F Delorme
email: fabiandelorme@yahoo.fr.

R Dujardin

F Schoenstein

B Pintault

P Belleville

C Autret

I Monot-Laffez

F Giovannelli

Nanostructuring of dense SnO2 ceramics by Spark Plasma Sintering

Keywords: Tin oxide, Spark Plasma Sintering, Thermoelectric, Nanostructuring, Thermal conductivity

The spark plasma sintering (SPS) behaviour of pure SnO2 has been studied. Two different SnO2 powders have been studied: a commercial 50-200 nm one and 4-6 nm nanoparticles obtained by precipitation. It has demonstrated that it is not possible to keep pure SnO2 above 1223 K by SPS. Indeed, at 1248 K, SnO appears whereas at higher temperatures, samples are composed by SnO2 and metal Sn. Three different cycles have been

INTRODUCTION

SnO2 exhibits a rutile-type structure (space group : P42/mnm, a = b = 4.7382 Å and c = 3.1871 Å, theoretical density = 6.994, PDF card 41-1445) [START_REF] Mccarthy | X-Ray diffraction data for SnO2. An illustration of the new powder data evaluation methods[END_REF]. It is an n-type semiconductor with a wide band gap of 3.6 eV [START_REF] Chen | Recent advances in tin dioxide materials: some developments in thin films, nanowires, and nanorods[END_REF]. It combines high electrical conductivity with high optical transparency in the visible range of the electromagnetic spectrum [START_REF] Batzill | The surface and materials science of tin oxide[END_REF]. Therefore, it is used for numerous applications such as transparent conducting oxide [START_REF] Kilic | Origins of coexistence of conductivity and transparency in SnO2[END_REF][START_REF] Singh | Sources of electrical conductivity in SnO2[END_REF], solid state gas sensor [START_REF] Batzill | The surface and materials science of tin oxide[END_REF][START_REF] Yamazoe | New approaches for improving semiconductor gas sensors[END_REF][START_REF] Göpel | SnO2 sensors: current status and future prospects[END_REF][START_REF] Comini | Metal oxide nano-crystals for gas sensing[END_REF], electrode [START_REF] Brousse | Thin-Film Crystalline SnO2-Lithium Electrodes[END_REF], catalyst material [START_REF] Miyauchi | Photocatalysis and photoinduced hydrophilicity of various metal oxide thin films[END_REF], photoluminescence [START_REF] Gu | Photoluminescence properties of SnO2 nanoparticles synthesized by sol-gel method[END_REF], varistor [START_REF] Pianaro | A new SnO2-based varistor system[END_REF][START_REF] Glot | Electrical properties of SnO2 ceramics for low voltage varistors[END_REF],

thermoelectric material [START_REF] Tsubota | Thermoelectric properties of Sn1-x-yTiySbxO2 ceramics[END_REF][START_REF] Yanagiya | Thermoelectric properties of SnO2 ceramics doped with Sb and Zn[END_REF][START_REF] Vo | Preparation and study of the thermoelectric properties of nanocrystalline Sn1-xTaxO2 (0 ≤ x ≤ 0.04)[END_REF] or for photovoltaic devices [START_REF] Mao | High efficiency dye-sensitized solar cells constructed with composites of TiO2 and the hotbubbling synthesized ultra-small SnO2 nanocrystals[END_REF].

It is well known that achieving high density SnO2 ceramics by conventional sintering is not possible due to non-densifying mechanisms such as the transformation of SnO2 into SnO and vaporization of tin monoxide which occurs at a high rate above 1373 K or surface diffusion and evaporation-condensation [START_REF] Kimura | Microstructure development in SnO2 with and without additives[END_REF][START_REF] Dolet | Densification of 0.99 SnO2-0.01 CuO mixture : Evidence for liquid phase sintering[END_REF]. Therefore, to overcome such a difficulty, one route has been to add sintering agents into SnO2, such as CoO, Co3O4, MnO2, CuO, ZnO, Sb2O3, … [START_REF] Kimura | Microstructure development in SnO2 with and without additives[END_REF][START_REF] Varela | Pore size evolution during sintering of ceramic oxides[END_REF][START_REF] Zuca | Contribution to the study of SnO2based ceramics. Part II Effect of various oxide additives on the sintering capacity and electrical conductivity of SnO2[END_REF][START_REF] Cerri | Effect of Cobalt(II) oxide and Manganese(IV) oxide on the sintering of tin(IV) oxide[END_REF][START_REF] Castro | Characterization of SnO2-varistors with different additives[END_REF][START_REF] Lalande | Sintering behaviour of CuO-doped SnO2[END_REF]. However, the sintering agent can also affect properties and especially transport properties that are very sensitive to small amounts of dopants in that kind of oxides [START_REF] Castro | Characterization of SnO2-varistors with different additives[END_REF][START_REF] Guilmeau | Tuning the transport and thermoelectric properties of In2O3 bulk ceramics through doping at In-site[END_REF].

Another route was to use high pressure. For instance, Park et al. [START_REF] Park | Densification of nonadditive SnO2 by hot isostatic pressing[END_REF] have obtained more than 97 % dense undoped SnO2 ceramics by hot isostatic pressing carried out under 150 MPa at 1673 K for 12 hours. However, such high temperature and long treatments leads also to grain growth and are not therefore a suitable solution for nanostructuring. Spark Plasma Sintering (SPS) is now a well-known technique to sinter, in a few minutes, dense ceramic bodies of oxide materials at low temperature [START_REF] Delorme | Effect of Ca substitution by Sr on the thermoelectric properties of Ca3Co4O9 ceramics[END_REF][START_REF] Imine | Bottom-up and new compaction processes: A way to tunable properties of nanostructured cobalt ferrite ceramics[END_REF][START_REF] Bah | High electromechanical performance with spark plasma sintering of undoped K0.5Na0.5NbO3 ceramics[END_REF][START_REF] Diaz-Chao | Textured Al-doped ZnO ceramics with isotropic grains[END_REF][START_REF] Guillon | Field-assisted sintering technology/spark plasma sintering: mechanisms, materials, and technology developments[END_REF][START_REF] Beynet | ZnO-based varistors prepared by spark plasma sintering[END_REF][START_REF] Delorme | Thermoelectric properties of oxygen deficient (K0.5Na0.5)NbO3 ceramics[END_REF][START_REF] Tõldsepp | Spark plasma sintering of ultra-porous γ-Al2O3[END_REF][START_REF] Delorme | Promising high temperature thermoelectric properties of dense Ba2Co9O14 ceramics[END_REF]. Therefore, SPS appears as a suitable technique to avoid or limit grain growth during sintering [START_REF] Imine | Bottom-up and new compaction processes: A way to tunable properties of nanostructured cobalt ferrite ceramics[END_REF].

Few controversial reports have been published on spark plasma sintering of undoped SnO2.

Park et al. [START_REF] Park | Enhanced densification of pure SnO2 by spark plasma sintering[END_REF] obtained dense ceramics (relative density > 95 %) from commercial and home-made SnO2 nanopowders at 1323 K under 37.5 MPa during 5 minutes under vacuum whereas at 1273 K in the same conditions, the relative density is only circa 72 %. Li et al. [START_REF] Li | Spark plasma sintering of undoped SnO2 ceramics[END_REF] have studied various sintering parameters on commercial SnO2 powders. At 1323 K under 37 MPa during 5 minutes under vacuum, they similarly found a relative density above 95 %. However, in the same conditions but at 1273 K, they already had a 95 % dense ceramic. They also reported that increasing the pressure (up to 40 MPa), or the heating rate (up to 200 K.min -1 ) leads to higher relative densities and that 3 minutes are enough to reach the plateau of highly dense materials.

Vo et al. [START_REF] Vo | Preparation and study of the thermoelectric properties of nanocrystalline Sn1-xTaxO2 (0 ≤ x ≤ 0.04)[END_REF] have reported a very different SPS cycle to sinter undoped SnO2 ceramics with relative density higher than 95 %. The sintering temperature is lower 1223 K and the pressure higher, 90 MPa during 5 minutes under an argon atmosphere. Moreover, they add first a dwell at 773 K during 2 minutes to reach the pressure. However, as their aim is to study thermoelectric properties of tantalum-doped SnO2 ceramics, they do not explain the reasons for such a cycle. Therefore, as the final aim of this article is to reach nanostructured dense SnO2 without sintering agent, the influence of spark plasma sintering cycle parameters on the density of undoped SnO2 ceramics will be first investigated. Then, grain size evolution will also be characterized as well as electrical and thermal conductivities of ceramics.

MATERIALS AND METHODS

Two kinds of SnO2 powders have been used for this study. The first one is from Aldrich (≥ 99.9 % purity, ≈ 325 mesh, 50-200 nm grain size) and is similar to the commercial powder used by Park et al. [START_REF] Park | Enhanced densification of pure SnO2 by spark plasma sintering[END_REF], and the second one was nanopowder synthesized according to the process described by Dujardin et al. [START_REF] Dujardin | A high yield one-pot aqueous synthesis of SnO2 nanoparticles[END_REF] (figure 1). Briefly, it consists in precipitating SnO2 nanoparticles (4-6 nm) from the dropwise addition of a 1 M NaOH aqueous solution into a 2 M SnCl4 aqueous solution at 373 K under reflux.

Sintering was performed by Spark Plasma Sintering (Syntex 515 S). The synthesised powders were placed in a 15 mm diameter graphite die (MERSEN -carbon grade 2333) in argon atmosphere. Samples were separated from punches and die by graphite foils. Several conditions have been tested, as detailed below. Note that for experiments with pressure higher than 100 MPa, high strength composite graphite dies were used (TOYO TANSO -carbon grade CX31V).

The obtained pellets were then polished to remove the graphite foils used during the SPS process and cut with a diamond wire saw as bars perpendicular to the pressure axis for electric properties measurements or thin 6 mm squares parallel to the pressure axis for thermal diffusivity measurements to prevent any orientation effect.

Thermoelectric properties of the sintered samples were determined from simultaneous measurement of resistivity and Seebeck coefficient in a ZEM-3 equipment (ULVAC Technologies) and thermal conductivity. Seebeck coefficient and electrical conductivity have been measured first from 323 to 1000 K. The thermal diffusivity was measured using the laser flash diffusivity technique (Netzsch LFA 457) from 373 to 1000 K in vacuum after graphite coating. The thermal diffusivity measurement of all specimens was carried out three times at each temperature. The heat capacity of the materials was measured from 300 to 1000 K, with a heating rate of 20 K.min -1 in platinum crucibles and in nitrogen atmosphere, using differential scanning calorimetry (Netzsch STA 449 F3 Jupiter).

Apparent density of the samples was calculated from the weight and dimensions of the bars cut from the pellets for ZEM-3 characterization and theoretical density [START_REF] Mccarthy | X-Ray diffraction data for SnO2. An illustration of the new powder data evaluation methods[END_REF].

Powder X-ray diffraction (XRD) patterns have been performed on a BRUKER D8 Advance θ/2θ diffractometer equipped with a Linxeye energy-dispersive one-dimensional detector, using Cu-Kα radiation and operating at 40 kV and 40 mA at room temperature. The scans have been recorded from 20 to 70°(2θ) with a step of 0.02° and a counting time of 0.5 s per step.

The scanning electron microscopy (SEM) observations have been performed using Tescan Mira 3 field emission microscope coupled with an Energy Dispersive Spectrometer (EDS Oxford INCA X-Act) without prior coating of the samples.

RESULTS AND DISCUSSION

As the first SnO2 nanopowder used in this study is similar to the one used by Park et al. [START_REF] Park | Enhanced densification of pure SnO2 by spark plasma sintering[END_REF], their SPS conditions were tried first, i.e. 1323 K under a pressure of 37.5 MPa during 5 minutes (heating and cooling rate of 100 K.min -1 ). At the end of the SPS cycle, small Sn beads appear on the top of the graphite die (figure 2). The XRD pattern measured on the pellet is shown in figure 3a) and also exhibits presence of metal Sn peaks. A cross section observed by SEM shows that metal tin (data not shown) can be observed on the surface of the pellet as well as in the core. The formation of metal tin is probably related to the reaction of SnO2 with graphite on the surface of the pellet. Moreover, it has already been noticed that graphite can diffuse within ceramics [START_REF] Meir | Synthesis and densification of transparent magnesium aluminate spinel by SPS processing[END_REF][START_REF] Morita | Distribution of carbon contamination in MgAl2O4 spinel occurring during spark-plasma-sintering (SPS) processing: I -Effect of heating rate and post-annealing[END_REF]. Indeed, replacing the graphite foil between the die and the sample by boron nitride or alumina powder allows to reduce the Sn amount but it is still possible to find some metal tin in the core of pellets. Moreover, similar results have been obtained for higher pressure (100 MPa) and with the nanoparticles synthesized using Dujardin et al.'s route [START_REF] Dujardin | A high yield one-pot aqueous synthesis of SnO2 nanoparticles[END_REF].

The influence of temperature decrease has been studied with the nanoparticles synthesized using Dujardin et al.'s route [START_REF] Dujardin | A high yield one-pot aqueous synthesis of SnO2 nanoparticles[END_REF]. The applied pressure is 100 MPa and the temperature dwell duration is 5 minutes with heating and cooling rate of 100 K.min -1 . PXRD patterns (figure 3b) show that at 1248 K, the peaks relative to Sn metal have disappeared. However, the phase is not pure SnO2 as some peak corresponding to SnO can be observed. At 1223 K, only the peaks related to SnO2 are present (figure 3c). This is the temperature used by Vo et al. [START_REF] Vo | Preparation and study of the thermoelectric properties of nanocrystalline Sn1-xTaxO2 (0 ≤ x ≤ 0.04)[END_REF].

Moreover, PXRD patterns also shows that when temperatures decrease, the peaks intensity decreases and the full-width half-maximum increases. According to SEM images (figure 4) no secondary phase was observed, confirming the XRD results. Moreover, a small porosity can be observed: this is consistent with a poor relative density of 76 %. On the contrary to the results presented by Vo et al. [START_REF] Vo | Preparation and study of the thermoelectric properties of nanocrystalline Sn1-xTaxO2 (0 ≤ x ≤ 0.04)[END_REF], the addition of a dwell at 773 K during 2 minutes before reaching 1223 K doesn't allow to achieve relative density higher than 95 %, but leads to unchanged density.

Therefore, the influence of the SPS parameters (pressure, duration of the high temperature dwell, heating/cooling rate), whereas the highest temperature remains constant (i.e. 1223 K) has been studied to reach higher densities. Figure 5 shows the influence of the duration of the high temperature dwell. It clearly appears that the density is not significantly improved even for long SPS experiments of 30 minutes. The best relative density achieved is 79 % for a 10 minutes dwell. Figure 6 shows the influence of the applied pressure: Density increases as pressure increases for low pressures (P < 100 MPa) but reach a plateau for higher values. The best density achieved is 81 % at 150 MPa. Figure 7 shows the influence of the heating/cooling rate: An optimum value clearly exists around 200 K.min -1 that allows reaching relative densities as high as 87 %. This effect of high heating rate on sintering has now been largely demonstrated [START_REF] Olevsky | Consolidation enhancement in spark-plasma sintering: Impact of high heating rates[END_REF][START_REF] Luo | Effects of surface diffusion and heating rate on first-stage sintering that densifies by grain-boundary diffusion[END_REF] but the reason for an optimum value remains unclear.

Individually, each optimized parameter does not allow to reach samples with relative densities of 95 % or higher. Therefore, they have been combined (Table 1). By combining the most influent heating/cooling rate and duration of the high temperature dwell, the following SPS cycle (Cycle A: 1223 K-100 MPa-10 min-200K.min -1 ) allows achieving 95 % relative density. One of the main interests of this sintering cycle is that working at a limited pressure, it is possible to use conventional graphite dies. Combining pressure and duration of the high temperature dwell, the following SPS cycle (Cycle B: 1223 K-150 MPa-10 min-100K.min -1 ) allows achieving 94 % density. However due to high pressure, a composite graphite die is required. Finally, Figure 8 As the aim is to reach nanostructured dense ceramics, the grain size of the different samples has been studied by measuring the grain size on SEM micrographs (figure 9) and estimating the size of coherent domain by applying the Scherrer formulae to the (101) peaks of the XRD patterns (figure 10). Figure 9 clearly shows that the samples are composed by grains smaller than 100 nm. Figure 10 shows that the value obtained from the Scherrer formulae is consistent with the median grain size measured from SEM micrographs. Moreover, as the density increases, the grain size increases. However, for the ceramics with the highest densities, the grain size is around 60-70 nm. Therefore, the dense ceramics obtained by SPS are nanostructured.

The Seebeck coefficient and electrical conductivity, from 330 to 1000 K, of the sample sintered at 1223 K-150 MPa-5 min-200K.min -1 are displayed in figure 11 a). The Seebeck coefficient shows a negative value which is consistent with the expected n-type conduction [START_REF] Chen | Recent advances in tin dioxide materials: some developments in thin films, nanowires, and nanorods[END_REF]. It improves from -234 to -290 μV.K -1 at 330 and 855 K, respectively. For higher temperatures, the absolute value slightly reduces. This is 30 % higher than the -195 µV.K -1 value measured by Vo et al. [START_REF] Vo | Preparation and study of the thermoelectric properties of nanocrystalline Sn1-xTaxO2 (0 ≤ x ≤ 0.04)[END_REF] at room temperature. The electrical conductivity increases on the whole temperature range which is characteristic of a semiconducting-like behaviour, from 550 to 1005 S.m -1 , at 330 and 1000 K, respectively. This is twice the value measured by Vo et al. [START_REF] Vo | Preparation and study of the thermoelectric properties of nanocrystalline Sn1-xTaxO2 (0 ≤ x ≤ 0.04)[END_REF]. The sample presents an unusual interesting feature: indeed, usually in semiconductors, when the electrical conductivity increases, the Seebeck coefficient decreases [START_REF] Guilmeau | Tuning the transport and thermoelectric properties of In2O3 bulk ceramics through doping at In-site[END_REF][START_REF] Diaz-Chao | Textured Al-doped ZnO ceramics with isotropic grains[END_REF][START_REF] Shakouri | Recent developments in semiconductor thermoelectric physics and materials[END_REF]. Similar behaviour can be observed in other compounds such as Ca3Co4O9 or delafossites [START_REF] Delorme | Effect of Ca substitution by Sr on the thermoelectric properties of Ca3Co4O9 ceramics[END_REF][START_REF] Koumoto | Thermoelectric properties of single crystal CuAlO2 with a layered structure[END_REF]. However, the origin of this behaviour in SnO2 remains unknown.

Therefore, the power factor (PF = S 2 σ) increases when temperature increases from 2.95 × 10 -5 W.m -1 .K -2 at 330 K to 7.75 × 10 -5 W.m -1 .K -2 at 1000 K (figure 11 b) ). This value at 1000 K is similar to the one reported by Yanagiya et al. [START_REF] Yanagiya | Thermoelectric properties of SnO2 ceramics doped with Sb and Zn[END_REF]. Temperature dependence of the thermal conductivity (κ) from 373 to 1000 K is shown in figure 11 c). The thermal conductivity of the SnO2 sample decreases when temperature increases from 6.59 W.m -1 .K -1 at 373 K to 3.99 W.m -1 .K -1 at 1000 K. These values can be compared to those reported for single crystals at room temperature that are 98 W.m -1 .K -1 when parallel to the c-axis and 55 W.m -1 .K -1 when perpendicular to c-axis [START_REF] Turkes | Thermal conductivity of SnO2 single crystals[END_REF] or in polycrystalline samples with micrometersized grains : between 25 to 40 W.m -1 .K -1 at room temperature for grains from 2.5 to 8 µm, and around 10 W.m -1 .K -1 at 1000 K [START_REF] Fayette | Influence of grain size on the thermal conductivity of tin oxide ceramics[END_REF]. This clearly demonstrates the ability of nanostructuring to significantly lower the thermal conductivity in oxides. Moreover, these values are slightly lower than the one recently reported in nanostructured Al-doped ZnO [START_REF] Giovannelli | Thermal conductivity and stability of Al-doped ZnO nanostructured ceramics[END_REF]. The study of the grain size shows that when the density increases the grain size increases to reach 60-70 nm for the high density samples. Therefore, SPS can be successfully used to produce dense nanostructured SnO2 ceramics without any sintering agent. The transport properties of the dense ceramics are the best reported for undoped SnO2. This is the first step to allow studies of electrical properties of doped SnO2 ceramics without the uncontrolled influence of the sintering agents. Moreover, nanostructuring is very efficient to lower thermal conductivity as values as low as 6.59 and 3.99 W.m -1 .K -1 at 373 and 1000 K respectively, are measured in SPS nanostructured ceramics.

FIGURE CAPTION

Table 1: Sintering parameters of the three cycles achieving high relative density. Relative density (%) Time (min)

  exhibits the three combined parameters (Cycle C: 1223 K-150 MPa-from 2 to 10 min-200K.min -1 ). It clearly allows reaching high relative densities even for time as short as 2 minutes. Finally, cycle A (1223 K-100 MPa-10 min-200K.min -1 ) has been used on the commercial powder and a density of 94 % has been achieved showing that these results are not only related to the very small size of the synthesized powders.

Figure 11 d

 11 Figure 11 d) exhibits the temperature dependence of the thermoelectric figure of merit ZT (= PF/κ): it increases when temperature increases and reach 0.02 at 1000 K.

Figure 1 :

 1 Figure 1: a) SEM image of the particles of the Aldrich SnO2 precursor, b) TEM image of

Figure 2 :

 2 Figure 2: Image of Sn beads appearing on the top of the graphite die after the SPS cycle.

Figure 3 :

 3 Figure 3: XRD pattern of the pellet sintered at a) 1323 K under a pressure of 37.5 MPa during 5 minutes (heating and cooling rate of 100K.min -1 ), b) 1248 K-100MPa-5min (heating and cooling rate of 100K.min -1 ), c) 1223 K-100MPa-5min (heating and cooling rate of 100K.min - 1 ).

Figure 4 :

 4 Figure 4: SEM image of the microstructure of the pellet sintered at 1223 K, under 100 MPa during 5 minutes.

Figure 5 :

 5 Figure 5: Relative density versus duration of the high temperature dwell of the SPS cycle (1223 K-100 MPa-100K.min -1 ).

Figure 6 :

 6 Figure 6: Relative density versus applied pressure during the SPS cycle (1223 K-5 min-100K.min -1 ).

Figure 7 :

 7 Figure 7: Relative density versus heating/cooling rate (1223 K-100 MPa-5 min).

Figure 8 :

 8 Figure 8: Relative density versus duration of the high temperature dwell of the SPS cycle (1223 K-150 MPa-200K.min -1 ).

Figure 9 :

 9 Figure 9: SEM micrograph of the sample sintered at 1223 K-150 MPa-2 min-200K.min -1 .

Figure 10 :

 10 Figure 10: Comparison of the grain size of the different samples obtained by SEM measurements and from the Scherrer formulae.

Figure 11 :

 11 Figure 11: Temperature dependence of a) the Seebeck coefficient and the electrical conductivity, b) the power factor, c) the thermal conductivity, d) the figure of merit, of the

Figure 1 22 Figure 2 Figure 3

 2223 Figure 1

  Figure 9

Table 1

 1

	Cycle	Maximum	Applied	Dwell	Heating/Cooling	Die	Relative
		Temperature	Pressure	Duration	Rates		Density
		(K)	(Mpa)	(min)	(K.min -1 )		(%)
	A	1223	100	10	200	Conventional Graphite	95
	B	1223	150	10	100	Composite Graphite	94
	C	1223	150	from 2 to 10	200	Composite Graphite	from 94 to 96

Acknowledgments

The authors acknowledge ADEME (Agence De l'Environnement et de la Maîtrise de l'Energie) for the financial support. The authors are grateful to use the SPS platform of Ile-de-France (France) and would like to thank B. Villeroy (ICMPE-CNRS, France) for his technical expertise. The authors acknowledge the technical assistance of P.Y. Raynal from the plateforme des microscopies (microscopy center of University of Tours) for SEM images.


Aleksandra Burdziej
email: aleksandra.burdziej@biol.uw.edu.pl

Cezary Pączkowski
email: c.pączkowskimyhacp@biol.uw.edu.pl

Agnes Destrac-Irvine
email: irvineagnes.destrac-irvine@inra.fr

Tristan Richard
email: richardtristan.richard@u-bordeaux.fr

Stéphanie Cluzet
email: s.cluzetstephanie.cluzet@u-bordeaux.fr

Anna Szakiel
email: szakal@biol.uw.edu.pl

Agnès Destrac-Irvine

Triterpenoid profiles of the leaves of wild and domesticated grapevines

Keywords: Vitis spp., Grapevine, Triterpenoids, Steroids, GC-MS/FID

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Grapevine (Vitis spp.) is one of the most ancient cultivated plants playing a primary role in culture and economics of many countries. Several crucial incidences in the history of its significantly contributed to the polymorphism of the present-day grapevines. A considerable diversity in the biosynthetic abilities of its varieties is linked to this heterogeneity. A majority of the studies on Vitis plants have focused on polyphenolic compounds, particularly the stilbenes which are considered as the key defensive secondary compounds (phytoalexins) produced by grapevine in response to microbial infections. In contrast, information on triterpenoids in grapevine is rather scarce. Nevertheless, several authors have reported the presence of triterpenoid compounds as constituents of cuticular waxes in different varieties of Vitis spp. leaves [START_REF] Batovska | Seasonal variations in the chemical composition of vinegrape leaf surface[END_REF][START_REF] Batovska | Biomarkers for the prediction of the resistance and susceptibility of grapevine leaves to downy mildew[END_REF][START_REF] Batovska | GC-MS analysis of the leaf surface components of four Bulgarian grapevines grown under different conditions[END_REF][START_REF] Berli | Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultravioletabsorbing compounds, antioxidant enzymes and membrane sterols[END_REF][START_REF] Özer | Investigations on the role of cuticular wax in resistance to powdery mildew in grapevine[END_REF]Pensec et al., 2016). A role of triterpenoids in plant defense responses to biotic and abiotic factors is suggested as the cuticular waxes are considered to be the first line of protection towards different stresses. The modifications in the content of the membrane-related triterpenoids, i.e. sterols, and some pentacyclic triterpenes like lupeol, α-and β-amyrins, were shown to be associated with environmental adaptation of grapevine during seasonal variations and to UV radiations as an indirect consequence of abscisic acid (ABA) signalling [START_REF] Berli | Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultravioletabsorbing compounds, antioxidant enzymes and membrane sterols[END_REF][START_REF] Batovska | Seasonal variations in the chemical composition of vinegrape leaf surface[END_REF][START_REF] Batovska | Biomarkers for the prediction of the resistance and susceptibility of grapevine leaves to downy mildew[END_REF][START_REF] Gil | Metabolism of terpenes in the response of grape (Vitis vinifera L.) leaf tissues to UV-B radiation[END_REF]. The seasonal accumulation of triterpene monohydroxy alcohols is suggested to be associated with the repulsion of microorganisms attacking in the autumn [START_REF] Batovska | Biomarkers for the prediction of the resistance and susceptibility of grapevine leaves to downy mildew[END_REF]. Indeed, the toxicity of αamyrin, lupeol, and taraxerol towards insects was reported and it may result from the ability of these molecules to inhibit acyl chain packing in lipid bilayers (Rodríguez et al., 1997). Some triterpenoids, as α-amyrin, lupeol, stigmasta-3,5-diene-7-one, are proposed as biomarkers for the prediction of resistance to grapevine pathogens, such as Plasmopara viticola or Erysiphe necator, responsible of downy and powdery mildews, respectively [START_REF] Batovska | Seasonal variations in the chemical composition of vinegrape leaf surface[END_REF][START_REF] Batovska | Biomarkers for the prediction of the resistance and susceptibility of grapevine leaves to downy mildew[END_REF]. However, triterpenoids are generally regarded as constitutive plant defense molecules (i.e. phytoanticipins) rather than phytoalexins (Pensec et al., 2016).

The aim of the present study was a comparison of triterpenoid content in the whole leaf tissue of wild grapevine crops used as progenitors or rootstocks of domesticated vines (Vitis aestivalis Michx., Vitis labrusca L., Vitis riparia Michx., and Vitis vinifera subsp. sylvestris (Gmelin) Hegi), and of typical cultivated Vitis vinifera varieties from different French vineyards (Cabernet Sauvignon, Gamay, Marselan, and Merlot). Crop wild relatives (CWRs) constitute a unique genetic resource, essential for the improvement and creation of the "new" grapevine thanks to their potential in production diversity. This material is also often at the origin of the rootstock varietal creations, as wild Vitis from North America for their ability to provide pathogen or pests resistance. The domesticated V. vinifera plants used in this study are cultivated in the frame of the "Vitadapt" project designed to an extensive phenotyping of a wide range of varieties in order to optimize the use of genetic diversity within the Vitis species as a tool for adaptation to a changing environment [START_REF] Van Leeuwen | Modified grape composition under climate change conditions requires adaptations in the vineyard[END_REF][START_REF] Cortázar-Atauri | Grapevine phenology in France: from past-observations to future evolutions in the context of climate change[END_REF]. The wild grapevines belong to a collection of INRA (Villenave d'Ornon) in order to preserve these species. The obtained results supplement existing data about steroid and triterpenoid profile of different Vitis spp., particularly in the whole leaf tissue, and can contribute to the explanation of variability among grapevine species in the context of their chemical characterization. The information about the steroid and triterpenoid content in grapevine leaves might help to better understand their role in plant defense and could support their application in traditional food and in folk medicine as a remedy for the treatment of diarrhea, vomiting, and some circulatory system diseases [START_REF] Orhan | Biological activities of Vitis vinifera L. leaves[END_REF][START_REF] Pacifico | Antioxidant polyphenolic constituents of Vitis x Labruscana cv. dipalmitoylphosphatidylcholine bilayers[END_REF]. The numerous biological activities of this nutritial matrix are mainly attributed to phenolic compounds, and the investigation of potential pharmacological effect of triterpenoids is neglected, although some of these molecules possess diverse antimicrobial and cytotoxic properties [START_REF] Shai | Four pentacyclic triterpenoids with antifungal and antibacterial activity from Curtisia dentata (Burm.f) C.A. Sm. Leaves[END_REF].

Results and discussion

Identification of steroids and triterpenoids occurring in leaves of Vitis spp.

A GC-MS analysis of the fractions containing steroids and neutral pentacyclic triterpenoids obtained from diethyl ether extracts of leaves of different Vitis plants was performed. Representative chromatograms are shown in Figure 1. They revealed rather similar general profile in "steroid and triterpenoid range" (retention time higher than 32 min in the applied program). The identification of the compounds associated with individual peaks revealed the occurrence of several steroids: cholesterol (1) (tR of 31.41 min), campesterol (2) (tR of 34.26 min), stigmasterol (3) (tR of 35.19 min), sitosterol (5) (tR of 37.28 min) as well as pentacyclic compounds of ursane-, oleanane-, lupane-and friedooleanane (taraxerane)-type skeletons, i.e. α-amyrin (11) (tR of 40.05 min), β-amyrin (8) (tR of 38.29 min), lupeol (10) (tR of 40.05 min) and taraxerol (7) (tR of 37.62 min), respectively. Lupeol (10) and α-amyrin (11) were identified as a mixture associated with one common peak, since they are not well separated on the applied GC-MS column, as described earlier (Pensec et al., 2016). According to the observed MS distribution, lupeol several times prevailed α-amyrin. One of the intermediates of sterol biosynthetic pathway, 24-methylenecycloartanol (12) (tR of 41.87 min), is also found in detectable amounts in all analyzed extracts. In two extracts obtained from wild grapevine species: V. labrusca L. and V. riparia Michx sitosterol was accompanied by its saturated form, sitostanol (6) (tR of 37.41 min). Two esters, which cofractionated with free triterpenoids, i.e. sitosterol and lupeol acetates (9 and 13, tR of 39.11 min and tR of 42.93 min, respectively), were identified in the majority of analyzed extracts. In turn, the sitosterol γisomer, clionasterol (4) (tR of 36.92 min), was found exclusively in V. vinifera subsp. sylvestris (Gmelin) Hegi. In some extracts, traces of ursane-type ketone, α-amyrenone, were detected, however, in amounts too low for FID quantitation. The chemical structures of the major identified compounds are shown in Figure 2. Other numerous peaks, with retention times up to 30 min and displaying various intensities, were associated with aliphatic or phenolic compounds. Two peaks of tR 30.03 and 32.05 min were identified as typical phenol/diterpenoid antioxidants: γ-and α-tocopherol, respectively.

The obtained results generally confirm the reported findings of several previous studies on triterpenoids occurring in grapevine leaves [START_REF] Batovska | Seasonal variations in the chemical composition of vinegrape leaf surface[END_REF][START_REF] Batovska | Biomarkers for the prediction of the resistance and susceptibility of grapevine leaves to downy mildew[END_REF][START_REF] Batovska | GC-MS analysis of the leaf surface components of four Bulgarian grapevines grown under different conditions[END_REF][START_REF] Berli | Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultravioletabsorbing compounds, antioxidant enzymes and membrane sterols[END_REF], Pensec et al., 2016). In the study performed on profiling of grapevine leaf cuticular waxes of eight V. vinifera cultivars from the Upper Rhine Valley (Pensec et al., 2016), additionally cycloartanol, oleanolic aldehyde as well as stigmasta-3,5-dien-7-one were found. However, the cited work had been done on cuticular waxes, which facilitated chromatographic fractionation and permitted easier and more precise identification of compounds due to lower levels of contaminants -by contrast to the present study made on extracts obtained from entire leaves. It was previously demonstrated that the compounds present in very low amounts, which could be found in cuticular wax extracts, were not detectable in extracts obtained from entire plant organs (Szakiel et al., 2012b).

Although the composition of steroids and triterpenoids was similar in all wild and domesticated grapevines studied, the various intensity of individual peaks associated with identified compounds suggested the significant differences in their content among the analyzed species and cultivars.

Quantitative analysis of triterpenoids occurring in leaves of Vitis spp.

The results of quantitative determination of steroids and triterpenoids in extracts of studied wild and domesticated grapevine leaves are presented in Tables 1 and2, respectively. In addition, Figure 3 shows quantitative profiles of pentacyclic triterpenoids occurring in grapevine leaves.

Generally, the total content of analyzed compounds ranged from 1.0 mg/g of dry leaf weight in domesticated Merlot cultivar to 2.7 mg/g in wild V. labrusca L. The average total content was lower in leaves of domesticated cultivars (approx. 1.7 mg/g, ranging from 1.0 to 2.4 mg/g) than in wild grapevines (approx. 2.0 mg/g, ranging from 1.6 to 2.7 mg/g).

The total content of sterols ranged from 0.6 mg/g of dry leaf weight in extract from Merlot to 1.7 mg/g in Marselan. The average sterol content was similar in leaves of wild and domesticated grapevines and equalled approx. 1 mg/g. However, the content of sterols in wild grapevines seems to be more stable and uniform among species, since the fluctuation between the lowest and the highest content did not exceed 0.2 mg, that is, 16% of the highest amount.

On the contrary, the difference between the lowest and the highest content of sterols in domesticated grapevines exceeded 1 mg, that is, 63% of the highest amount. Sitosterol was the most abundant compound in the sterol profile of all analyzed grapevines, constituting from 63% of the total sterol content in V. labrusca L. to 80% in Gamay cultivar. The smallest amount of sitosterol was detected in V. vinifera subsp. sylvestris (Gmelin) Hegi (only 41% of the total sterol content), however, as it was described above, in this species also the sitosterol γ-isomer, clionasterol, was found in remarkable amount, and together these two isomers constituted 72% of the total sterol content. The second abundant sterol in the wild species was stigmasterol, with the exception of V. labrusca L., in which 24-methylenecycloartanol was prevailing. In the domesticated species, the second abundant compound among steroids was 24-methylenecycloartanol, whereas stigmasterol was prevailing only in Cabernet Sauvignon cultivar. The content of the third phytosterol, campesterol, was in average more than twice higher in wild grapevines (approx. 0.037 mg/g) than in domesticated cultivars (0.015 mg/g).

The total content of pentacyclic triterpenoids ranged from 0.36 mg/g of dry leaf weight in Merlot cultivar to 1.5 mg/g in wild V. labrusca L., and it was generally much lower in the domesticated grapevines (approx. 0.7 mg/g) than in the wild species (approx. 0.9 mg/g). In the majority of the wild grapevines analyzed in this study, lupeol was the predominant pentacyclic compound, constituting (as a mixture with α-amyrin) from 45% of total pentacyclic triterpenoids in V. vinifera subsp. sylvestris (Gmelin) Hegi to 75% in V. labrusca L. The only exception among wild grapevines was V. riparia Michx., with only 13% of lupeol, and β-amyrin as the prevailing pentacyclic compound (82% of total pentacyclic triterpenoids).

Among the domesticated grapevines, lupeol is prevailing only in one cultivar, Gamay (the mixture of lupeol and α-amyrin constituting 67% of total pentacyclic triterpenoids). In Cabernet Sauvignon, Marselan and Merlot, taraxerol was predominating (46, 58 and 62% of the sum of pentacyclic triterpenoids, respectively). The content of β-amyrin ranged from approx. 0.11 mg/g in Merlot to 0.19 mg/g in Cabernet Sauvignon, and it was not the dominant compound in any of domesticated cultivars. Interestingly, a similar level of taraxerol was found in domesticated Vitis vinifera cultivars and in their oldest wild ancestor V. vinifera subsp. sylvestris (Gmelin) Hegi.

Conclusions

The dendrogram of hierarchical clustering on principal components (HCPC) of the quantified steroids and triterpenoids in all samples (Fig. 4) suggests that the analyzed group of molecules may be an important phenotypic trait of Vitis plants. Indeed, two main types of clusters can be observed: the first one grouped the domesticated grapevines together, and the other showed strong similarities of steroid and triterpenoid profiles among the wild ones.

Furthermore, the results seem to predict phylogenetic relationships of domesticated V. vinifera cultivars. As instance, Cabernet Sauvignon and Merlot were clustered together which may be due to their common parent Cabernet Franc [START_REF] Boursiquot | Parentage of Merlot and related winegrape cultivars of southwestern France: discovery of the missing link[END_REF]. Unexpectedly, V. vinifera cv. Marselan was grouped apart from Merlot and Cabernet Sauvignon, although the latter is involved in its parentage. Perhaps, the genetic background of Grenache, which is another parent of Marselan, prevails in terms of steroids and triterpenoids against the remaining cultivars of its origin (http://www.vivc.de). In turn, Gamay (Pinot Noir x Heunisch Weiss (also named Gouais Blanc)) was grouped along with Cabernet Sauvignon and Merlot, even if a close kinship of these cultivars was undetected [START_REF] Terral | European cultivars[END_REF]). An advanced study of hybrid domesticated grapevines varieties and their respective parents is needed in order to verify an importance of steroid and triterpenoid profile in the determination of grapevines origins.

Moreover, the results obtained in the current study confirm that the leaves of numerous domesticated grapevines showed the profile of pentacyclic triterpenoids, which are generally considered as secondary metabolites, with predominating lupeol or taraxerol. For example, the division of eight cultivars from the Upper Rhine Valley into lupeol-or taraxerolrich groups was reported previously (Pensec et al., 2016). These differential triterpene contents could not be directly correlated to differential susceptibility to some pathogens, since the two groups show similar levels of susceptibility to P. viticola and E. necator, however, some observations could indicate that the cultivars with high lupeol contents were more resistant to Esca, a grapevine trunk disease, than those with a high taraxerol content (Pensec et al., 2016). Concerning sterols, in some species the profile of these compounds were demonstrated to be an important feature in the context of tolerance to abiotic stress as they are substantial constituents of cells membranes playing function in their structure and dynamics [START_REF] Wang | Differential profiles of membrane proteins, fatty acids, and sterols associated with genetic variations in heat tolerance for a perennial grass species, hard fescue (Festuca Trachyphylla)[END_REF]Posé et al., 2009). A relatively higher amount of sterols in the wild grapevines comparing with the domesticated ones reported herein, may partially explain their general insusceptibility to drought, high/low temperature, UV radiation, or salinity.

The content of steroids and triterpenoids in the grapevines leaves is comparable with those from other plant species. However, the amount of these molecules can be significantly higher, e.g., in leaves of evergreen plants like lingonberry (Vaccinium vitis-idaea, Ericaceae), or much lower, e.g., in leaves of herbal plants like marigold (Calendula officinalis, Asteraceae) (Szakiel et al., 2012a;[START_REF] Niżyński | The content of free and esterified triterpenoids of the native marigold (Calendula officinalis) plant and its modifications in in vitro cultures[END_REF]. Thus, the biosynthetic ability in the production of triterpenoids varies in plants which can reflect the participation of these compounds in defenses. Nevertheless, a possible direct correlation between steroid and triterpenoid profile of Vitis plants and their resistance to common pathogens and extreme environmental conditions is worth further investigations.

Experimental

Plant material

The four wild Vitis spp. cultivars of interest (V. aestivalis Michx., V. labrusca L., V. riparia For each cultivar, ten mature fully expanded leaves (approximately leaves at the sixth position from the apex, and located always in the same orientation in the vines) were collected in October 2016 from plants with no evidence of disease or stress symptoms. The samples were freezed at -80°C for at least 72 h, and then lyophilized before extraction.

Extraction and fractionation

The preparation of samples was performed according to the method used by Szakiel et al. (2012a,b). The freeze-dried plant material (1 g) was grounded to a fine powder in a grinding mortar, and extracted with 100 ml of diethyl ether (99.5%, POCH S.A., Gliwice, Poland) in Soxhlet apparathus for 8 h. The obtained extracts were evaporated to dryness at 40°C under reduced pressure, then fractionated by prep. TLC on 20 cm x 20 cm glass plates coated with a 0.25 mm layer of silica gel 60G (Merck, Darmstadt, Germany) in a solvent system CHCl3/MeOH (97:3, v/v). The fractions containing free (non-esterified) neutral steroids and triterpenoids (alcohols, aldehydes and ketones) were localized on plates by comparison with standard of sitosterol and α-amyrin. The standards were visualized by spraying the relevant part of the plate with 50% H2SO4, followed by heating with a hot-air stream. Subsequently, the fraction was eluted from the gel with diethyl ether and directly analyzed by GC-MS/FID.

Identification and quantification of steroids and triterpenoids by GC-MS/FID

The analyses were performed using an Agilent Technologies 7890A gas chromatograph (Perlan Technologies, Warsaw, Poland) coupled to 5975C mass spectrometric detector and equipped with a flame ionization detector, FID. All the samples were dissolved in diethyl ether/methanol (5:1, v/v) and a volume of 1-4 μl was applied by split injection 1:10. The GC separation was made on a HP-5MS UI column of 30 m x 0.25 mm i.d., film thickness 0.25 μm (Agilent Technologies) in the temperature programme: initial temperature of 160°C held for 2 min, then increased to 280°C at 5°C/min and the final temperature of 280°C held for further 44 min with constant helium flow rate of 1 ml/min. For GC-MS/FID detection the following parameters were employed: inlet and a flame ionization detector (FID) temp. 290 ºC, MS transfer line temp. 275 ºC, quadrupole temp. 150 ºC, ion source temp. 230 ºC, EI 70eV, m/z range 33-500; FID-H2 flow 30 ml/min (hydrogen generator), air flow 400 ml/min. The individual compounds were identified by comparing their mass spectra with library data from Wiley 9th ED. & NIST 2008Lib. SW (Version 2010) or data from the literature, and by comparison of their retention times and corresponding mass spectra with those of authentic standards: α-amyrin (Roth, Karlsruhe, Germany); β-amyrin, campesterol, sitosterol, and stigmasterol (Sigma-Aldrich Steinheim, Germany). The identified compounds were quantified with the use of a flame ionization detector (FID). In case of coelution, the manual peak integration was applied for precise measurement of peak areas. Quantitation was made using the external standard method based on calibration curves determined for typical representatives of each triterpenoid group: α-amyrin for monohydroxyalcohols, and stigmasterol for steroids. Calibration curves were drawn between the peaks areas versus the concentration of α-amyrin (linear regression equation y=45800x-300) and stigmasterol (y=35900x-550) in the range of 0.002-2.0 mg/ml. The correlation coefficient (r 2 ) values of αamyrin was 0.999 and for stigmasterol 0.998. RSD values of peak areas were less than 4%.

The LOD (Limit of Detection) was determined by analysis of samples with decreasing level of concentration of analyte, value for α-amyrin was 0.8 μg/ml, for stigmasterol 1.0 μg/ml. A 2.5 μg/ml and 3.2 μg/ml limit of quantification (LOQ) was attained for α-amyrin and stigmasterol, respectively.

Statistical analysis of data

The results presented consist of the mean values ± standard deviation (s. Results are referenced to leaf dry weight and expressed in µg g -1 as the means ± S.D. of three independent samples analysed in triplicate. Results are referenced to leaf dry weight and expressed µg g -1 as the means ± S.D. of three independent samples analysed in triplicate.

  Michx. and V. vinifera subsp. sylvestris (Gmelin) Hegi were introduced from North America and France between 1963 and 2000 at St Louis experimental field in Villenave d'Ornon, France. The vineyard belongs to INRA-Bordeaux germplasm collection and is managed by the UMR EGFV (Villenave d'Ornon, France). Descriptive sheets (e.g. voucher specimens) of wild Vitis plants can be found at https://www.bordeaux.inra.fr/ampelobase. The four V. vinifera varieties (Cabernet Sauvignon, Gamay, Marselan and Merlot) were planted in 2009 in the "Vitadapt" experimental field and cultivated by the INRA and the ISVV, Villenave d'Ornon, France. All plants were similarly grafted and are managed by conventional methods.

  d.) obtained from samples analyzed in triplicates. Hierarchical clustering on principal components (HCPC) with Manhattan distance measure was performed in R software version 3.4.3 using the FactoMineR plugin.

Fig. 1 .

 1 Figure captions: Fig. 1. Representative GC-FID chromatograms of the fraction containing steroids and neutral triterpenoids from diethyl ether leaf extracts of grapevines. Cholesterol (1); campesterol (2); stigmasterol (3); clionasterol (4); sitosterol (5); sitostanol (6); taraxerol (7); β-amyrin (8); sitosterol acetate (9); lupeol/α-amyrin (10+11); 24-methylenecycloartanol (12); lupeol acetate (13).

Fig. 2 .

 2 Fig. 2. Chemical structures of steroids and triterpenoids identified in Vitis spp. leaf diethyl ether extracts: cholesterol (1), campesterol (2), stigmasterol (3), clionasterol (4), sitosterol (5), sitostanol (6), taraxerol (7), β-amyrin (8), sitosterol acetate (9), lupeol (10), α-amyrin (11), 24methylenecycloartanol (12), lupeol acetate (13).

Fig. 3 .

 3 Fig. 3. Quantification of pentacyclic triterpenoids in grapevine leaf diethyl ether extracts based on GC-MS/FID analysis.

Fig. 4 .

 4 Fig. 4. Cluster dendrogram performed by hierarchical clustering on principal components (HCPC) showing the discrimination of wild and domesticated grapevines Vitis spp. based on the composition of triterpenoids in leaves diethyl ether extracts.

Table 1 .

 1 Triterpenoid content in the leaves of Vitis aestivalis Michx., Vitis labrusca L., Vitis riparia Michx. and Vitis vinifera subsp. sylvestris (Gmelin) Hegi

	Compound	V.	V.	V. riparia	V.
		aestivalis	labrusca	Michx.	vinifera
		Michx.	L.		subsp.
					sylvestris
					(Gmelin)
					Hegi
			(µg g -1 d.w. ± S.D.)
	Steroids:
	Cholesterol (1)	37.51 ±	44.41 ±	29.31 ±	49.12 ±
		5.63	6.66	4.40	7.37
	Campesterol (2)	45.14 ±	42.90 ±	24.62 ±	38.25 ±
		4.51	4.29	2.46	3.82
	Stigmasterol (3)	134.58 ±	85.74 ±	90.70 ±	106.93 ±
		12.11	7.72	8.16	9.62
	Clionasterol (4)				310.14 ±
					46.52
	Sitosterol (5)	735.69 ±	714.83 ±	822.07 ±	419.73 ±
		73.57	71.48	82.21	41.97
	Sitostanol (6)		55.02 ±	32.77 ±
			8.25	4.92
	Sitosterol acetate (9)	10.30 ±		21.01 ±	70.66 ±
		0.67		3.15	10.60
	24-Methylenecycloartanol	49.57 ±	179.46 ±	52.00 ±	20.94 ±
	(12)	5.95	21.53	6.24	2.51
	Sum	1 012.79	1 122.35	1 072.48	1015.77
	Pentacyclic triterpenoids:
	Taraxerol (7)	49.70 ±	14.23 ±	26.33 ±	269.35 ±
		7.45	2.13	3.95	40.40
	β-Amyrin (8)	143.23 ±	363.23 ±	546.48 ±	218.82 ±
		12.89	32.69	49.18	19.69
	Lupeol/α-Amyrin (10+11)	369.16 ±	1 172.94	88.37 ±	397.19 ±
		40.61	± 129.02	9.72	43.69
	Lupeol acetate (13)	3.04 ±	8.00 ±	3.75 ±	6.95 ±
		0.24	0.64	0.30	0.56
	Sum	565.13	1 558.39	664.93	892.31
	Total	1 577.92	2 680.75	1 737.41	1 908.08

Table 2 .

 2 Triterpenoid content in the leaves of Vitis vinifera cv. Cabernet Sauvignon, Gamay, Marselan,

	and Merlot
	Compound	V. vinifera	V. vinifera	V. vinifera	V. vinifera
		cv.	cv.	cv.	cv. Merlot
		Cabernet Sauvignon	Gamay	Marselan
			(µg g -1 d.w. ± S.D.)
	Steroids:
	Cholesterol (1)	20.17 ±	33.91 ±	50.50 ±	31.90 ±
		3.02	3.05	7.57	4.78
	Campesterol (2)	11.72 ±	13.60 ±	23.42 ±	12.47 ±
		1.17	1.22	2.34	1.25
	Stigmasterol (3)	54.77 ±	50.74 ±	77.15 ±	45.66 ±
		4.93	4.57	6.94	4.11
	Sitosterol (5)	537.24 ±	643.68 ±	1 141.06 ±	486.77 ±
		53.72	64.37	114.11	48.68
	Sitosterol acetate (9)	58.45 ±	11.43 ±	156.56 ±	16.41 ±
		2.92	0.86	14.09	1.64
	24-Methylenecycloartanol	33.48 ±	52.45 ±	267.11 ±	49.03 ±
	(12)	4.02	4.72	8.16	5.88
	Sum	715.83	805.80	1 715.80	642.24
	Pentacyclic triterpenoids:
	Taraxerol (7)	366.49 ±	145.77 ±	384.14 ±	226.66 ±
		54.97	14.58	57.62	34.00
	β-Amyrin (8)	193.28 ±	168.84 ±	145.03 ±	105.15 ±
		17.40	16.88	13.05	9.46
	Lupeol/α-Amyrin (10+11)	220.14 ±	651.78 ±	91.65 ±	23.83 ±
		24.22	65.18	10.08	2.62
	Lupeol acetate (13)	16.42 ±		46.48 ±	10.18 ±
		1.31		3.72	0.81
	Sum	796.34	966.40	667.30	365.82
	Total	1 512.17	1 772.20	2 383.09	1 008.06

Acknowledgements

We thank the Institut National de la Recherche Agronomique (INRA) Bordeaux-Aquitaine for their kind permission for collecting wild grapevine leaves in their vineyards.

The study was performed owing to the financial support of the French Government and the French Embassy in Poland.

Analyses were carried out with the use of CePT infrastructure financed by the European Union-the European Regional Development Fund (Agreement POIG.02.02.00-14-024/08-00).

Conflict of interest

All authors declare that they have no conflict of interest.


Anna-Lena Herbig
email: anna-lena.herbig@mail.de

Nicolas Delchier
email: nicolas.delchier@gmail.com

Lisa Striegel
email: lisa.striegel@tum.demichael.rychlik@tum.de

Michael Rychlik

Catherine M G C Renard
email: catherine.renard@inra.fr

Stability of 5-methyltetrahydrofolate in fortified apple and carrot purées

Keywords: folate, fortification, food matrix, degradation, impacts

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.

Introduction

Folic acid fortification is linked to a reduced risk of neural tube defects in newborns-and thus became mandatory in the United States and other countries for cereal products (Eichholzer, Tonz, & Zimmermann, 2006;Grosse & Collins, 2007). In addition, the insufficient intake of folates is associated with a higher colorectal cancer risk (Kim, 2003). Folic acid, a synthetic vitamer, is predominantly used for fortification and is transformed in the body into naturally occurring forms.

When ingested in high doses, however, it also circulates in its unmetabolised form [START_REF] Smith | Is folic acid good for everyone?[END_REF]. Furthermore, folic acid fortification in excess (>1000µg/day) can mask the vitamin B 12 deficiency. Thus 5-methyltetrahydrofolate is naturally abundant form in the body, it has been proposed as an alternative fortificant [START_REF] Pietrzik | Folic acid and L-5-methyltetrahydrofolate[END_REF][START_REF] Scaglione | Folate, folic acid and 5-methyltetrahydrofolate are not the same thing[END_REF].

Concerning its chemical structure, 5-methyltetrahydrofolate differs from folic acid by being reduced in positions N(5), C(6) and C(7), and N(8), while hydrogen in position N(10) is substituted by a methyl group (Delchier, Herbig, Rychlik, & Renard, 2016;[START_REF] Scott | Folic acid and folates: The feasibility for nutritional enhancement in plant foods[END_REF]. This structural difference has an enormous effect on stability. Compared to folic acid, 5-methyltetrahydrofolate is highly susceptible to oxidation. In the absence of oxygen, 5-methyltetrahydrofolate is completely stable (Delchier et al., 2014). The stability of 5-methyltetrahydrofolate under aerobic conditions decreases with rising temperatures (Indrawati et al., 2004;[START_REF] Nguyen | Model studies on the stability of folic acid and 5methyltetrahydrofolic acid degradation during thermal treatment in combination with high hydrostatic pressure[END_REF][START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF] and depends on pH. It is more stable in a neutral rather than in an acidic medium (Indrawati et al., 2004;Liu et al., 2012). At the same pH, its stability depends additionally on the buffer solution used (Indrawati et al., 2004;[START_REF] Paine-Wilson | Thermal destruction of folacin -effect of pH and buffer ions[END_REF]).

The degradation of 5-methyltetrahydrofolate is accelerated by fructose but not by glucose [START_REF] Verlinde | Influence of reducing carbohydrates on (6S)-5-methyltetrahydrofolic acid degradation during thermal treatments[END_REF]. The impact is independent of the sugar concentration in the range from 1.6 mmol/L to 1.5 mol/L and is linked to a new product that is formed by the glycation of the exocyclic amino group of 5-methyltetrahydrofolate (Figure 1). This product is formed along with the three degradation products that are also generated in the absence of fructose (Figure 1). The acceleration of degradation by a fructose is counteracted by ascorbic acid, probably by reduction of the first oxidation product, 5methyl-7,8-dihydrofolate. Liu et al. (2012) and [START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF] reported that ascorbic acid decreases the degradation of 5methyltetrahydrofolate . [START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF] related the concentration of ascorbic acid necessary to protect 5-methyltetrahydrofolate to the initial oxygen concentration. The concentration of ascorbic acid that led to a complete protection between 50 °C and 170 °C for 15 minutes was claimed to be twice as high as the molar initial oxygen concentration (Liu et al., 2012;[START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF]. However, the relationship can be supposed to be valid only in the analysed time range as ascorbic acid is also susceptible to degradation and thus, the protection effect will be lost once the ascorbic acid is degraded.

Almost all groups who studied the stability of 5-methyltetrahydrofolate, used model solutions: Paine-Wilson and Chen (1979) worked with McIlvaine, HCl/KCl and citrate buffers; [START_REF] Nguyen | Model studies on the stability of folic acid and 5methyltetrahydrofolic acid degradation during thermal treatment in combination with high hydrostatic pressure[END_REF] with phosphate buffer; [START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF] with phosphate buffer; Verlinde et al. (2010) with water andLiu et al. (2012) with HEPES/CHES buffer. [START_REF] Mnkeni | Thermal destruction of 5-methyltetrahydrofolic acid in buffer and model food systems[END_REF] reported a faster degradation of 5-methyltetrahydrofolate in tomato and apple juices than in a citrate buffer. When comparing buffers and different foods, Indrawati et al. (2004) observed but did not explain different behaviours. Delchier et al. (2014) used spinach and green bean purées but only modulated oxygen.

Therefore, the mutual influence of factors (pH, oxygen, ascorbic acid, sugars) with the complex composition and physico-chemistry of foods has hardly been taken into account. In particular, dissolved oxygen in real food products may also be consumed by other oxidisable compounds, which may change the relationship. For example, 500 mg/kg of ascorbic acid increase the stability of 5methyltetrahydrofolate in carrot juice but not in asparagus when heat-treated for 40 minutes at 120 °C (Indrawati et al., 2004). The stability difference of 5-methyltetrahydrofolate in skim milk and soy milk has been linked to the different antioxidant capacities of the two food systems (Liu et al., 2012).

For the present study, apple purée and carrot purée were used as fortified food matrices since they are usually less heat-treated than cereal products and are well appreciated all over Europe. They are representative fruit and vegetable foods with different pHs, fructose contents, and antioxidants. The aim was to find suitable reheating conditions in the temperature range of 60-80 °C to impart high stability to 5-methyltetrahydrofolate in apple purée and carrot purée.

Material and methods

Chemicals

Formic acid, 2-(N-morpholino)-ethanesulfonic acid (MES) hydrate, 2,2′-Bipyridyl, ascorbic acid, trichloroacetic acid, DL-dithiothreitol, Na 2 HPO 4 , NaH 2 PO 4 •H 2 0, N-Ethylmaleimide, folic acid and citric acid monohydrate were purchased from Sigma Aldrich (Steinheim, Germany), and acetonitrile, methanol, ortho-phosphoric acid (85%), iron (III) chloride hexahydrate and ascorbic acid from VWR (Darmstadt, Germany). Applichem GmbH (Darmstadt, Germany) provided 2,3-Dihydroxybutan-1,4dithiol; and [ 13 C 5 ]-5-methyltetrahydrofolate, [ 13 C 5 ]-folic acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium acetate trihydrate and acetic acid were supplied by Merck (Darmstadt, Germany). Sodium hydroxide and sodium chloride were obtained from J.T. Baker (Deventer, the Netherlands) and ethanol from Fisher Scientific (Fair Lawn, NJ, USA). 5-Methyltetrahydrofolate was purchased from Schircks (Jona, Switzerland).

Fortification and thermal treatment

The formate buffer was attained by diluting formic acid (10 mL/L) with distilled water and adding sodium hydroxide to the solution until a pH of 3.5. Apple purée (brand: ALNATURA) and carrot purée (brand: HIPP) without additives were purchased at a supermarket in Freising, Germany. In order to attain a concentration of 4.0 µmol/kg of 5-methyltetrahydrofolate and folic acid, the dilution steps were carried out as follows: The 5-methyltetrahydrofolate and folic acid were pre-dissolved in a phosphate buffer (pH 7.0) and diluted with distilled water to a volume of 100 mL; Subsequently, 10 mL of this solution were added to 40 g of apple purée and carrot purée and thoroughly vortexed.

Homogeneity was tested by comparing the recovery amount in two aliquots of two independently prepared fortification mixtures. Amber glass vials (4 mL) were filled with 2 mL of a formate buffer solution containing 4.0 µmol/kg of 5-methyltetrahydrofolate, or up to the corresponding height in the case of fortified apple and carrot purées, and closed with screw caps. Thermal treatments were carried out in a stirred silicon oil bath. Temperatures at the bottom and in the middle were verified and showed a homogenous temperature distribution over time. After taking the tubes out of the silicon oil bath, the tubes were immediately deep-frozen (≤ -18 °C). Before analysis, glass vials were put in a water bath for rapid thawing. The first point of every time curve was omitted from the heat treatment.

Folate analysis

The stable isotope dilution assay of [START_REF] Ringling | Analysis of seven folates in food by LC-MS/MS to improve accuracy of total folate data[END_REF] was employed with the modification that only the vitamer 5-methyltetrahydrofolate was analysed and 13 C-instead of 2 H-labelled standards were taken for quantification. Shortly after an equilibration of the isotopic marked internal standards with the sample, the extraction of folates followed. Before analysis by HPLC-MS/MS, the samples were purified.

Instrumental conditions were identical. A HyperClone Reversed Phase Column C18 (Phenomenex, Aschaffenburg, Germany) was used for separations. Values were first calculated as calcium salt. The percental aberration of a fixed standard concentration of 200 µg/100 g was then calculated and was on average 7 ±7 %. Each point of a time curve was subsequently corrected by the percental aberration of the first value to the standard concentration, to avoid parallelism of kinetics. The values were then transferred to their actual 5-methyltetrahydrofolate content by taking into consideration the calcium proportion, and converted to their molar concentration by respecting a molar mass of 459 g/mol.

Ascorbic acid analysis

Ascorbic acid quantification was carried out by means of the colorimetric method of [START_REF] Stevens | Technique for rapid, smallscale analysis of vitamin C levels in fruit and application to a tomato mutant collection[END_REF]. Modifications were the following: approximately 500 mg of the food sample were taken for extraction, and absorption was measured at 525 nm on a spectrophotometer (Safas Xenius, Monaco).

Results and discussion

Natural folate concentration, extraction yield and test of homogeneity

Apples and carrots are food matrices that contain inherently low amounts of folates. Raw apples do not contain any measurable folates amounts and raw carrots only contain 19 µg/100 g of folates (USDA, 2016). The amount of 5-methyltetrahydrofolate monoglutamate in the purchased apple purée was under the limit of quantification for the analytical method, which is 0.22 µg/100 g [START_REF] Ringling | Analysis of seven folates in food by LC-MS/MS to improve accuracy of total folate data[END_REF]. The concentration of 5-methyltetrahydrofolate in carrot purée was 2.5 ± 0.1 µg/100 g which was in the range listed on the USDA platform for raw carrot (19 µg/100 g, USDA, 2018).

Natural amounts of 5-methyltetrahydrofolate in both matrices were thus negligible compared to the target fortification level of 185 µg/100 g corresponding to 4.0 µmol/kg. Folic acid is a synthetic, notnaturally-occurring vitamer, and is thus not present in apple and carrots.

In order to verify the homogeneity of fortification preparations, the apple purée was supplemented with folic acid. Recovery rates accounted to 107 ± 5 % and 109 ± 1 %, which were determined by analysing two aliquots of two independently prepared fortification mixtures. Extraction was thus quantitative and homogeneity was acceptable, indicated by low standard deviations.

Stability in buffer solution and apple purée

The stability of the folic acid and 5-methyltetrahydrofolate was first evaluated in a formate buffer (10 mL/L, pH 3.5) heated at 80 °C. The daily folate amount recommended by the European Food Safety Authority (EFSA) depends on the origin of the folate, as natural folates are less bioavailable than folic acid (EFSA, 2014). A dietary folate equivalent (DFE) is usually applied to convert folic acid amounts to the concentration that would be ingested if natural folates were absorbed. One µg of DFE is defined as 1 µg food folate, which in turn corresponds to 0.6 µg of folic acid from fortified food. This conversion factor was considered when preparations were carried out and it results in differing initial values depending on the vitamer used (Figure 2). Consistent with literature results (Delchier et al., 2016;[START_REF] O'broin | Nutritional stability of various naturally occurring monoglutamate derivatives of folic acid[END_REF][START_REF] Paine-Wilson | Thermal destruction of folacin -effect of pH and buffer ions[END_REF], folic acid was more stable than 5-methyltetrahydrofolate (Figure 2A). Folic acid dissolved in a formate buffer was completely stable at 80 °C for the duration of 180 minutes while in turn 5-methyltetrahydrofolate degraded entirely within 15 minutes. The stability of folates depends on ions (Indrawati et al., 2004;[START_REF] Paine-Wilson | Thermal destruction of folacin -effect of pH and buffer ions[END_REF]); hence direct comparison of degradation rates with literature results is not possible as other buffer solutions were used. However, the degradation of 5-methyltetrahydrofolate occurred within the same time range as previously reported, registering a complete loss within some minutes [START_REF] O'broin | Nutritional stability of various naturally occurring monoglutamate derivatives of folic acid[END_REF][START_REF] Paine-Wilson | Thermal destruction of folacin -effect of pH and buffer ions[END_REF]).

According to literature, the stability of 5-methyltetrahydrofolate for 15 minutes up to 170 °C can be obtained by adding ascorbic acid [START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF]. In the present study, the addition of 2840 µmol/kg of ascorbic acid induced complete stability of 5-methyltetrahydrofolate in the formate buffer for three hours at 80 °C (Figure 2A). The apple purée contains fructose, which is known to accelerate the degradation of folates [START_REF] Verlinde | Influence of reducing carbohydrates on (6S)-5-methyltetrahydrofolic acid degradation during thermal treatments[END_REF], but also polyphenols that may potentially exhibit a protective effect as a result of their anti-oxidative properties. Therefore, the stability of folic acid and 5-methyltetrahydrofolate was also assessed in apple purée (Figure 2B). As in the formate buffer, folic acid was completely stable over the investigated time range and 5-methyltetrahydrofolate degraded rapidly (Figure 2B). [START_REF] Mnkeni | Thermal destruction of 5-methyltetrahydrofolic acid in buffer and model food systems[END_REF] found that 5-methyltetrahydrofolate is also fairly unstable in apple juice. When 2840 µmol/kg of ascorbic acid were added, the complete stability of 5methyltetrahydrofolate was also attained in the apple purée. Naturally present polyphenols in the apple purée did not lead to the same stability as the addition of 2840 µmol/kg of ascorbic acid. Ascorbic acid completely protected the 5-methyltetrahydrofolate in the apple purée even in the presence of a high amount of fructose. This is coherent with the result of [START_REF] Verlinde | Influence of reducing carbohydrates on (6S)-5-methyltetrahydrofolic acid degradation during thermal treatments[END_REF], who studied the stability of 5-methyltetrahydrofolate (0.04 mmol/L) in the presence of fructose (0.04 mmol/L) and ascorbic acid (1.13 mmol/L) in a model solution which was heated for 45 minutes at 100 °C.

Thus, 5-methyltetrahydrofolate, in the presence of 2840 µmol/kg of ascorbic acid is equally as suitable for fortification as folic acid. Both vitamers withstood long periods of heating that can be encountered when food is reheated and kept warm for several hours. Until now, flour products have been exclusively chosen for mandatory folic acid fortification in the United States. However, folic acid, a fairly stable folate vitamer, is lost during the bread baking process (Gujska & Majewska, 2005).

Neither folic acid nor 5-methyltetrahydrofolate in the presence of 2840 µmol/kg of ascorbic acid were lost in the apple purée under the reported conditions. Thus, this study suggests that with regard to stability, apple purée exhibits an advantageous fortification matrix compared to flour that is baked into bread.

Concentration effect of ascorbic acid on the stability of 5-methyltetrahydrofolate tested in apple and carrot purées

The amount of ascorbic acid (2840 µmol/kg) used for the assessment above was added in excess when compared to the content of 5-methyltetrahydrofolate (4.0 µmol/ kg). It was also much higher than the concentration of dissolved oxygen, which was not measured but is of 258 µmol/kg in water at 25 °C and is probably even lower under the studied conditions, given the matrices and temperatures used [START_REF] Penicaud | Oxygen quantification methods and application to the determination of oxygen diffusion and solubility coefficients in food[END_REF]. In the next phase, lower amounts of ascorbic acid were added to the apple purée so as to verify if stability was still maintained. However, the addition of neither 570 µmol/kg nor 1420 µmol/kg led to the absolute stability of 5-methyltetrahydrofolate within the investigated time range (Figure 3A). When adding 570 µmol/kg of ascorbic acid, degradation started within 60 minutes and amounted to 30 ± 1 % after 180 minutes and in the case of 1420 µmol/kg, it initiated within 120 minutes and reached 17 ± 3 % after 180 minutes. However, once degradation started, the depletion pace for both ascorbic acid concentrations was still slower than when no vitamin C was added. In order to examine whether the loss of complete preservation of 5methyltetrahydrofolate was linked to a loss of ascorbic acid, the concentration of the latter was followed over the time period analysed (Figure 3B). However, the amounts of ascorbic acid still present when the degradation of 5-methyltetrahydrofolate started were quite high. [START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF] reported that even low amounts of ascorbic acid (284 µmol/L) are sufficient to completely protect 5-methyltetrahydrofolate (4 µmol/L) at 80 °C. From these results, it can be concluded that besides the amount of ascorbic acid present in the medium, another time-dependent factor seems to crucially influence the stability of 5-methyltetrahydrofolate. It might be that dissolved oxygen is involved in this phenomenon. Dissolved oxygen was consumed within one hour in apple purée heated at 80 °C under the conditions of Herbig, Maingonnat, and Renard (2017), but this was not tested in the present work due to the difficult access of the oxygen sensor to the medium in the experimental set-up. The degradation of 5-methyltetrahydrofolate does not proceed when the medium is deprived of oxygen before the heat treatment (Delchier et al., 2014). It may also be that the generation of reactive oxygen species might as well be implicated in this phenomenon. Boatright (2016) measured up to 32.5 µmol/L of hydrogen peroxide during the oxidation of 100 µmol/L of ascorbic acid. The formation of hydrogen peroxide and other reactive oxygen species during the oxidation of ascorbic acid and possibly also other components in the apple purée medium, might explain the time-limited protective effect of ascorbic acid. A longer protection with increasing amounts of ascorbic acid is consistent with the assumption of reactive oxygen species being involved, as the probability rises that reactive oxygen species encounter an ascorbic acid molecule before a 5methyltetrahydrofolate one.

Another explanation might also be an implication of sugar derivatives. In model solutions, ascorbic acid protects 5-methyltetrahydrofolate in the presence of fructose (100 °C, 45 minutes) by reducing 5methyldihydrofolate, the first oxidation product (Figure 1) [START_REF] Verlinde | Influence of reducing carbohydrates on (6S)-5-methyltetrahydrofolic acid degradation during thermal treatments[END_REF]. In an apple purée, the protective effect might, however, be counteracted by an accumulation of sugar degradation products in the course of time and consequently explain the shifted degradation initiation of 5methyltetrahydrofolate. In addition, since some ascorbic acid degradation products, namely dehydroascorbic acid and 2,3-diketogulonic acid, are also dicarbonyls that are even more reactive Maillard precursors than sugars, they might participate in the shifted degradation beginning [START_REF] Ortwerth | Ascorbic acid induced crosslinking of lens proteins -evidence supporting a Maillard reaction[END_REF][START_REF] Reihl | Characterization and detection of lysine-arginine cross-links derived from dehydroascorbic acid[END_REF][START_REF] Roig | Studies on the occurrence of nonenzymatic browning during storage of citrus juice[END_REF][START_REF] Slight | Glycation of lens proteins by the oxidation products of ascorbic acid[END_REF].

In future studies, it might therefore be interesting to study the combined effect of the amount of oxygen in the headspace, dissolved oxygen, the appearance of reactive oxygen species, sugar derivatives and ascorbic acid derivatives, in order to understand the interactions more profoundly.

Until these interactions are understood in detail, degradation kinetics cannot be extrapolated to other experimental set-ups.

As the composition of the food matrix may potentially influence the stability of 5methyltetrahydrofolate, the latter was subsequently supplemented to carrot purée so as to verify possible stability changes. As with the apple purée, when no ascorbic acid was added, the 5methyltetrahydrofolate was very fragile and degraded rapidly. Its stability in carrot purée in the presence of 570 µmol/kg and 2840 µmol/kg of ascorbic acid was not different from that of the apple purée when the same amount of ascorbic acid was added (Figure 3C). Stability was attained with the presence of 2840 µmol/kg of ascorbic acid but not when only 570 µmol/kg were used for fortification.

Coherently, [START_REF] Mnkeni | Thermal destruction of 5-methyltetrahydrofolic acid in buffer and model food systems[END_REF], who studied the stability of fortified folic acid in apple and tomato juices (100-140 °C), did not observe any difference between degradation paces in the two studied food media despite their different compositions. In accordance with results obtained for the apple purée and with those of Indrawati et al. ( 2004), who worked with carrot juice (500 mg/kg of ascorbic acid) heated at 120 °C for 40 minutes, the presence of ascorbic acid in carrot purée was linked to a high stability of 5-methyltetrahydrofolate.

Temperature effect (60-80 °C)

Temperature diminishes the stability of 5-methyltetrahydrofolate in a model solution according to the Arrhenius equation (Indrawati et al., 2004;[START_REF] Nguyen | Model studies on the stability of folic acid and 5methyltetrahydrofolic acid degradation during thermal treatment in combination with high hydrostatic pressure[END_REF][START_REF] Oey | Temperature and pressure stability of Lascorbic acid and/or [6S] 5-methyltetrahydrofolic acid: A kinetic study[END_REF]. However, the extent depends on the pH used and on the buffer ions (Indrawati et al., 2004;[START_REF] Paine-Wilson | Thermal destruction of folacin -effect of pH and buffer ions[END_REF]. It was hypothesised that stability for 3 h could be reached in apple purée with only 570 µmol/kg of ascorbic acid by decreasing temperature. However, when the temperature was decreased from 80 °C to 70 °C or to 60 °C, the stability did not increase at the same time (Figure 4). Apparently, the supply of energy was not the limiting factor for the degradation pace in this temperature range. Indrawati et al. ( 2004) studied the impact of temperature on the stability of 5-methyltetrahydrofolate in orange juice, kiwi purée, carrot juice and asparagus. In the mentioned study, orange juice and kiwi purée contained 500 mg/kg of ascorbic acid i.e. 2840 µmol/kg, and carrot juice and asparagus contained 100 mg/kg of ascorbic acid, corresponding to 570 µmol/kg. Consistent with the results of this study, they observed no marked impact between the amount that degraded at 70 °C or at 80 °C in the different food matrices.

Conclusion

The heat susceptibility of 5-methyltetrahydrofolate was confirmed in apple and carrot purées.

Polyphenols and carotenoids that could, due to their anti-oxidative properties, potentially enhance the stability of 5-methyltetrahydrofolate are far from being as effective as the addition of vitamin C. Stability can only be obtained in the presence of vitamin C. The vitamin C amount which is necessary to entirely protect 5-methyltetrahydrofolate depends on the intended duration of the heat treatment.

The length of time of complete stability can be prolonged by increasing the vitamin C concentration.

The molar vitamin C concentration is not directly related to the concentration of 5methyltetrahydrofolate. Even if the amount of ascorbic acid is still in excess considering the amount of 5-methyltetrahydrofolate or dissolved oxygen, degradation starts in the course of time. A timedependent factor seems to intervene. Decreasing temperature in the range of 60-80 °C does not have a marked impact on the stability of 5-methyltetrahydrofolate. Thus, by heating purées at 80 °C, the risk of microbial growth can be decreased while the same nutritional value is maintained as if heated at 60 °C. Apple purée and carrot purée in the presence of an adequate amount of vitamin C are thus suitable food matrices for folate fortification. (1: 5-methyl-5,6,7,8-tetrahydrofolate, 2: 5-methyl-7,8-dihydrofolate, 3: p-aminobenzoyl glutamic acid, 4: 2-amino-8-methyl-4,9-dioxo-7-methyl-p-aminobenzoylglutamate-6,7,8,9- A: Formate buffer (10 mL/L, pH 3.5)

Figure captions

Fig. 1 .

 1 Fig. 1. Degradation mechanism of 5-methyltetrahydrofolate in the presence sugars.

  tetrahydro-4Hpyrazino(1,2-a)-s-triazine, 5 : unknown reaction products, 6 : N(2α)-[1-(carboxyethyl)]-5-methyl-5,6,7,8-tetrahydrofolic acid.) Postulated and adapted from the version from Verlinde et al. (2010).

Fig. 2 .

 2 Fig. 2. Degradation kinetics at 80 °C in a formate buffer and apple purée.

  Figure 1 :

  Figure 3:

Figure 4 :

 4 Figure 4:

Acknowledgement

This project has received funding from the European Union's Seventh Framewok Programme OPTIFEL for research, technological development and demonstration under grant agreement n°311754.