific production.

Colour or antioxidant capacity are properties relatively easy to assess, which may explain why they constitute the main fields of application for extracts from fruit and vegetables. Having sufficient data for a health claim or nutraceutical use is a totally different proposition as the cost of demonstrating the claim becomes high.

All of this may explain why, in spite of much research, few fruit and vegetable co-products are actually used at industrial scale for production of bioactives (Galanakis, 2012): anthocyans from grape skin, (oenological) tannins from grape seeds, lycopene from tomato waste (Strati & Oreopoulou, 2014), polyphenols from olive mill waste water and flavonoids from citrus peel. Production of oil from grape seeds or kernels of plums, apricots and peaches, dietary fibers from vegetable wastes and pectin extraction (from citrus peel, apple pomace or sugar-beet pulp) may also be counted in this valorization of fruit and vegetable co-products. The few successful examples
## Outline

### Introduction

### Bioactive compounds in fruit and vegetables

### Fruit and vegetable co-products and their stabilisation

### Extraction technologies

- Traditional methods

- New extraction methods

- Alternative solvents

### Research ... and organisation needs

17

## What are the potential sources ?

### Co-products ... if there are products

- Grape, apple, red fruit pomaces

- Citrus peels

- Mango and other tropical fruit peels

- Tomato by-products

- Olives processing water

- Onion peels or salad outer leaves

- Sugar-beet pulp, potato pulp

Juices, wine

Tomato products

Oil

Fresh cut

Sugar, starch

### The co-products must be stabilised

- Availability limited in time plus highly fermentescible

- Generally by drying, which can impact bioactives

- Sometimes pasteurization (storage)

18
need to be carefully analysed, not from a technological point of view but from a socio-economic and legislative point of view to identify the factors of their success.

## References

Almeida, M. R., H. Passos, et al. (2014). "Ionic liquids as additives to enhance the extraction of antioxidants in aqueous two-phase systems." Separation and Purification Technology 128: 1-10.

Ameer, K., H. M. Shahbaz, et al. (2017). "Green Extraction Methods for Polyphenols from Plant Matrices and Their Byproducts: A Review." Comprehensive Reviews in Food Science and Food Safety 16(2): 295-315.

Amiot M.J., V. Coxam et al. (2012) "Les Phytomicronutriments". TechetDoc Lavoisier, 386 pp.

Baiano, A. and M. A. Del Nobile (2016). "Antioxidant Compounds from Vegetable Matrices: Biosynthesis, Occurrence, and Extraction Systems." Critical Reviews in Food Science and Nutrition 56(12): 2053-2068.

Banerjee, J., R. Singh, et al. (2017). "Bioactives from fruit processing wastes: Green approaches to valuable chemicals." Food Chemistry 225: 10-22.

Barba, F. J., Z. Z. Zhu, et al. (2016). "Green alternative methods for the extraction of antioxidant bioactive compounds from winery wastes and by-products: A review." Trends in Food Science & Technology 49: 96-109.

Brewer, M. S. (2011). "Natural Antioxidants: Sources, Compounds, Mechanisms of Action, and Potential Applications." Comprehensive Reviews in Food Science and Food Safety 10(4): 221-247.

Corrales, M., A. F. Garcia, et al. (2009). "Extraction of anthocyanins from grape skins assisted by high hydrostatic pressure." Journal of Food Engineering 90(4): 415-421.

Dai, Y., J. van Spronsen, et al. (2013). "Natural deep eutectic solvents as new potential media for green technology." Analytica Chimica Acta 766: 61-68.

Dangles, O. (2012). "Antioxidant Activity of Plant Phenols: Chemical Mechanisms and Biological Significance." Current Organic Chemistry 16(6): 692-714.

Filly, A., A. S. Fabiano-Tixier, et al. (2015). "Alternative solvents for extraction of food aromas. Experimental and COSMO-RS study." LWT - Food Science and Technology 61(1): 33-40.

Galanakis, C. M. (2012). "Recovery of high added-value components from food wastes: Conventional, emerging technologies and commercialized applications." Trends in Food Science & Technology 26(2): 68-87.

Garcia, Y. D., B. S. Valles, et al. (2009). "Phenolic and antioxidant composition of by-products from the cider industry: Apple pomace." Food Chemistry 117(4): 731-738.

Gaucher, M., T. D. de Bernonville, et al. (2013). "Histolocalization and physico-chemical characterization of dihydrochalcones: Insight into the role of apple major flavonoids." Phytochemistry 90: 78-89.

Gil-Chavez, G. J., J. A. Villa, et al. (2013). "Technologies for Extraction and Production of Bioactive Compounds to be Used as Nutraceuticals and Food Ingredients: An Overview." Comprehensive Reviews in Food Science and Food Safety 12(1): 5-23.
<figure><img src="image_48.png" /></figure>

Bioactive compounds are a
minor part of the co-products

Fibers represent > 50% of F&V co-products

- Dietary fibers: hydration, texturants

- Apple and citrus: production of pectins - 10 - 25%

- A major residue after extraction of bioactives

Seeds & pips can be rich in oils and proteins

Some typical concentrations

lycopene in tomato co-product: 0,1% - 1 g/kg ms Kalogeropoulos et al., 2012

carotenoids in mango peel: 0,3% - 3 g/kg ms Ajila et al., 2010

carotenoids in carrot pomace: 0,015% - 150 mg/kg ms Pieszka et al., 2015

polyphenols in apple pomace: 0,5-3 % 5-30 g/kg ms

polyphenols in blackcurrant pomace: 1,2% 12 g/kg ms Juskiewicz et al., 2015

procyanidins in perry pear pomace: 1 % 10 g/kg mf Brahem et al., 2016

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

Retention in pomace is selective

And oxidation during processing also impacts compositions

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>
Heras-Ramirez, M. E., A. Quintero-Ramos, et al. (2012). "Effect of Blanching and Drying Temperature on Polyphenolic Compound Stability and Antioxidant Capacity of Apple Pomace." Food and Bioprocess Technology 5(6): 2201-2210.

Kolodziejczyk, K., M. Kosmala, et al. (2009). "Characterisation of the chemical composition of scab-resistant apple pomaces." Journal of Horticultural Science & Biotechnology: 89-95.

Lavelli, V. and S. Corti (2011). "Phloridzin and other phytochemicals in apple pomace: Stability evaluation upon dehydration and storage of dried product." Food Chemistry 129(4): 1578-1583.

Lavelli, V. and M. C. Torresani (2011). "Modelling the stability of lycopene-rich by-products of tomato processing." Food Chemistry 125(2): 529-535.

Lu, C. X., X. L. Luo, et al. (2013). "Preliminary extraction of tannins by 1-butyl-3-methylimidazole bromide and its subsequent removal from Galla chinensis extract using macroporous resins." Journal of Separation Science 36(5): 959-964.

Lu, M. W., C. T. Ho, et al. (2017). "Extraction, bioavailability, and bioefficacy of capsaicinoids." Journal of Food and Drug Analysis 25(1): 27-36.

Mandal, V. and R. Tandey (2016). "A critical analysis of publication trends from 2005–2015 in microwave assisted extraction of botanicals: How far we have come and the road ahead." TrAC Trends in Analytical Chemistry 82: 100-108.

Martins, N. and I. Ferreira (2017). "Wastes and by-products: Upcoming sources of carotenoids for biotechnological purposes and health-related applications." Trends in Food Science & Technology 62: 33-48.

Mounir, S., T. Allaf, et al. (2014). "Instant Controlled Pressure Drop technology: From a new fundamental approach of instantaneous transitory thermodynamics to large industrial applications on high performance-high controlled quality unit operations." Comptes Rendus Chimie 17(3): 261-267.

Mustafa, A. and C. Turner (2011). "Pressurized liquid extraction as a green approach in food and herbal plants extraction: A review." Analytica Chimica Acta 703(1): 8-18.

Poojary, M. M., F. J. Barba, et al. (2016). "Innovative Alternative Technologies to Extract Carotenoids from Microalgae and Seaweeds." Marine Drugs 14(11).

Puertolas, E., E. Luengo, et al. (2012). Improving Mass Transfer to Soften Tissues by Pulsed Electric Fields: Fundamentals and Applications. Annual Review of Food Science and Technology, Vol 3. M. P. Doyle and T. R. Klaenhammer. 3: 263-282.

Radosevic, K., N. Curko, et al. (2016). "Natural deep eutectic solvents as beneficial extractants for enhancement of plant extracts bioactivity." Lwt-Food Science and Technology 73: 45-51.

Rajha, H. N., W. Ziegler, et al. (2014). "Effect of the Drying Process on the Intensification of Phenolic Compounds Recovery from Grape Pomace Using Accelerated Solvent Extraction." International Journal of Molecular Sciences 15(10): 18640-18658.

Rapinel, V., N. Rombaut, et al. "An original approach for lipophilic natural products extraction: Use of liquefied n-butane as alternative solvent to n-hexane." LWT - Food Science and Technology, in press DOI: https://doi.org/10.1016/j.lwt.2016.10.003

Rastogi, N. K. (2003). "Application of high-intensity pulsed electrical fields in food processing." Food Reviews International 19(3): 229-251.

Renard C.M.G.C., A. Hyardin A. et al. : « Sources, consommation et principaux facteurs de variation des microconstituants bioactifs des végétaux ». In : « Les
<figure><img src="image_58.png" /></figure>

Drying can modify the molecules
and their extractability

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

Outline

R Introduction

R Bioactive compounds in fruit and vegetables

R Fruit and vegetable co-products and their stabilisation

R **Extraction technologies**

- Traditional methods

- New extraction methods

- Alternative solvents

R Research ... and organisation needs

<figure><img src="image_64.png" /></figure>
phytomicronutriments », Eds MJ Amiot, V. Coxam & F. Strigler, Tec&Doc Lavoisier, Paris, 2012, pp 27-47

Renard, C., A. A. Watrelot, et al. (2017). "Interactions between polyphenols and polysaccharides: Mechanisms and consequences in food processing and digestion." *Trends in Food Science & Technology* 60: 43-51.

Roohinejad, S., M. Koubaa, et al. (2016). "Negative pressure cavitation extraction: A novel method for extraction of food bioactive compounds from plant materials." *Trends in Food Science & Technology* 52: 98-108.

Strati, I. F. and V. Oreopoulou (2014). "Recovery of carotenoids from tomato processing by-products – a review." *Food Research International* 65, Part C: 311-321.

Tiwari, B. K. (2015). "Ultrasound: A clean, green extraction technology." *TrAC Trends in Analytical Chemistry* 71: 100-109.

Verdu, C. F., N. Childebrand, et al. (2014). "Polyphenol variability in the fruits and juices of a cider apple progeny." *Journal of the Science of Food and Agriculture* 94(7): 1305-1314.

Wijngaard, H., M. B. Hossain, et al. (2012). "Techniques to extract bioactive compounds from food by-products of plant origin." *Food Research International* 46(2): 505-513.

Xi, J. (2017). "Ultrahigh pressure extraction of bioactive compounds from plantsA review." *Critical Reviews in Food Science and Nutrition* 57(6): 1097-1106.

Yan, H. T. and W. L. Kerr (2013). "Total phenolics content, anthocyanins, and dietary fiber content of apple pomace powders produced by vacuum-belt drying." *Journal of the Science of Food and Agriculture* 93(6): 1499-1504.

Zhang, H. F., X. H. Yang, et al. (2011). "Microwave assisted extraction of secondary metabolites from plants: Current status and future directions." *Trends in Food Science & Technology* 22(12): 672-688.
# The « traditional » extraction methods

<figure><img src="image_65.png" /></figure>

## By simple contact between plant matter and its solvent

- Maceration - followed by filtration - with ethanol

- Decoction - cold water or infusion - hot (boiling) water

- Percolation - renewed solvent

## Expression

- Mechanical effect, liquid/solid separation

- Citrus essential oils, oils

## Steam distillation

- Essential oils plus hydrosols

## Enfleurage

- Extraction by contact with odorless fats

## Soxhlet

- Constant renewal of solvent plus heat

## Grinding or micronization

- Traditional pretreatments to accelerate extraction

- Solid/liquid separation may become more difficult

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

23

# Supercritical fluid extraction

<figure><img src="image_69.png" /></figure>

## Pressure and temperature

- CO₂: > 31.1°C, 7.39 MPa

- But also ethane, propane...

- Advantages: low temperature, easy extract recovery

- CO₂: GRAS, low cost, non explosive

- Low polarity but it can be modulated by co-solvents

- Ethanol

<figure><img src="image_70.png" /></figure>

## Large scale industrial uses

- Decaf, hop extract

- Essential oils and pharmaceuticals

- Carotenoids
New extraction methods

<figure><img src="image_71.png" /></figure>

Intensification: increase yield, decrease solvent use and extraction duration

- Extraction itself « one pot »

- Pretreatment: degradation of plant tissues followed by extraction

<figure><img src="image_72.png" /></figure>

Various physical principles have been proposed

- Most often « athermal » - except microwaves

- Increased tissue destructuration and facilitated transfer

- Grinding or micronization

- Pressure / vacuum

- Directly or using ultrasound

- Electrotechnologies

- Mechanical, enzymic...

<figure><img src="image_73.png" /></figure>

Microwaves-assisted
extraction

<figure><img src="image_74.png" /></figure>

Electromagnetic waves (2,45 GHz) which generate heat by
interacting with polar molecules (such as water)

High temperature: hydrodistillation « without added water »

<figure><img src="image_75.png" /><figcaption>Extraction of pomelo essntial oil</figcaption></figure>

Chen et al., 2016

<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>
Ultrasound-assisted
extraction

The underlying mechanism is cavitation created by ultrasound
waves in the solvent

Explosion of cavitation bubbles erodes the surface of the plant
tissues and desaggregates particles while facilitating
homogeneisatio

ACOUSTIC CAVITATION

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>

rarefacion

ranefaction

ranefaction

ranefaction

ranefaction

<figure><img src="image_80.png" /></figure>

ACOUSTIC CAVITATION
in a homogeneous liquid medium

<figure><img src="image_81.png" /></figure>

<figure><img src="image_82.png" /></figure>

At the surfaces

<figure><img src="image_83.png" /></figure>

<figure><img src="image_84.png" /></figure>

<figure><img src="image_85.png" /></figure>

Application to extraction of
polyphenols from apple pomace

Experimental planning to optimize yield as function of temperature,
ultrasound, extraction duration, S/L ratio, solvent composition

<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>

The (statistical) optimum corresponds to max
conditions

Generally a very limited effect of ultrasound power

Pilot to industrial-scale equipments

<figure><img src="image_88.png" /></figure>
## Pressurized solvent extraction

PSE or ASE™: solvents under pressure and high temperature
Subcritical conditions but solvent polarity (hence specificity) is modified

Facilitated penetration in the pores of the material

Mostly analytical use

<figure><img src="image_89.png" /></figure>

<figure><img src="image_90.png" /></figure>

Pinero et al., 2004

29

## Pulsed electric fields

Electroporation i.e. opening of pores in the
cell membranes by an electric field

Used to facilitate extraction from
intracellular liquids - juices rather than
extraction of bioactives from co-products

Electrode +

<figure><img src="image_91.png" /></figure>

Electrode - Tension : U (V)
Distance : d (cm)

Ec : critical
electric field

Membraen compression

<figure><img src="image_92.png" /></figure>

Membrane

No extraction

Electroporation

<figure><img src="image_93.png" /></figure>

Membrane

Selective extraction

Electroplasmolysis

<figure><img src="image_94.png" /></figure>

Membrane

Intensive extraction

30
## Apple juice extraction

<figure><img src="image_95.png" /></figure>

Pilot-scale extraction on a belt press, with different apple fragmentation sizes

<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>

31

## Enzymes that degrade the plant cell walls

<figure><img src="image_98.png" /></figure>

To facilitate diffusion by tissue degradation and decrease of adhesion

<figure><img src="image_99.png" /></figure>

Incubation with pectinases and cellulases

32
## Negative pressure cavitation

Cavitation by depression of a gas-saturated liquid to generate intense erosion of solid particles and increase turbulence and mass transfer from the solid matrix to the solvent

<figure><img src="image_100.png" /></figure>

Roohinejad et al., 2016

Cavitation is due to gas expansion, and the bubbles also contribute to a turbulent flow and intense mass transfer

33

## Détente instantanée controlée

In instant pressure drop the sample is treated in a stream of saturated steam then the water is vaporized by sudden depression, resulting in cell wall expansion

Essential oils can be recovered with the cooled vapors while other compounds are easily extracted from the fine solid powder

<figure><img src="image_101.png" /></figure>

<figure><img src="image_102.png" /></figure>

<figure><img src="image_103.png" /></figure>

Optnein of orange essential oil glands by DIC treatment

Allaf et al., 2013

34
## High pressure homogeneization

The (wet) plant particles are disintegrated by high intensity
mechanical stresses as the liquid flows through an
homogeneization chamber

<figure><img src="image_104.png" /></figure>

<figure><img src="image_105.png" /><figcaption>Extraction of phenolics from grape by-products</figcaption></figure>

<figure><img src="image_106.png" /></figure>

Corrales et al., 2008

35

## Alternative solvents

The incentive is the need to replace petrochemical solvents with
negative evaluation according to REACH legislation

In choosing a solvent its solution properties as well as recovery /
elimination have to be assessed

Different options have been proposed:

- « classical », GRAS solvents such as water (removal...) or ethanol

- Natural « green » solvents: terpenes (limonene, alpha-pinene...)

- Liquefied but not supercritical gases (n-propane, n-butane)

- Ionic liquids notably natural deep eutectic solvents

36
## Computer-aided solvent choice

### Solvent and solutes compatibility using Cosmo-RS

### 3D distribution of the polarization charges on the molecule surface

<figure><img src="image_107.png" /></figure>

Rapinel et al., 2017

<figure><img src="image_108.png" /></figure>

## Ionic liquids and NADES

<figure><img src="image_109.png" /></figure>

### Ionic liquids:

- salts in the liquid state

<figure><img src="image_110.png" /></figure>

### Natural deep eutectic solvents:

- Approximate the medium in which the compounds were dissolved in the cell

- Mixtures of sugars and amino-acids - and water

<figure><img src="image_111.png" /></figure>

Glucose : choline

<figure><img src="image_112.png" /></figure>

Dai et al., 2013

<figure><img src="image_113.png" /></figure>
## Outline

- Introduction

- Bioactive compounds in fruit and vegetables

- Fruit and vegetable co-products and their stabilisation

- Extraction technologies

- Traditional methods

- New extraction methods

- Alternative solvents

- Research ... and organisation needs

39

## Needs in research... And organisation

- Extraction itself is the easy part...

- Extraction intensification has been the focus of recent research

- Validated methods, some available at pilot / industrial scale

- Research perspectives

- Go beyond optimization

- Improve pre-extraction (drying, stabilisation) and post-extraction treatments (purification, stabilisation...)

40
<figure><img src="image_114.png" /></figure>

A need of organisation of
value chains

<figure><img src="image_115.png" /></figure>

Extraction of WHAT from WHICH material: co-product or
dedicated production?

- Choice / cost / concentration / « food first »

- Can we reconcile those two very different questions:

- I have a co-product, what can I do with it?

- I want this molecule/property, how can I get it?

<figure><img src="image_116.png" /></figure>

Which properties are relevant?

- Nutraceutical niche markets?

- Replacement of synthetic colorants or antioxidants?

- Legislation?

<figure><img src="image_117.png" /></figure>

Stabilisation

- Extraction during a large part of the year

- Versatility to adapt to different raw materials/co-products or stabilization to expand period of use?

- What about the residues? Solvent recovery?

- What about contamination by pesticides / mycotoxins?

<figure><img src="image_118.png" /></figure>

<figure><img src="image_119.png" /></figure>

Thank you for your attention

<figure><img src="image_120.png" /></figure>

Tempantiox

<figure><img src="image_121.png" /></figure>

ISA

FRUIT

<figure><img src="image_122.png" /></figure>

PRO

JECT

Thanks to CBB Capbiotek for the invitation

Thanks to M. Kosmala, A. Cendrès, M. Turk, M. Virot,
N. Pingret, P. Sanoner, T. Allaf and many other students

Thanks to W. Plocharski, A. Baron, K. Kotodziejczyk,
F. Chemat, K. Allaf, E. Vorobiev,