Jany Dandurand

Vale ´rie Samouillan
email: valerie.samouillan@univ-tlse3.fr

Colette Lacabanne

Antonietta Pepe

Brigida Bochicchio

Á V Samouillan

Á C Lacabanne

Á B Bochicchio

Water structure and elastin-like peptide aggregation A differential calorimetric approach

Keywords: Elastin, Hydrophilic hydration, Hydrophobic hydration, Clathrate, Differential scanning calorimetry

For the first time, calorimetric studies performed at very low temperature highlighted the preliminary and required conditions for further aggregation/coacervation of peptides from elastin in solution through the structural water reorganization around the peptides. For this purpose, we firstly characterized by turbidimetry and differential scanning calorimetry the synthetic S4 fragment peptide containing the XGGZG motif (where X and Z correspond to Valine or Leucine) which is able to form amyloid fibres under certain conditions. We also investigated two mediumsized elastin-related model elastin peptides containing the VGVPG motif (E50 and E18) as well as their analogues where proline is hydroxylated in hydroxyproline. These peptides were shown to coacervate, and a close correlation was found between the inverse transition temperature obtained by turbidimetry and the clathrate-like structures evidenced at low temperature by the calorimetric method.

Introduction

Surface hydration is closely connected to the structural stability and flexibility of proteins, and its knowledge is essential for a better understanding of biological activities and pathologies. An important goal concerning elastin research is to explain its ability to extend and contract in repetitive motion in the hydrated state. Forty years of research on the dynamics of elastin are reviewed by Muiznieks et al. [START_REF] Muiznieks | Structural disorder and dynamics of elastin[END_REF] emphasizing the crucial role of the structural disorder of both the peptide chain and the surrounding water in the mechanism of elasticity that can be well described by the entropy-driven model [START_REF] Debelle | Elastin: molecular description and function[END_REF][START_REF] Urry | Elastin: a representative ideal protein elastomer[END_REF]. Because of the extreme insolubility of elastin, most of the investigations are carried out on tropoelastin, its soluble precursor. The primary structure of tropoelastin consists of crosslinking regions-where the lysyl residues are locatedalternating with large hydrophobic domains [START_REF] Wise | Tropoelastin[END_REF] that were shown to be responsible for elasticity [START_REF] Tamburro | Dissection of human tropoelastin : exon-by-exon chemical synthesis and related conformational studies[END_REF]. Tamburro's group synthesized a large series of polypeptides sequences encoded by the distinct exons of human tropoelastin and evidenced labile conformations by circular dichroism (CD) and nuclear magnetic resonance (NMR) depending on the experimental conditions and environment. These polypeptides alternate between different conformations in equilibrium, giving rise to a conformational ensemble that includes a-helices (favoured in structuring solvent like TFE), folded sequences b turns and b strands [START_REF] Bisaccia | The amino acid sequence coded by the rarely expressed exon 26A of human elastin contains a stable beta-turn with chemotactic activity for monocytes[END_REF] and the more extended polyproline II (PPII) [START_REF] Tamburro | Localizing alpha-helices in human tropoelastin: assembly of the elastin ''puzzle[END_REF][START_REF] Bochicchio | Dissection of human tropoelastin: solution structure, dynamics and self-assembly of the exon 5 peptide[END_REF]. PPII structures are defined by an absence of intramolecular and intermolecular hydrogen bonds and can, therefore, interconvert between conformations. Nevertheless, even if the great majority of these polypeptides sequences form organized structures, only a few of them are able to coacervate [START_REF] Urry | Temperature of polypeptide inverse temperature depends on mean residue hydrophobicity[END_REF][START_REF] Reiersen | Short elastin-like peptides exhibit the same temperature-induced structural transitions as elastin polymers: implications for protein engineering[END_REF] or to irreversibly aggregate into amyloid structure [START_REF] Pepe | Dissection of human tropoelastin: supramolecular organization of polypeptide sequences coded by particular exons[END_REF][START_REF] Ostuni | Molecular and supramolecular structural studies on human tropoelastin sequences[END_REF][START_REF] Bochicchio | Investigating the amyloidogenic nanostructured sequences of elastin: sequence encoded by exon 28 of human tropoelastin gene[END_REF]. Coacervation, the initial step of fibrillogenesis in native tropoelastin [START_REF] Wise | Tropoelastin: a versatile, bioactive assembly module[END_REF], is an entropy-driven process due to the cooperative interactions between hydrophobic domains [START_REF] Luan | Differential scanning calorimetry studies of NaCl effect on the inverse temperature transition of some elastin-based[END_REF][START_REF] Luan | Hydrophobicity of amino acid residues: differential scanning calorimetry and synthesis of the aromatic analogues of the polypentapeptide of elastin[END_REF]. Below the coacervation temperature, hydrophobic peptides are certainly shielded by clathrate-like water which prevents the self-aggregation. At the coacervation temperature, these clathrate-like structures would be disrupted, and the self-interaction of hydrophobic domain becomes possible [START_REF] Yeo | Coacervation of tropoelastin[END_REF]. Coacervation phenomenon and the associated inverse temperature transition are affected by the amino acid sequences [START_REF] Ribeiro | Influence of the amino-acid sequence on the inverse temperature transition of elastin-like polymers[END_REF], the residue hydrophobicity [START_REF] Urry | Temperature of polypeptide inverse temperature depends on mean residue hydrophobicity[END_REF], the number of proline residues and their spacing in the elastin-like polypeptides [START_REF] Muiznieks | Proline periodicity modulates the self-assembly properties of elastin-like polypeptides[END_REF], and by the introduction of electronegative groups [START_REF] Pepe | Effect of proline analogues on the conformation of elastin peptides[END_REF]. Nevertheless, if these structural requirements are essential for the coacervation process [START_REF] Maeda | Structural requirements essential for elastin coacervation: favorable spatial arrangements of valine ridges on the threedimensional structure of elastin-derived polypeptide (VPGVG)n[END_REF], the coacervation temperature is also largely influenced by experimental parameters such as pH, solution concentration and NaCl content [START_REF] Vrhovski | Coacervation characteristics of recombinant human tropoelastin[END_REF][START_REF] Reguera | Effect of NaCl on the exothermic and endothermic components of the inverse temperature transition of a model elastin-like polymer[END_REF]. Moreover, the hydroxylation of proline in (2S,4R)-4-hydroxyproline (Hyp) is one of the most frequent post-translational modification in the proteins of the extracellular matrix-like collagen and elastin. In elastin, the exact role of this modification is not yet understood, and a very recent work showed that the self-assembly of some elastin polypeptides was significantly altered by the presence of Hyp, evidencing different supramolecular structures [START_REF] Bochicchio | Investigating the role of (2S,4R)-4-hydroxyproline in elastin model peptides[END_REF].

If hydrophobic elastin sequences are in general optimized to avoid an amyloid fate, studies have evidenced the propensity of some elastin sequences to form amyloid fibres [START_REF] Miao | Sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin[END_REF]. These authors suggested that the substitution of the rigid proline amino acids with the more flexible glycine residues accounts for this irreversible precipitation [START_REF] Rauscher | Proline and glycine control protein self-organization into elastomeric or amyloid fibrils[END_REF]. This precipitation depends on some parameters such as temperature, time and hydration level, and the main structures observed before aggregation are the b conformations which act as seeds for b sheet structures [START_REF] Bochicchio | Investigating the amyloidogenic nanostructured sequences of elastin: sequence encoded by exon 28 of human tropoelastin gene[END_REF][START_REF] Pepe | Formation of nanostructures by self-assembly of an elastin peptide[END_REF]. To highlight the propensity of Glycine-rich motif to amyloid forming, Salvi et al. have extensively studied the influence of amino acid specificities or solvation on the molecular and supramolecular organization by CD, NMR, AFM and XPS [START_REF] Salvi | Influence of amino acid specificities on the molecular and supramolecular organization of glycine-rich elastin-like polypeptides in water[END_REF][START_REF] Salvi | Combined effects of solvation and aggregation propensity on the final supramolecular structures adopted by hydrophobic, glycinerich, elastin-like polypeptides[END_REF]. Elastin-like or amyloid-like peptides adopt complex supramolecular structures, showing either hydrophobic or hydrophilic surfaces. Molecular simulations of the monomeric and aggregated states strongly evidence that elastin-like and amyloid-like peptides have distinctive backbone hydration and peptide-peptide hydrogen bonding [START_REF] Rauscher | Proline and glycine control protein self-organization into elastomeric or amyloid fibrils[END_REF]. More generally, the self-assembly molecules domain is of rising interest, involving the construction of smart materials able to perform specific functions at the molecular scale. If the self-assembly of organic nanostructures is mainly driven by weak intermolecular forces (van der Waals forces, hydrogen bonding and hydrophobic effect), strong intermolecular interaction such as ionic covalent and coordination bonds can also play a central role in the formation of self-assembled materials, enhancing the dynamic properties of the final supramolecule [START_REF] Dhotel | Molecular motions in functional self-assembled nanostructures[END_REF].

Differential scanning calorimetry (DSC) has proven to be a useful technique to study phase transitions in protein mixtures [START_REF] Ostrowska-Lige ˛za | An assessment of various powdered baby formulas by conventional methods (DSC) or FT-IR spectroscopy[END_REF], protein denaturation in biological compounds [START_REF] Briere | Measurement of protein denaturation in human synovial fluid and its analogs using differential scanning calorimetry[END_REF], aggregation and self-assembly of proteins and peptides [START_REF] Suh | Detection and characterization of hemoglobin dissociation and aggregation using microcalorimetry[END_REF][START_REF] Tine | Effect of temperature on self-assembly of an ionic tetrapeptide[END_REF]; it is well suited for quantifying freezable water in hydrated elastin [START_REF] Samouillan | Effect of water on the molecular mobility of elastin[END_REF][START_REF] Samouillan | Hydrated elastin: dynamics of water and protein followed by dielectric spectroscopies[END_REF], for revealing the physical structure of collagen and elastin in cardiovascular tissues [START_REF] Samouillan | Analysis of the molecular mobility of collagen and elastin in safe, atheromatous and aneurysmal aortas[END_REF] and for analyzing recurring sequences of elastin such as pentapeptide GLGGV [START_REF] Megret | Phase transitions and chain dynamics, in the solid state, of a pentapeptide sequence of elastins[END_REF], poly(GVGVP) [START_REF] Rodriguez-Cabello | Structural investigation of the poly(pentapeptide) of elastin, poly(GVGVP), in the solid state[END_REF] and the synthetic S4 fragment [START_REF] Dandurand | Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: signature of the amyloid fibers[END_REF] in the condensed state. Finally, it also runs in solution, evidencing for example the hydrophobic hydration of poly(VPGVG) [START_REF] Rodrıguez-Cabello | Differential scanning calorimetry study of the hydrophobic hydration of the elastin-based polypentapeptide poly(VPGVG), from deficiency to excess of water[END_REF] or the thermal behaviour of exon 6 [START_REF] Tintar | Human tropoelastin sequence: dynamics of polypeptide coded by exon 6 in solution[END_REF].

In this work, we propose to compare the thermal behaviour of water in the solutions of distinct peptides from elastin that either coacervate or form amyloid-like structures to gain insight into the role of water in the mechanism of aggregation.

Experimental

Polypeptide synthesis and purification

Five polypeptides (Table 1) were synthesized by solid-phase methodology using an automatic synthesizer APPLIED BIOSYSTEM model 431 A. Fmoc/DCC/HOBT chemistry was used starting from (0.25 mmol) Wang resin (Nova Biochem Laufelfingen, Switzerland) for S4, E50, E50H polypeptide syntheses. In the case of E18 and E18H polypeptides having a proline at the C-terminus, the amidated polypeptides were synthesized using (0.25 mmol) Rink-amide resin (Novabiochem, Laufelfingen, Switzerland) to avoid side reactions. The Fmoc-amino acids were purchased from Novabiochem (Laufelfingen, Switzerland) and from Inbios (Pozzuoli, Italy). The cleavage of the peptides from resin was achieved by an aqueous mixture of 95 % trifluoroacetic acid (TFA). The peptides were lyophilised and purified by reversed-phase high-performance liquid chromatography (HPLC). A binary gradient was used, and the solvents were H 2 O (0.1 % TFA) and CH 3 CN (0.1 % TFA). The purity of peptides was assessed by electrospray mass spectrometry.

Turbidimetry

Experiments were performed on a Cary 50 UV spectrophotometer equipped with a Peltier temperature controller using quartz cells of 1 cm path length and reported as turbidimetry on apparent absorbance (TAA). The solution temperature was increased from 10 to 90 °C with 2 °C every 5 min and then decreased back to 10 °C, monitoring the absorbance under stirring after 5 min to reach the temperature equilibrium. S4 peptides were dissolved in TBS buffer [Tris (50 mmol L -1 ), NaCl (1.5 mol L -1 ) and CaCl 2 (1.0 mmol L -1 ) (pH 7.0)] to a concentration of 2 g L -1 , and the absorbance was monitored at 440 nm. The E50, E50H, E18 and E18H peptides were dissolved in TBS buffer (pH 7.5) to a concentration of 100 g L -1 , and the absorbance was monitored at 400 nm.

Differential scanning calorimetry (DSC)

The DSC curves were recorded with a Pyris Diamond calorimeter (PERKIN ELMER). The calorimeter was calibrated using cyclohexan and indium as standards. Solution samples (15 lL, corresponding to a sample mass of 15 mg) were sealed in hermetic aluminium pans, and an empty pan was used as reference. All the curves were recorded with a cooling or heating rate of 10 °C min -1 . The concentration of S4 peptide was 30 g L -1 (*17 mmol L -1 ) in TBS buffer. The concentration of polypeptides E50, E50H, E18 and E18H was 100 g L -1 (*25 mmol L -1 ) in TBS buffer.

Results and discussion

TBS buffer

In order to determine the origin of the different transitions of the peptides/TBS buffer systems, a preliminary thermal characterization of the buffer alone was performed. The DSC cooling and heating curves of the TBS buffer in the -80/60 °C temperature range are presented in Fig. 1.

On the cooling scan are observed 2 exothermic peaks at -20 and -45 °C. As previously observed in saline solutions, these events are ascribed to the two-phase transitions of the TBS buffer [START_REF] Kamasa | DSC approach for the investigation of mobile water fractions in aqueous solutions of NaCl and Tris buffer[END_REF], namely the freezing of primary ice (intense signal) and the freezing of the eutectic phase, respectively. On the heating scan are observed two endothermic transitions, namely the melting of the eutectic phase at -22 °C and the melting of primary ice at 0 °C [START_REF] Kamasa | DSC approach for the investigation of mobile water fractions in aqueous solutions of NaCl and Tris buffer[END_REF]. It is noteworthy that the twophase transitions recorded on cooling are shifted towards low temperature when compared with the transitions recorded on heating due to the supercooling of the liquids.

S4 in TBS buffer

In order to investigate the aggregation properties of the S4 peptide, turbidimetry experiments were carried out as a function of temperature (Fig. 2). The aggregation is experimentally measured by spectrophotometry as a sharp increase in the apparent absorbance value concomitant with heating to a critical temperature. These experiments are generally performed in TBS buffer, since the presence of ions makes the repulsive force between peptides charges decrease and promotes association, resulting in an increased tendency of the peptides to aggregate by agitation [START_REF] Sasahara | Thermal response with exothermic effects of beta2-microglobulin amyloid fibrils and fibrillation[END_REF].

As previously reported [START_REF] Bochicchio | Amyloidogenesis of proteolytic fragments of human elastin[END_REF], the turbidity measurements showed that S4 peptide undergoes an irreversible aggregation triggered by the temperature. The temperature at which the aggregation started was 60 °C and reached the highest value at 90 °C. After that, cooling the solution did not reduce the TAA. Combined with microscopy and conformational studies [START_REF] Dandurand | Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: signature of the amyloid fibers[END_REF][START_REF] Bochicchio | Amyloidogenesis of proteolytic fragments of human elastin[END_REF], these findings strongly suggest for S4 an irreversible phase transition compatible with an amyloid-like behaviour.

DSC Curves of S4 in TBS buffer (30 g L -1 ) were recorded in the cooling mode (Fig. 3) after a previous heating ramp up to the indicated temperature. On the whole, cooling scans are observed 3 exothermic peaks at -70, -50 and -20 °C. As previously shown, the events occurring at -20 and -50 °C originate from the TBS buffer.

As for the exothermic peak observed at -70 °C, undetectable in TBS buffer alone, it arises from the combination between solvent and S4 peptide. The low-temperature value of this transition is rather unusual when compared with literature data on the thermal characterization of hydrated biomolecules. It is also noteworthy that a very restricted number of DSC studies have been performed in this temperature range for biological systems, and none of the studies have been presented in this temperature range on such peptides/solution systems. Nevertheless, a quite similar thermal transition is detected for the crystallization of water adsorbed in clays [START_REF] Kozlowski | Low temperature exothermic effects on cooling of homoionic clays[END_REF] or activated carbon [START_REF] Vm | Interfacial behavior of polar, weakly polar, and nonpolar compounds bound to activated carbons[END_REF] in the -60/-50 °C temperature range. In the case of hydrated hydrophilic polymers, loosely bound water exhibits a crystallization phenomenon with considerable supercooling in the -50/-40 °C range [START_REF] Hatakeyama | Interaction between water and hydrophilic polymers[END_REF]. In cellulose, the freezing of water bound to hydroxyl groups was shown to decrease with the structural organization of the polymer [START_REF] Nakamura | Studies on bound water of cellulose by differential scanning calorimetry[END_REF]. By analogy with these previous studies, the exothermic event recorded at -70 °C could be ascribed to the crystallization of freezing bound water in the hydration shell of S4 and could be interpreted in terms of hydrophilic hydration. It is noteworthy that this peculiar transition is in general better evidenced in the cooling scans than in the heating ones, where it can be completely absent because of its continuous character [START_REF] Kozlowski | Low temperature exothermic effects on cooling of homoionic clays[END_REF].

This peak increases when the solution is heated above 70 °C, decreases when the solution is heated at 80 °C and completely disappears when the sample is maintained at 80 °C during 3 h. As shown by turbidimetry experiments, S4 peptide in TBS solution self-aggregates into amyloid fibres with increasing temperature. Moreover, CD studies [START_REF] Bochicchio | Amyloidogenesis of proteolytic fragments of human elastin[END_REF][START_REF] Bochicchio | Polyproline II structure in proteins: identification by chiroptical spectroscopies, stability, and functions[END_REF] evidenced that the dominant conformation of S4 peptide in its initial state was the PPII helix, extended and flexible structure devoid of intramolecular hydrogen bonds, which interconverts into folded b-sheet conformation through hydrophobic interaction when the temperature increases, reaching the irreversible, cross-b amyloid-like structure above 80 °C. The hydrophilic hydration of S4 peptide can be thus associated with the PPII conformation; once formed, the cross-b structure is devoid of such a hydrophilic hydration, leading to the disappearance of the exothermic event at -70 °C in the DSC curves. As shown in Fig. 3, this hydrophilic hydration disappears after complete aggregation into amyloid fibrils that are characterized by an ordered, waterexcluding core-containing extensive secondary structures [START_REF] Rauscher | Proline and glycine control protein self-organization into elastomeric or amyloid fibrils[END_REF]. Nevertheless, it was also evidenced by molecular simulations [START_REF] Rauscher | Proline and glycine control protein self-organization into elastomeric or amyloid fibrils[END_REF][START_REF] Dubay | Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains[END_REF] on (GVPGV) 7 and (GVAGGV) 6 that hydrophobic forces drive the emergence of collapsed waterswollen conformations of polypeptide chains which precede formation of the hydrophobic core; this fact could explain the reminiscent and increase of hydrophilic hydration in the curves corresponding to the prefibrillar states of S4 as shown in Fig. 3 (after a first heating up to 60 and 70 °C).

Influence of ageing on S4 solutions

We have superimposed the cooling curves of S4 in solution in TBS recorded in the low-temperature zone in the initial state and after storage at 4 °C for 15 and 30 days (Fig. 4).

The thermal signature of the hydrophilic hydration at -70 °C increases with storage of 15 days-which can considered as the optimal time to maximize the polypeptide chains/water hydrogen bonds-and slowly decreases with further ageing. These slow changes show that the hydration behaviour-connected to the self-assembly behaviour of the peptide-is able to vary with storage, in good agreement with the suggested multistate processes of protein hydration dynamics [START_REF] Halle | Protein hydration dynamics in solution: a critical survey[END_REF], and with the role of water observed in proteins [START_REF] Bochicchio | Investigating the amyloidogenic nanostructured sequences of elastin: sequence encoded by exon 28 of human tropoelastin gene[END_REF]. The influence of the time and/or temperature on the structure is corroborated by the work of Salvi et al. showing by CD and FTIR investigations that preferential conformations can be affected by time of analyses or storage [START_REF] Salvi | Combined effects of solvation and aggregation propensity on the final supramolecular structures adopted by hydrophobic, glycinerich, elastin-like polypeptides[END_REF][START_REF] Flamia | AFM study of the elastin-like biopolymer poly(ValGlyGlyValGly)[END_REF] and pointing out by AFM experiments that the propensity of peptides to form amyloid fibres depends on the water activity in the medium.

E50, E50H, E18 and E18H polypeptides in TBS buffer

In order to correlate results from turbidimetry and calorimetry, the four elastin-like peptides were studied in TBS solution. We reported in Fig. 5 the turbidimetry experiments performed for E50 and E50H (heating mode). The same experiments (not shown) were performed for E18 and E18H.

In this case, the sharp increase of the apparent absorbance was associated with a coacervation phenomenon [START_REF] Bochicchio | Investigating the role of (2S,4R)-4-hydroxyproline in elastin model peptides[END_REF] since the processus is reversible on cooling. The coacervation temperature was determined as the temperature value at which 50 % of coacervation took place. As expected, the coacervation temperatures of the peptides containing hydroxyproline are higher than the ones of the parent polypeptides containing the proline residues. In particular, E50 and E18 have coacervation temperatures of 35 and 50 °C, respectively. Conversely, E50H and E18H have coacervation temperatures of 60 and 85 °C, respectively. The introduction of the hydroxyl group on the pyrrolidine ring of proline reduces the hydrophobicity of the peptides, reducing propensity to coacervate.

The successive DSC heating scans of E50H polypeptides with different final temperatures are reported in Fig. 6. The first DSC scan performed between -100 and 80 °C is characterized by the two-phase transitions of the TBS solution at -22 and 0 °C as previously shown in Fig. 1. In the low-temperature range is detected a small exothermic event at -50 °C. Such a type of event has been already detected in the curves of synthetic polymers and associated with the cold crystallization phenomenon occurring in the heating mode. This mode is reversible on the second scan performed after the first heating up to 80 °C. Nevertheless, this mode is not detected on the third and fourth scans performed after the heating up to -30 °C. It is detected again on the fifth scan, recorded after the heating up to -10 °C associated with to the melting of the eutectic phase at -22 °C. A part of this peculiar ordered structure formed at -50 °C must disrupt at the melting of the eutectic phase, and in this way it is intimately connected with water. By analogy with previous DSC studies on CCl 3 F in a water-in-oil emulsion showing that clathrate compounds could be formed during DSC heating scans [START_REF] Fouconnier | Study of CCl3F hydrate formation and dissociation in W/O emulsion by differential scanning calorimetry and X-ray diffraction[END_REF], we propose to associate this low-temperature exothermic event with the formation of clathrate-like forms of water at the vicinity of the hydrophobic residues of the polypeptidic chains. In these structures, water molecules are supposed to align themselves parallel to the non-polar side chains, interacting with each other rather than with protein atoms and would shield the hydrophobic residues from self-interactions [START_REF] Yeo | Coacervation of tropoelastin[END_REF]. Therefore, this exothermic event can be considered as the thermal answer of the hydrophobic hydration of the polypeptides E50 and E50H; such a type of hydration was previously estimated at 100 or more molecules of water per pentamers in hydrophobic sequences from tropoelastin [START_REF] Yeo | Coacervation of tropoelastin[END_REF]. The DSC heating scans of E50 and E50H polypeptides are reported in Fig. 7. Between -30 and 10 °C, DSC curves are characterized by the two-phase transitions of the TBS solution, namely the melting of the eutectic phase at -22 °C and the melting of primary ice at 0 °C [START_REF] Kamasa | DSC approach for the investigation of mobile water fractions in aqueous solutions of NaCl and Tris buffer[END_REF]. It is noteworthy that the two-phase transitions recorded on heating are shifted towards high temperature when compared with the transitions recorded on cooling as done in Fig. 3; the shift towards low temperature during cooling is due to the supercooling of the liquids. At low temperature (enlarged zone of Fig. 7) is detected a small exothermic event at -75 and -50 °C for E50 and E50H, respectively. Such a type of event has been already detected in the curves of synthetic polymers and associated with the cold crystallization phenomenon occurring in the heating mode. Comparing the temperature of this exothermic event in E50 and E50H, it is patently obvious that hydroxylation of proline leads to a significant shift towards high temperature of the clathrate water formation. To confirm the effect of the proline hydroxylation on the clathrate water formation, similar DSC experiments have been performed on E18 and E18H, and the resulting curves are presented in Fig. 8. In the two successive scans performed for E18 and E18H, the exothermic peaks associated with clathrate-like water appeared at -73 °C and at -43 °C, respectively, with a good reproducibility indicative of the same kinetics behaviour. In a similar way as for E50 and E50H, hydroxylation of proline leads to a shift towards high temperature of the clathrate formation monitored by DSC. These DSC experiments confirm that the presence of the hydroxyl group of hydroxyproline in the polypeptidic chain, which can undergo hydrogen bonding with water molecules, alters the equilibrium among hydrophobic and hydrophilic hydration [START_REF] Bochicchio | Investigating the role of (2S,4R)-4-hydroxyproline in elastin model peptides[END_REF]. It was previously argued [START_REF] Yeo | Coacervation of tropoelastin[END_REF] that hydrogen bonds between clathrate water molecules are disrupted when the temperature increases, exposing the hydrophobic domains for self-interaction and thus leading to coacervation phenomenon. In order to correlate the clathrate water behaviour with the coacervation phenomenon, we plotted in Fig. 9 the temperatures of clathrate formation and coacervation for the four peptides. As shown in Fig. 9, the gap between the cold crystallization temperatures of E50/E50H and E18/E18H from DSC experiments is exactly equal to the gap between the coacervation temperatures from turbidimetry experiments, confirming the close relationship between these two thermally induced events. According to Cirulis and Keeley [START_REF] Cirulis | Kinetics and morphology of self-assembly of an elastin-like polypeptide based on the alternating domain arrangement of human tropoelastin[END_REF], we can wonder if the conformational changes in the polypeptide sequence are the cause or the result of the change in hydration. We can assume that the clathrate-like water whose formation is monitored by DSC in the solid state remains stable in the liquid state until the coacervation temperature, keeping the peptide unfolded [START_REF] Vrhovski | Coacervation characteristics of recombinant human tropoelastin[END_REF]. At the coacervation temperature, the clathrate water molecules become as disordered as the surrounding bulk ones, and the hydrophobic residues are submitted to Van der Waals interactions, leading to the folded conformation. The presence of hydroxyproline in the polypeptidic chains alters the clathrate formation, enhancing hydrophilic hydration and leading to less-aligned water molecules around the hydrophobic residues. As a consequence, it induces a reducing propensity of the polypeptidic chain to coacervate: a higher temperature is required to induce coacervation, because a higher energy is required to destabilize the hydrophilic hydration shell. Moreover, a previous study showed that the reduced hydrophobicity of hydroxyproline alters the ultrastructure of aggregates, which show a more polymorphic aggregation pattern when compared with the

Conclusions

This work shows that DSC can evidence distinct lowtemperature behaviours for different elastin-like peptides solutions. On the one hand, the amyloidogenic S4 peptides possess in pure water a great level of disorder. In the presence of salts, this disordered phase leads to a hydrophilic hydration detectable by DSC. Once aggregated into their amyloid form, these S4 peptides do not exhibit either amorphous phase or hydrophilic hydration, confirming the ordered character of the water-excluding cross-b structure. On the other hand, a hydrophobic hydration is revealed for the series of peptides that coacervate, confirming that elastin self-assembly is mainly triggered by hydrophobic interactions. The temperature of the clathrate-like water formation is closely connected to the amino acid sequence, with an important influence of proline hydroxylation, and can be used to plan the ability of the peptide to coacervate.

Fig. 1 Fig. 2

 12 Fig. 1 DSC curves of pure TBS buffer in the cooling (open squares) and heating (full squares) modes at 10 °C min -1

Fig. 3

 3 Fig. 3 DSC curves of S4 in TBS (30 g L -1 ) in the cooling mode, enlarged zone. Inset whole curves

Fig. 4 Fig. 5

 45 Fig. 4 DSC curves of S4 in TBS (30 g L -1 ) in the cooling mode recorded after different times of storage at 4 °C

Fig. 6 Fig. 7

 67 Fig. 6 DSC successive curves of E50H in TBS (100 g L -1 ) in the heating mode with different final temperatures

Fig. 8 Fig. 9

 89 Fig. 8 DSC curves of E18 and E18H in TBS (100 g L -1 ) in the heating mode, enlarged zone. Inset whole curves

Table 1

 1 Polypeptides characteristics (amino acid sequence and molecular weight)

	Peptide	Sequence


A Troegeler-Meynadier

C Palagiano

F Enjalbert

Effects of pH and fermentative substrate on ruminal metabolism of fatty acids during short-term in vitro incubation

Keywords: ruminal biohydrogenation, trans fatty acids, linoleic acid, pH, hay, starch

The ruminal biohydrogenation of c9,c12-18:2 can be affected by the fibre/starch ratio of the diet and the ruminal pH. The objectives of this study were to examine independently in vitro the effects of fermentation substrate (hay vs. corn starch) and buffer pH (6 vs. 7) on the biohydrogenation of c9,c12-18:2 carried out by grape seed oil, focusing on its t11 and t10 pathways, using 6-h ruminal incubations. The experimental design was a 2 9 2 factorial arrangement. Fermentation substrate and pH affected the C18 fatty acid balance in incubated media, but few interactions were observed. Compared with starch, hay as the fermentation substrate favoured the production of 18:0 (92.3), all trans-18:1 isomers (912.6) and CLA (96.1), except c9,t11-CLA, and the disappearance of unsaturated C18 fatty acids, but decreased the production of odd and branched chain fatty acids. Compared with pH 6 buffer, pH 7 buffer resulted in higher c9,c12-18:2 disappearance and CLA production. For c9,t11-CLA, an interaction was noticed between the two factors, leading to the highest production in cultures incubated on hay with the 7 pH buffer. Compared with starch, hay as fermentation substrate favoured the activity of t11 producers, which are fibrolytic bacteria, and the production of t10 isomers, possibly due to the presence of potential t10 producers in hay. Low pH resulted in a decreased t11 isomers production and in a slightly increased t10 isomers production, probably due to a modulation of enzymatic or bacterial activity.

Introduction

Although early studies showed potential beneficial effects for humans of a mixture of conjugated linoleic acid (CLA) isomers containing c9,t11-CLA and t10, c12-CLA as major components, the physiological effects are isomer specific [START_REF] Pariza | The biologically active isomers of conjugated linoleic acid[END_REF]. Specifically, t10,c12-CLA has been shown to exhibit possible detrimental effects on human health [START_REF] Tricon | Opposing effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid on blood lipids in healthy humans[END_REF][START_REF] Wilson | Conjugated linoleic acid isomers reduce blood cholesterol levels but not aortic cholesterol accumulation in hypercholesterolemic hamsters[END_REF][START_REF] Ip | The t10, c12 isomer of conjugated linoleic acid stimulates mammary tumorigenesis in transgenic mice overexpressing erbB2 in the mammary epithelium[END_REF]. Conversely, no detrimental effects have been demonstrated for c9, t11-CLA or its metabolic precursor (via tissue desaturation) t11-18:1. On the other hand, dairy milk fat content is specifically and strongly affected by t10, c12-CLA, which inhibits mammary fatty acid (FA) uptake and de novo synthesis, resulting in a milk fat depression [START_REF] Shingfield | Role of biohydrogenation intermediates in milk fat depression[END_REF]. The highest CLA contents in human food are found in ruminant products, mainly dairy products. Indeed, CLA and t11-18:1 are formed during biohydrogenation (BH) of dietary unsaturated FA in the rumen [START_REF] Enjalbert | Biosynthesis of trans fatty acids in ruminants[END_REF], and some of them are secreted into milk. Moreover, most milk c9, t11-CLA originates in a mammary desaturation of t11-18:1 [START_REF] Enjalbert | Biosynthesis of trans fatty acids in ruminants[END_REF].

Attempts to increase milk CLA should focus on t11 isomers, as t10 isomers can be detrimental for human health and dairy cow milk fat production. In vivo, the t10 pathway is favoured by high-concentrate lowfibre diets [START_REF] Flachowsky | Influence of roughage/concentrate ratio and linseed oil on the concentration of trans-fatty acids and conjugated linoleic acid in duodenal chyme and milk fat of late lactating cows[END_REF] inducing a low rumen pH, and the shift from t11 to t0 pathways, known as t10 shift, occurred mainly with fat-enriched diets [START_REF] Loor | Biohydrogenation, duodenal flow, and intestinal digestibility of trans-fatty acids and conjugated linoleic acids in response to dietary forage:concentrate ratio and linseed oil in dairy cows[END_REF]. Although t10 shift is widely accepted to occur with high-concentrate diets supplemented with oils, as demonstrated in vivo [START_REF] Roy | Examination of the persistency of milk fatty acid composition responses to plant oils in cows fed different basal diets, with particular emphasis on trans-C18:1 fatty acids and isomers of conjugated linoleic acid[END_REF][START_REF] Zened | Starch plus sunflower oil addition to the diet of dry dairy cows results in a trans-11 to trans-10 shift of biohydrogenation[END_REF] and in vitro [START_REF] Zened | Starch and oil in the donor cow diet and starch in substrate differently affect the in vitro ruminal biohydrogenation of linoleic and linolenic acids[END_REF], the key factor for its occurrence is not clear, as dietary starch and rumen pH are closely linked. Starch was identified as the main factor for t10 shift in a long-term fermentation system [START_REF] Maia | Is the trans-10 shift that sometimes occurs in the ruminal biohydrogenation of linoleic caused by low pH or starch? A rusitec study[END_REF], but starch and pH effects could not be completely separated. A low pH has been reported to induce t10 shift in batch [START_REF] Choi | Effect of pH and oxygen on conjugated linoleic acid (CLA) production by mixed rumen bacteria from cows fed high concentrate and high forage diets[END_REF] and continuous culture systems [START_REF] Fuentes | Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture[END_REF], although starch was also found to promote t10,c12-CLA formation in this latter study. The t10 isomers are thought to be produced from c9,c12-18:2 by Megasphera elsdenii [START_REF] Kim | The enrichment of a ruminal bacterium (Megasphaera elsdenii YJ-4) that produces the trans-10, cis-12 isomer of conjugated linoleic acid[END_REF] and/or Propionibacter acnes [START_REF] Verhulst | Isomerisation of polyunsaturated fatty acids by Propionibacteria[END_REF], which are lactate-consuming bacteria, but the involvement of other bacteria that grow best on starch substrate and/or at low pH is possible. On the other hand, high-fibre diets favour the t11 pathway of BH by fibrolytic bacteria, among them Butyrivibrio fibrisolvens and Butyrivibrio proteoclasticus are reported to produce t11 isomers [START_REF] Maia | Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen[END_REF].

The objectives of this study were to investigate independently in vitro the effects of buffer pH (6 vs. 7) and fermentation substrate (starch vs. hay) and their interaction on the BH of linoleic acid (c9,c12-18:2), focusing on its disappearance and the production of t10 and t11 isomers, to reveal the contribution of each factor, since each could be corrected independently at the farm level, by dietary bicarbonate for example for rumen pH, and variation of the concentrates/forage ratio in the diet for starch and fibre levels.

Material and methods

In vitro cultures

The experiment was conducted in a 2 9 2 factorial arrangement (2 pH buffers 92 fermentation substrates), using two ruminally fistulated dry dairy cows as donors of ruminal content. Cows received 8 kg dry matter (DM) per day of corn silage and concentratebased diet (37/67 on a DM basis). Table 1 shows the detailed composition of the diet, distributed in two equal meals per day.

Incubation substrates consisted of either 3.0 g of ground (1 mm screen) meadow hay (89% of DM; 8% of crude protein, 3% of crude fat, 8% of ash, 37% of crude fibre, 76% of neutral detergent fibre, 39% of acid detergent fibre and 3% of acid detergent lignin on DM basis) or 1.5 g of corn starch (purity 99%; Merck, Darmstadt, Germany). In both cases, substrates also contained 200 mg of grape seed oil, containing mainly c9,c12-18:2 (60% of total FA) and oleic acid (c9-18:1; 19% of total FA), and 0.15 g of urea (purity 99.5%; Prolabo, Nogent-sur-Marne, France). Fatty acids composition of the different media before incubation is shown in Table 2. The two buffer solutions were based on phosphate and bicarbonate, as described in a previous paper [START_REF] Troegeler-Meynadier | Effects of pH and concentrations of linoleic and linolenic acids on extent and intermediates of ruminal biohydrogenation in vitro[END_REF]; after saturation with CO 2 at 39 °C, they had a pH near 6 (LpH, low pH buffer, pH = 6.15 on average, 64.12 mM of KH 2 PO 4 , 2.60 mM of Na 2 HPO 4 and 27.50 mM of NaHCO 3 ) and 7 (HpH, high pH buffer, pH = 7.07 on average, 66.73 mM of Na 2 HPO 4 and 11.00 mM of NaHCO 3 ) respectively. Buffers also contained minerals to sustain microbial growth: 12.06 mM of NaCl, 9.05 mM of KCl, 0.73 mM of CaCl 2 and 0.73 mM of MgSO 4 . For each combination of fermentation substrate and pH buffer, six replicates with added oil were incubated for each of the two donor cow, for a total of 12 repetitions per treatment, and four control flasks, without added oil and not incubated, were prepared per donor cow (i.e. eight control flasks in total), which needed four series of in vitro incubations. Control flasks were immediately frozen.

Ruminal fluid was taken from each cow before each series of incubation with a vacuum pump 30 min after feeding and strained through a metal sieve (1.6-mm mesh). It was transferred to the laboratory under anaerobic conditions at 39 °C and then centrifuged (150 g, 5 min, 39 °C, CO 2 ) to produce low-fibre inoculums (<1% of NDF on a DM basis). The pH was measured, and a sample of centrifuged ruminal fluid was taken for FA analysis. Centrifuged ruminal fluid (80 ml) and 80 ml of the LpH or HpH solution, prewarmed at 39 °C and saturated with CO 2 , were placed in 250-ml Erlenmeyer flasks containing the fermentation substrates. Initial pH was measured, and flasks were placed in a water-bath rotary shaker (Aquatron; Infors AG, Bottmingen, Germany) at 39 °C and gassed for 1 h with CO 2 . Flasks were then closed with a rubber cap, with a plastic tube leading into water to vent fermentation gas without oxygen ingress. Flasks were stirred at 130 rpm and kept in the dark during the 6-h incubation. Thereafter, incubations were stopped by placing the flasks in ice water and final pH was measured. The contents of the flasks were frozen, freezedried (Virtis Freezemobile 25; Virtis, Gardiner, NY, USA), weighed, ground and homogenized in a ball mill (Dangoumau, distributed by Prolabo), and kept at À18 °C until analysis.

Chemical analysis

The FAs were extracted and methylated in situ with the procedure of [START_REF] Park | In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods[END_REF], except that the solution of 14% of boron trifluoride in methanol was replaced by a solution of methanol-acetylchloride (10:1). Nonadecanoic acid (C19:0) was used as the internal standard at a dose of 0.8 mg. The FA methyl esters were then quantified by gas chromatography (Agilent 6890N, equipped with a model 7683 auto injector; Network GC System, Palo Alto, CA, USA), using a fused silica capillary column (CPSil88, 100 m 9 0.25 mm ID, 0.20 lm film thickness; Varian, Middelburg, The Netherlands).

For the analysis of samples, flame ionization detector temperature was maintained at 260 °C and the injector at 255 °C, the split ratio was 1:75. Hydrogen was the carrier gas with a constant flow of 1 ml/min. The samples were injected in 0.5ll of hexane. Initial temperature of the oven was 60 °C, held for 1 min, and then increased by 20 °C/min to 150 °C, held at 150 °C for 15 min, then increased by 2 °C/min to 175 °C, held at 175 °C for 20 min, then increased by 10 °C/min to 225 °C, maintained at 225 °C for 10 min, and increased by 10 °C/min to reach a final temperature of 240 °C. This first method did not allow 16:1 to be separated from anteiso-17:0, t13+t14-18:1 from c9-18:1 and alpha-linolenic acid (c9,c12,c15-18:3) from 20:1, so a second analysis was carried out. Flame ionization detector temperature was maintained at 260 °C and the injector at 255 °C; the split ratio was 1:50. Hydrogen was the carrier gas with a constant pressure of 160 kPa. The samples were injected in 0.5ll of hexane. Initial oven temperature was 60 °C, held for 3 min, increased by 8 °C/min to 190 °C, held at 190 °C for 13 min, increased by 5 °C/ min to 225 °C, held at 225 °C for 10 min, increased by 10 °C/min to a final temperature of 230 °C and maintained there for 10 min.

Peaks were identified and quantified by comparison with commercial standards (Sigma, St. Louis, MO, USA), except 18:1 other than t9-18:1, t11-18:1 and c9-18:1, which were identified by order of elution.

Calculations and statistical analysis

The FA balance (expressed as mg per flask) during incubation of each flask was calculated as the amount assayed after incubation (final quantity) minus the initial amount, calculated as the quantity in the in the non-incubated flasks plus that carried out by the 200 mg of grape seed oil. Sums of CLA and trans-18:1 were obtained by adding productions of all isomers measured; sums of t10 and t11 isomers were obtained by adding t10 and t11 isomers originating from c9,c12-18:2 BH: CLA = t10,c12-CLA + c9,c11-CLA + c9,t11-CLA + t9,t11-CLA trans-18:1 = t4-18:1 + t5-18:1 + t6+t7+t8-18:1 + t9-18:1 + t10-18:1 + t11-18:1 + t12-18:1 + t13+t14-18:1 + t15-18:1 + t16-18:1 t10 isomers = t10,c12-CLA + t10-18:1 t11 isomers = c9,t11-CLA + t9,t11-CLA + t11-18:1

The rates (mg/l/h) and efficiencies of the c9,c12-18:2 isomerization (v1, E1), CLA reduction (v2, E2) and trans-18:1 reduction (v3) were calculated as previously described by Troegeler-Meynadier et al. [trans-18:1]e represented the concentration of total trans-18:1 isomers measured at the beginning and end of the 6-h incubation period. As c9,c12,c15-18:3 was a minor unsaturated FA at the beginning of incubation (0.6% and 6.0% of total FA for cultures with starch and hay substrates respectively) due to the great addition of c9,c12-18:2 by grape seed oil, the production of trans-18:1 from c9,c12,c15-18:3 was ignored [START_REF] Troegeler-Meynadier | Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations[END_REF].

All data were analysed using the GLM procedure of SYSTAT (version 9; SPSS, Chicago, IL, USA) and reported as mean values and their standard errors: variable = mean + day of incubation effect + cow effect + buffer effect + substrate effect + buffer 9 substrate interaction + e.

Differences were considered significant at p ≤ 0.05, and trends were declared at p ≤ 0.10.

Results

Ruminal cultures initially contained numerous trans-18:1 and CLA isomers, including 2.58 mg of t11 isomers and 1.62 mg of t10 isomers, on average. The most abundant FA was 18:0 (43.07 mg, on average), and c9-18:1 and c9,c12-18:2 quantities were similar among media (40.59 mg and 126.44 mg, on average respectively), but the quantity of c9,c12,c15-18:3 was higher in cultures with hay than in cultures with starch (6.13 mg vs. 1.30 mg on average respectively; Table 2). Hay also carried some other FA. Buffer addition had no effect on FA initial quantities of cultures.

The initial pH of incubates was significantly lower (by 0.5 units) in LpH than in HpH flasks (Table 3). The final pH of incubated media depended on the pH buffer but also on the fermentation substrate, with an interaction between these two factors, leading to the lowest final pH value (pH = 5.8) for cultures with corn starch and LpH (Table 3).

Considering FA with less than 18 carbons, iso-17:0 production was increased and 13:0 production was decreased by LpH. Compared with hay, starch as fermentation substrate favoured the production of more C10 to C17 FA (Table 3), including odd and branched chain FA. In particular, the productions of 12:0, 14:0 and 16:0 were 5.7, 2.9 and 2.4 times higher with starch than with hay as fermentation substrate respectively. A significant interaction between pH buffer and fermentation substrate was only observed for the 13:0 and iso-15:0 productions; however, no large differences between values were noticed. A trend towards an interaction between pH buffer and fermentation substrate was observed for anteiso-15:0 and 14:0, leading to the highest production for cultures incubated on starch with HpH.

For C18 FA, compared with starch, hay as fermentation substrate reduced c11-18:1 production but favoured the production of 18:0, c15-18:1, trans-18:1 (all isomers, Tables 3 and4) and CLA (all isomers except c9,c11-CLA, Table 4). Notably, the production of 18:0, trans-18:1 and CLA was 2.3, 12.6 and 6.1 times higher respectively, with hay than with starch. The productions of t10 and t11 isomers were 3.4 and 12.2 times higher with hay than with starch as substrate respectively. The quantity of unsaturated C18 FA that disappeared was significantly lower with starch than with hay, and the percentage of disappearance was also lower with starch than with hay except for c9,c12,c15-18:3.

Buffer pH significantly affected some C18 FA balances during incubation, although to a lesser extent than substrate (Table 3). The production of CLA was 1.2 times greater with HpH than with LpH. Moreover, the effects of buffer pH on CLA and trans-18:1 production concerned major isomers (Table 4): t9-18:1, t11-18:1, t16-18:1 and c9,t11-CLA productions were higher with HpH, and t10-18:1 production was higher with LpH. This resulted in a significant 1.5 times increase in t11 isomers and a 1.4 times decrease in t10 isomers with HpH compared with LpH. HpH slightly but significantly increased c9-18:1 and c9,c12-18:2 disappearance (Table 3). Few significant interactions were observed (Tables 3 and4): they concerned mainly CLA isomers, in particular c9t11-CLA production, which was highest in cultures with hay and HpH, and lowest in cultures with starch and LpH (Table 4).

Kinetic parameters were affected by fermentation substrate and buffer pH (Table 5), but no interaction between these two factors was observed. Rate and efficiency of c9,c12-18:2 isomerization were increased by hay as substrate and by HpH. The rate of the first reduction was increased by single effects of hay and HpH, but the efficiency of this reduction was increased by single effects of starch and LpH. The last reduction was only slightly affected by fermentation substrate: hay as substrate increased its rate but decreased its efficiency.

Discussion

Balance of FA with less than 18 carbons

In ruminants, C10 to C17 FAs are of interest because they could give some information about the bacterial community, in particular odd and branched FA, because these originate from bacterial synthesis (Vlaeminck et al., 2006a,b), and bacteria species differ in the FA they mainly produce [START_REF] Ifkovits | Cellular fatty acid composition and identification of rumen bacteria[END_REF][START_REF] Fievez | Milk odd-and branched-chain fatty acids as biomarkers of rumen function -An update[END_REF]. Moreover, some of them, that is, branched chain FA, may have a beneficial role in human health [START_REF] Ran-Ressle | Branched chain fatty acid content of United States retail cow's milk and implications for dietary intake[END_REF], so that an increase in their ruminal production could increase their milk and meat content for human consumers. Production of C10 to C17 FA, mainly 12:0, 14:0, 16:0 and anteiso-15:0, was higher with starch than hay as fermentation substrate. This is in agreement with the study of [START_REF] Dewhurst | Apparent recovery of duodenal odd-and branched-chain fatty acids in milk of dairy cows[END_REF] in which duodenal flows of 10:0, 12:0, 14:0, 16:0, and some odd and branched chain FA, that is, anteiso-15:0, iso-15:0, 17:0 and iso-17:0, increased linearly with the addition of concentrate to the diet. Indeed, some of these FAs predominate in amylolytic and lactate-utilizing bacteria, but are scarce in fibrolytic ones [START_REF] Ifkovits | Cellular fatty acid composition and identification of rumen bacteria[END_REF]Vlaeminck et al., 2006a). In our experiment, all these FAs were produced in larger amounts in starch cultures, where amylolytic and lactate-consuming bacteria would be able to develop rapidly. On the other hand, iso-FAs are reported to be merely linked to fibrolytic bacteria [START_REF] Fievez | Milk odd-and branched-chain fatty acids as biomarkers of rumen function -An update[END_REF]. In the present study, iso-17:0 production was not affected by fermentation substrate, and iso-C15:0 production was higher with starch than with hay. These FAs are both present in some amylolytic and fibrolytic bacteria [START_REF] Ifkovits | Cellular fatty acid composition and identification of rumen bacteria[END_REF]). Hence, iso-FAs are difficult to attribute to specific bacterial species, as suggested by differences between studies, in particular between studies relative to digestive content and studies relative to milk FA [START_REF] Dewhurst | Apparent recovery of duodenal odd-and branched-chain fatty acids in milk of dairy cows[END_REF][START_REF] Fievez | Milk odd-and branched-chain fatty acids as biomarkers of rumen function -An update[END_REF]. This could be explained by an important effect of the diet of cows on the microbiota, which makes comparisons between studies difficult, and by the origin of milk odd and branched chain FA, that could result from tissue synthesis and not only from rumen metabolism [START_REF] Berthelot | Effect of dietary propionate on intake patterns and fatty acid composition of adipose tissues in lambs[END_REF]. The buffer pH had little effect on C10 to C17 FA production in our study, except 13:0 and iso-17:0 productions that were only slightly affected. The pH range for bacterial growth is very wide: for example, 4.5-7.7 for the amylolytic bacteria S. bovis (with an optimum at 6.4), 4.6-7.8 for the lacticolytic bacteria M. elsdenii (with an optimum at 6.1), or 5.4-7.7 for the fibrolytic bacteria B. fibrisolvens (with an optimum at 6.3-6.5; [START_REF] Therion | Effect of pH on growth rates of rumen amylolytic and lactilytic bacteria[END_REF]. So in our range of pH, a great variety of bacteria could develop, probably resulting in no clear selection of species by buffer pH.

Biohydrogenation steps of unsaturated C18 fatty acids

The efficiency of unsaturated C18 FA disappearance was significantly affected by fermentation substrate and pH buffer. The disappearances of c9-18:1 and c9, c12-18:2 were significantly higher with hay than starch substrate and were significantly greater with HpH than LpH. The percentage of disappearance of c9, c12,c15-18:3 was not affected by pH or by substrate, and its disappeared quantity was not affected by pH, but was higher in cultures with hay. This could be explained by the higher initial quantity of c9,c12,c15-C18:3 in these cultures, that is 6.1 mg per flask, compared with 1.6 mg in cultures with starch (Table 2). Hay cultures initially contained nearly the same quantities of c9-18:1 (41.0 vs. 40.2 mg per flask) and c9, c12-18:2 (127.3 vs. 125.6 mg) than starch cultures, so that the 2.0 and 3.4 times higher disappearances observed, respectively, for these FAs with hay than starch were due to higher BH efficiencies. Values obtained in the present study for kinetic parameters of c9,c12-18:2 BH (Table 5) were of the same order of magnitude as those obtained in a previous study [START_REF] Troegeler-Meynadier | Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations[END_REF].

The greater disappearance of unsaturated FA with hay than starch resulted in increased production of all c9,c12-18:2 BH intermediates or final products: CLA, trans-18:1 and 18:0. In fact, BH is mainly due to B. fibrisolvens, a fibrolytic bacterium that carries out the two first reactions of c9,c12-18:2 BH, and secondly to Butyrivibrio hungatei (Van de [START_REF] Van De Vossenberg | Biohydrogenation of C18 unsaturated fatty acids to stearic acid by a strain of Butyrivibrio hungatei from the bovine rumen[END_REF] and B. proteoclasticus [START_REF] Wallace | Clostridium proteoclasticum: a ruminal bacterium that forms stearic acid from linoleic acid[END_REF], other fibrolytic bacteria responsible for the last reduction to 18:0. Fibrolytic bacteria, including biohydrogenating ones, were more likely to develop on hay as the fermentation substrate than on starch, and so could achieve the three steps of c9,c12-18:2 BH, leading to higher rates for the three BH reactions and a higher efficiency for the first reaction of isomerization with hay.

In the present study, the accumulation of trans-18:1 and CLA obtained with cultures incubated on hay could not be linked to an inhibition of the reductases, as hay increased the rates of both reductions. The rapid disappearance of c9,c12-18:2, due to a high rate of isomerization observed in hay cultures, led to a great production of CLA, trans-18:1 and 18:0. In hay cultures compared with starch cultures, accumulation of CLA and trans-18:1 resulted from a saturation of the two reductions, leading to a decrease in their efficiencies in spite of increased rates. Such a saturation of the second reduction by a large quantity of its substrate or CLA had previously been hypothesized [START_REF] Troegeler-Meynadier | Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations[END_REF]. In starch cultures, the productions of CLA and trans-18:1 were limited by a low isomerization rate.

Buffer pH did not affect c9,c12,c15-18:3 balance, as previously reported with low c9,c12,c15-18:3 level cultures [START_REF] Fuentes | Effect of pH on ruminal fermentation and biohydrogenation of diets rich in omega-3 or omega-6 fatty acids in continuous culture of ruminal fluid[END_REF]. However, LpH slightly decreased the disappearance of c9-C18:1 and strongly decreased that of c9,c12-18:2. Such a protection of c9, c12-18:2 from BH by a low pH could firstly be explained by an inhibition of lipolysis as the growth of the main lipolytic bacteria, that is, Anaerovibrio lipolytica, has been shown to be decreased at low pH [START_REF] Fuentes | Effect of pH on ruminal fermentation and biohydrogenation of diets rich in omega-3 or omega-6 fatty acids in continuous culture of ruminal fluid[END_REF]. Nevertheless, these authors observed that lipolysis was barely reduced at low pH as opposed to a strong BH reduction. In our study, the reduction in BH by low pH was linked to an inhibition of c9,c12-18:2 isomerization (Table 5), as previously reported [START_REF] Troegeler-Meynadier | Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations[END_REF]. As a result, HpH resulted in a greater CLA production and a slight increase in trans-18:1 production. The reduction in CLA to trans-C18:1 had a higher rate but a lower efficiency with HpH than LpH, suggesting an inhibition by excess of substrate, originating in a high isomerization rate. Buffer pH did not significantly affect the kinetic parameters of the second reduction, so it did not affect 18:0 production. The slight accumulation of trans-C18:1 observed with HpH was probably due to the slowness of the third reaction compared with the previous reactions. Such results contrast with our previous study [START_REF] Troegeler-Meynadier | Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations[END_REF], reporting an inhibition of the second reduction by low pH, but in this study, this inhibition only became significant at pH ≤ 5.5, that is, lower than in the present study. Moreover, HpH cultures could have maintained a higher protozoa population [START_REF] Dehority | Effect of pH on viability of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei in vitro[END_REF], which could have participated in the accumulation of trans-18:1 and CLA as they have high contents of CLA and trans-18:1 [START_REF] Devillard | Rumen ciliate protozoa contain high concentrations of conjugated linoleic acids and vaccenic acid, yet do not hydrogenate linoleic acid or desaturate stearic acid[END_REF].

Biohydrogenation pathways of unsaturated C18 fatty acids

Regarding the two main pathways of c9,c12-C18:2 BH, the t11 isomers production was much higher with hay than starch and higher with HpH than LpH buffer. Among fibrolytic bacteria, B. fibrisolvens is the main t11 isomers producer [START_REF] Maia | Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen[END_REF] and is able to develop on hay but not on starch [START_REF] Klieve | Establishing populations of M. elsdenii YE 34 and B. fibrisolvens YE 44 in the rumen of cattle fed high grain diets[END_REF], so that hay as fermentation substrate offered better conditions for its growth and/or activity than starch. [START_REF] Choi | Effect of pH and oxygen on conjugated linoleic acid (CLA) production by mixed rumen bacteria from cows fed high concentrate and high forage diets[END_REF], [START_REF] Fuentes | Effect of pH on ruminal fermentation and biohydrogenation of diets rich in omega-3 or omega-6 fatty acids in continuous culture of ruminal fluid[END_REF][START_REF] Troegeler-Meynadier | Effects of pH and concentrations of linoleic and linolenic acids on extent and intermediates of ruminal biohydrogenation in vitro[END_REF], 2006) also noticed that t11 isomers production was favoured by a high pH. As the incubation duration in the present study was probably not sufficient to induce a notable modification of the microflora, the lower production of t11 isomers in cultures with LpH was probably due to an inhibition of the D12 isomerase, whose optimal pH is between 7 and 7.2 [START_REF] Kepler | Biohydrogenation of unsaturated fatty acids: purification and properties of a linoleate D12 cis -D11 trans isomerase from Butyrivibrio fibrisolvens[END_REF]. This inhibition begins at pH 6.3 [START_REF] Choi | Effect of pH and oxygen on conjugated linoleic acid (CLA) production by mixed rumen bacteria from cows fed high concentrate and high forage diets[END_REF] and would be very important below pH 6.0 [START_REF] Troegeler-Meynadier | Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations[END_REF]. Indeed, a major inhibition of t11 isomers production was observed in the cultures with LpH. An interaction between pH and fermentation substrate was observed for c9,t11-CLA production: the HpH + hay combination led to the highest value and the LpH + starch combination to the lowest value. Hay was a good substrate for t11 isomers-producing bacteria and favoured an elevation of pH, and HpH favoured the activity of these bacteria, in particular that of the D12 isomerase they synthesized, unlike starch and LpH.

The surprising finding in the present study, contradicting previous research, was the higher values of t10 isomers production in cultures with hay as the fermentation substrate than in cultures with starch. Similarly, [START_REF] Fuentes | Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture[END_REF] observed a higher t10 isomers production with their low-concentrate diet compared with their high-concentrate diet. Usually, t10 isomers are favoured by high-starch diets, whilst t11 isomers are favoured by high-fibre diets [START_REF] Flachowsky | Influence of roughage/concentrate ratio and linseed oil on the concentration of trans-fatty acids and conjugated linoleic acid in duodenal chyme and milk fat of late lactating cows[END_REF][START_REF] Abughazaleh | Production of trans C18:1 and conjugated linoleic acid in continuous culture fermenters fed diets containing fish oil and sunflower oil with decreasing levels of forage[END_REF].

Moreover, starch has been identified as the main factor for t10 isomers production in a long-term fermentation system [START_REF] Maia | Is the trans-10 shift that sometimes occurs in the ruminal biohydrogenation of linoleic caused by low pH or starch? A rusitec study[END_REF]. Among bacteria favoured by a high-starch diet, [START_REF] Kim | The enrichment of a ruminal bacterium (Megasphaera elsdenii YJ-4) that produces the trans-10, cis-12 isomer of conjugated linoleic acid[END_REF] reported that M. elsdenii, a lactate-utilizing bacterium, is able to produce t10,c12-CLA from c9,c12-18:2. Nevertheless, the isomerization of c9,c12-18:2 into t10, c12-CLA by M. elsdenii still remains questionable, because [START_REF] Maia | Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen[END_REF] did not observe a capacity of this bacterium to isomerize c9,c12-18:2. Moreover, in our experiment, the production of t10,c12-CLA by M. elsdenii in cultures with hay was probably low as this bacterium is unable to develop on a medium without grain [START_REF] Klieve | Establishing populations of M. elsdenii YE 34 and B. fibrisolvens YE 44 in the rumen of cattle fed high grain diets[END_REF]. Other species like P. acnes and Propionibacter freudenreichii are able to produce t10,c12-CLA from c9,c12-18:2 [START_REF] Verhulst | Isomerisation of polyunsaturated fatty acids by Propionibacteria[END_REF]. P. acnes is also a lactate-consuming bacterium. As reported above, lactate-consuming and amylolytic bacteria probably grew and acted better on starch substrates, as suggested by the high production of some specific short and medium FA in starch cultures. However, hay could have provided these bacteria, as Propionibacteria that are similar to those isolated from rumen fluid of cattle can be found in hay (6.10 8 per gram of timothy hay, for example) as reported by [START_REF] Gutierrez | Numbers and characteristics of lactate utilizing organisms in the rumen of cattle[END_REF]. Moreover, this author showed that Propionibacteria have the capacity to grow on hay. So in our experiment, P. acnes could have been responsible for the high t10 isomers production in the cultures incubated for 6 h on hay.

As expected, production of t10 isomers was higher with LpH than with HpH. On the other hand, [START_REF] Maia | Is the trans-10 shift that sometimes occurs in the ruminal biohydrogenation of linoleic caused by low pH or starch? A rusitec study[END_REF] did not observe an effect of pH on t10 isomers, but their low pH was 6.0, probably insufficiently acidic to promote t10 isomers production. Previous in vitro studies also reported a significant increase in t10 isomers [START_REF] Troegeler-Meynadier | Effects of pH and concentrations of linoleic and linolenic acids on extent and intermediates of ruminal biohydrogenation in vitro[END_REF] or t10,c12-CLA [START_REF] Choi | Effect of pH and oxygen on conjugated linoleic acid (CLA) production by mixed rumen bacteria from cows fed high concentrate and high forage diets[END_REF] when pH decreased from 6.6-6.5 to 5.6-5.4. However, as in the present study, values obtained for t10 isomers were very low and were largely below those of t11 isomers. [START_REF] Abughazaleh | Production of trans C18:1 and conjugated linoleic acid in continuous culture fermenters fed diets containing fish oil and sunflower oil with decreasing levels of forage[END_REF] and [START_REF] Fuentes | Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture[END_REF][START_REF] Fuentes | Effect of pH on ruminal fermentation and biohydrogenation of diets rich in omega-3 or omega-6 fatty acids in continuous culture of ruminal fluid[END_REF] obtained in vitro a ratio of t10/t11 isomers above 1, but the comparison with our experiment is difficult because they used continuous ruminal cultures over 10, 8 and 7 day at pH 5.6. The t10-producing bacteria probably had enough time to develop and be selected by the low pH and/or the high starch content of the substrates used in their study, but not in ours. As our 6-h incubation duration probably was not sufficient to induce a notable modification of the relative abundances of bacteria, the effect of low pH in our experiment was more probably due to a greater D9 isomerase production by t10 biohydrogenating bacteria, or to the enhancement of the activity of the D9 isomerase. A greater activity of the D9 isomerase could be due to a better activity at low pH than D12 isomerase (optimal pH around 7.0; [START_REF] Kepler | Biohydrogenation of unsaturated fatty acids: purification and properties of a linoleate D12 cis -D11 trans isomerase from Butyrivibrio fibrisolvens[END_REF] and/or to an increased availability of its substrate, c9,c12-18:2, because of the inhibition of the D12 isomerase.

Conclusion

Ruminal c9,c12-18:2 BH was greatly affected in vitro by the fermentation substrate and buffer pH, but these factors essentially acted independently, and buffer pH, within physiological limits, had smaller effects than fermentation substrate. So, to promote ruminal production of t11 isomers for human consumer, a highfibre diet is necessary. Fiber mainly acts as a growth substrate for t11-producing bacteria. Low pH slowed D12 isomerase activity and so resulted in a decreased t11 isomers production, but addition of bicarbonate in high-starch diet was not efficient enough to restore t11 isomers production.

  (2006): v1 = ([c9,c12-18:2]bÀ[c9,c12-18:2]e)/6, with 6 being the incubation time E1 = ([c9,c12-18:2]bÀ[c9,c12-18:2]e)/[C18:2]b, where [c9,c12-18:2]b and [c9,c12-18:2]e represented the concentration of c9,c12-18:2 at the beginning and at the end of the 6 h incubation respectively. v2 = ([c9,c12-18:2]bÀ[c9,c12-18:2]e + [CLA]bÀ [CLA]e)/6, E2 = ([c9,c12-18:2]bÀ[c9,c12-18:2]e + [CLA]bÀ [CLA]e)/([c9,c12-18:2]bÀ[c9,c12-18:2]e + [CLA]b) where [CLA]b and [CLA]e represented the concentration of total CLA isomers measured at the beginning and end of the 6 h incubation respectively. v3 = ([c9,c12-18:2]bÀ[c9,c12-18:2]e + [CLA]bÀ [CLA]e + [trans-18:1]bÀ[trans-18:1]e)/6, E3 = ([c9,c12-18:2]bÀ[c9,c12-18:2e + [CLA]bÀ [CLA]e + [trans-18:1]bÀ[trans-18:1]e)/([c9,c12-18:2]bÀ[c9,c12-18:2]e + [CLA]bÀ[CLA]e + [trans-18:1]b), where [trans-18:1]b and

Table 1

 1

	Ingredients and chemical composition of the cows' diet (% of
	dry matter)
	Ingredients (% of dry matter)
	Corn silage	37.3
	Wheat flour	55.7
	Soybean meal	5.6
	Minerals and vitamins*	1.4
	Nutrient content (% of dry matter)
	Crude protein	11.4
	Neutral detergent fibre	18.1
	Acid detergent fibre	7.2
	Starch	39.2
	Total fatty acids	2.3
	*Contained: 50 g/kg of P, 140 g/kg of Ca, 60 g/kg of Na, 4 g/kg of Zn,
	3.2 g/kg of Mn, 3 g/kg of Fe, 0.8 g/kg of Cu, 250 800 IU/kg of vitamin A,
	62 700 IU/kg of vitamin D 3 and 112 IU/kg of vitamin E.

Table 2

 2 Initial quantities (mg) of fatty acids in the different media before incubation (n = 48)

Table 3

 3 Initial and final pH, and production or disappearance (mg) of fatty acids (FA) assayed in the media after 6-h incubation with 200 mg of grape seed oil (n = 48)

		Corn starch		Hay			Effect, p-value
	Fermentation substrate
	Buffer pH	LpH	HpH	LpH	HpH	SEM	FS	pH	FS 9 pH
	Initial pH	6.25	6.78	6.22	6.75	0.02	0.60	<0.01	-
	Final pH	5.82	6.56	6.21	6.71	<0.01	<0.01	<0.01	0.02
	Fatty acid production or disappearance (mg per flask)
	10:0	0.02	0.01	ND	0.01	<0.01	0.17	0.99	0.07
	11:0	0.01	0.02	À0.01	0.01	<0.01	0.03	0.05	0.46
	12:0	0.35	0.34	0.05	0.07	0.02	<0.01	0.86	0.72
	13:0	0.06	0.08	<0.01	0.08	0.01	0.06	<0.01	0.05
	14:0	0.71	0.86	0.31	0.23	0.03	<0.01	0.52	0.06
	15:0	0.49	0.54	0.40	0.31	0.02	<0.01	0.68	0.13
	anteiso-15:0	0.66	0.99	0.46	0.38	0.06	<0.01	0.29	0.08
	iso-15:0	0.20	0.24	0.17	0.12	0.01	<0.01	0.75	0.03
	16:0	2.54	2.75	1.09	1.07	0.12	<0.01	0.70	0.62
	16:1+ anteiso-17:0	0.59	0.84	0.34	0.42	0.05	<0.01	0.08	0.37
	17:0	0.35	0.35	0.30	0.28	0.01	<0.01	0.58	0.61
	iso-17:0	0.15	0.10	0.12	0.09	0.01	0.22	0.02	0.73
	18:0	8.84	10.65	20.62	24.91	0.94	<0.01	0.12	0.51
	c9-18:1	À4.83	À5.28	À9.47	À10.98	0.23	<0.01	0.04	0.25
	c11-18:1	1.82	2.02	1.38	1.35	0.03	<0.01	0.18	0.09
	c15-18:1	<0.01	0.01	0.03	0.08	<0.01	<0.01	<0.01	0.09
	Sum of trans-18:1	2.01	2.89	26.68	30.83	0.81	<0.01	0.11	0.37
	c9,c12-18:2	À13.49	À18.69	À48.61	À60.83	1.21	<0.01	<0.01	0.16
	Sum of CLA	0.57	3.03	8.39	13.65	0.41	<0.01	<0.01	0.10
	t11,c15-18:2	À0.02	À0.01	0.05	À0.06	0.01	0.70	0.05	0.01
	c9,c12,c15-18:3	À0.40	À0.49	À2.30	À2.03	0.07	<0.01	0.55	0.19
	Percentage of disappearance of unsaturated fatty acids (%)
	c9-18:1	À12.00	À13.21	À23.14	À26.75	1.13	<0.01	0.03	0.27
	c9,c12-18:2	À10.75	À14.91	À38.12	À47.84	1.95	<0.01	<0.01	0.15
	c9,c12,c15-18:3	À30.95	À38.49	À36.08	À33.32	2.35	0.97	0.31	0.03

HpH, high pH buffer; LpH, low pH buffer; FS, fermentative substrate; ND, non-detected.

Table 4

 4 Production of trans-18:1 and CLA isomers (mg) in the media after 6-h incubation with 200 mg of grape seed oil (n = 48)

		Corn starch	Hay			Effect, p-value
	Fermentation substrate
	Buffer pH	LpH	HpH	LpH	HpH	SEM	FS	pH	FS 9 pH
	Trans-18:1 isomers
	t4-18:1	À0.01	0.01	0.11	0.17	0.01 <0.01	0.03 0.39
	t5-18:1	<0.01 <0.01	0.11	0.14	0.01 <0.01	0.27 0.10
	t6+t7+t8-18:1	0.09	0.13	1.04	1.18	0.03 <0.01	0.12 0.44
	t9-18:1	0.07	0.11	0.61	0.72	0.02 <0.01	0.04 0.31
	t10-18:1	0.79	0.41	2.61	1.86	0.12 <0.01	0.03 0.46
	t11-18:1	0.40	1.64 17.49 21.52	0.83 <0.01	0.10 0.45
	t12-18:1	0.27	0.25	1.55	1.61	0.05 <0.01	0.79 0.66
	t13+t14-18:1	0.15	0.13	2.04	2.24	0.06 <0.01	0.43 0.41
	t15-18:1	0.16	0.14	0.63	0.71	0.04 <0.01	0.68 0.50
	t16-18:1	0.07	0.08	0.50	0.67	0.03 <0.01	0.10 0.13
	CLA isomers
	t10,c12-CLA	0.14	0.28	0.66	0.49	0.03 <0.01	0.62 0.02
	c9,c11-CLA	ND	0.02	0.02	0.01 <0.01	0.09	0.38 0.06
	c9,t11-CLA	0.39	2.52	7.34 12.87	0.39 <0.01 <0.01 0.04
	t9,t11-CLA	0.03	0.22	0.37	0.27	0.03 <0.01	0.45 0.03
	Sum of t11 isomers	0.83	4.38 25.19 34.67	1.08 <0.01 <0.01 0.20
	Sum of t10 isomers	0.94	0.68	3.27	2.35	0.14 <0.01	0.04 0.24

HpH, high pH buffer; LpH, low pH buffer; FS, fermentative substrate; ND, non-detected.

Table 5

 5

	Rates (mg/l/h) and efficiencies of the
	three reactions of linoleic acid biohydrogena-	Fermentation substrate	Corn starch	Hay			Effect, p-value
	tion for 6-h incubation with 200 mg of grape seed oil (n = 48)	Buffer pH	LpH	HpH	LpH	HpH	SEM	FS	pH	FS 9 pH
		Isomerization of c9,c12-18:2
		Rate v1	14.06	19.47	50.63	63.37	1.26	<0.01	<0.01	0.16
		Efficiency E1	0.11	0.15	0.38	0.48	0.01	<0.01	<0.01	0.15
		Reduction in CLA
		Rate v2	13.46	16.31	41.90	49.15	1.02	<0.01	0.02	0.30
		Efficiency E2	0.93	0.82	0.82	0.77	0.01	<0.01	<0.01	0.10
		Reduction in trans-18:1
		Rate v3	11.37	13.30	14.11	17.04	0.92	0.01	0.22	0.73
		Efficiency E3	0.59	0.52	0.26	0.25	0.02	<0.01	0.21	0.37
		HpH, high pH buffer; LpH, low pH buffer; FS, fermentative substrate.

Acknowledgements

The authors thank M.L. Chemit and the staff at the experimental station (UMR 1289 Tandem).