Mohsen Dahesh

Amélie Banc

Agnès Duri

Marie-Hélène Morel

Laurence Ramos
email: laurence.ramos@umontpellier.fr

Spontaneous gelation of wheat gluten proteins in a food grade solvent

Structuring wheat gluten proteins into gels with tunable mechanical properties would provide more versatility for the production of plant protein-rich food products. Gluten, a strongly elastic protein material insoluble in water, is hardly processable. We use a novel fractionation procedure allowing the isolation from gluten of a water/ethanol soluble protein blend, enriched in glutenin polymers at an unprecedented high ratio (50%). We investigate here the viscoelasticity of suspensions of the protein blend in a water/ethanol (50/50 v/v) solvent, and show that, over a wide range of concentrations, they undergo a spontaneous gelation driven by hydrogen bonding. We successfully rationalize our data using percolation models and relate the viscoelasticity of the gels to their fractal dimension measured by scattering techniques. The gluten gels display self-healing properties and their elastic plateaus cover several decades, from 0.01 to 10000 Pa. In particular very soft gels as compared to standard hydrated gluten can be produced.

Introduction

The worldwide rising consumption of meat is not sustainable and access to a wider range of proteinrich foods derived from plant proteins is desirable. Because of their availability at large scale, grain storage proteins issued from wheat (gluten) and corn (zein) are good candidates in the quest for food products based on plant proteins. These proteins share many similarities. They both contain a high amount of proline, and are insoluble in water but soluble in aqueous alcohol. Gluten proteins differ however from zein and exhibit remarkable elastomeric properties. Those properties, which are ascribed to the ability of gluten proteins to form very long polymers, can be regarded as a real asset in the optics of producing protein-based food-products and biomaterials. The water-insolubility of gluten proteins has precluded their rational study by standard physicochemical approaches and contrarily to globular proteins their structure and phase behavior remain largely unknown. They display moreover a high polymorphism and an extremely broad polydispersity. As several animal elastomeric proteins (elastin from vertebrates, abductin from arthropods, and dragline and flagelliform silks from spiders), they include repeated sequences rich in proline and glycine. The repetitive domains of gluten proteins are also rich in glutamine, which is prone to hydrogen-bonding [START_REF] Tatham | Wheat seed proteins exhibit a complex mechanism of protein elasticity[END_REF]. Gluten proteins include two classes of polypeptides differing in their propensity to form intermolecular disulphide bonds (Fig. 1A). Gliadin, which accounts for half of the gluten proteins are monomeric species and glutenin, the other half, consists in Comment citer ce document : Dahesh, M., Banc, A., Duri, A., Morel, M. H., Ramos, L. (2016). Spontaneous gelation of wheat gluten proteins in a food grade solvent. Food Hydrocolloids, 52, 1-10. DOI : 10.1016/j.foodhyd.2015.06.014 2 a concatenation of disulfide bond-stabilized polypeptides whose size can reach several millions of kDa [START_REF] Wrigley | Giant proteins with flour power[END_REF]. So far, the complexity of gluten proteins and the lack of knowledge on their structure are clearly scientific bottlenecks that restrict the use of gluten.

In its water hydrated form, gluten is a viscoelastic cohesive mass comprising typically 1.7 g of water per 1 g of protein with an elastic plateau modulus of typically 1 kPa [START_REF] Cornec | Characterisation of gluten subfractions by SE-HPLC and dynamical rheological analysis in shear[END_REF][START_REF] Létang | Characterization of wheat flour-water doughs. Part I: Rheometry and microstructure[END_REF][START_REF] Ng | Power law gels at finite strains[END_REF][START_REF] Pruska-Kedzior | Comparison of viscoelastic properties of gluten from spelt and common wheat[END_REF]. Making a food product from this matrix is challenging, as gluten displays too high viscosity and elasticity to be easily shaped. Hence fibrous structures could only be obtained at a high energy cost (high temperature and pressure) by extrusion, according to a processing route difficult to control. In addition the negative effects of harsh treatments on the nutritional properties of the gluten based food begin to be recognized [START_REF] Abe | Metabolic transit of lysinoalanine in the rat-body -Observation of feeding tests with alkali-treated casein and gluten[END_REF][START_REF] Rombouts | Heat-induced cross-linking and degradation of wheat gluten, serum albumin, and mixtures thereof[END_REF]. We will show however that alternative routes might be achievable to make gluten suitable for food processing in mild conditions. In this optics, our strategy is to take one step back and bring a gluten gel to a sol state, which will subsequently gel in a fully controlled fashion. To reach this goal, we have developed an original fractionation procedure yielding an ethanol/water soluble gluten protein mixture comprising a 1:1 weight ratio of gliadin, the monomeric gluten proteins, and glutenin, the polymeric ones. Because the largest most insoluble glutenin polymers are discarded in our process, homogeneous and stable dispersions are obtained for a large range of protein concentrations up to several hundred of mg/ml [START_REF] Dahesh | Polymeric assembly of gluten proteins in an aqueous ethanol solvent[END_REF]. The superior quality of samples prepared using ethanol/water mixture as compared to standard gluten is illustrated in fig. 1B-C: dispersing the proteins in water leads to highly heterogeneous samples comprising a viscoelastic mass in an excess of water, whereas homogeneous dispersions and gels are produced using an ethanol/water mixture solvent, a food-grade solvent.

The paper is organized as follows. We first describe the linear viscoelastic properties of the gluten dispersions in an ethanol/water mixture and show that, above a critical concentration, the dispersions exhibit a spontaneous concentration-and time-dependent gelation. We then demonstrate that the solgel transition can be quantitatively rationalized using percolation theories and exhibit the hallmarks of critical gelation conventionally obtained for synthetic polymers, and we successfully relate the sample viscoelasticity to structural measurements. Thanks to biochemical assays, we show that the gelation mainly results from slow hydrogen-bonds formation. Finally, we describe the unique rheological properties of the as-prepared gels.

Materials and Methods

Materials

Gluten fractionation. Native gluten powder (81.94% protein, dry basis) was courtesy of Tereos-Syral (France). A protein fraction representative of gluten in composition (gliadin/glutenin ratio = 1.1), soluble in ethanol/water (50/50, v/v), was extracted according to a protocol previously published by us [START_REF] Dahesh | Polymeric assembly of gluten proteins in an aqueous ethanol solvent[END_REF]. In brief, gluten powder (20g) was placed in a centrifuge bottle (volume 250 mL) with 200 mL of 50 % (v/v) ethanol/water gluten and submitted to a continuous rotating agitation (60 rpm at 20°C) for 19 hours. After a 30 minutes centrifugation at 15000g at 25°C, the clear supernatant (yield ~50%) was recovered and placed at 4°C for 24 hours. This led to a phase separation between a light phase (yield ~25%) including gliadin at an unprecedented level of purity and a dense phase (yield ~ 25%) consisting in a 50/50 blend of gliadin and glutenin. The dense phase is the fraction of interest in this study. This fraction was immediately frozen at -18°C before being freeze-dried and grinded. For this fraction, the amount of free thiol groups (0.648 µmoles/g of protein) and of total thiol equivalent groups (106 µmoles/g of protein) were measured according to [START_REF] Morel | Protein insolubilization and thiol oxidation in sulfite-treated wheat gluten films during aging at various temperatures and relative humidities[END_REF] Sample preparation. Samples were prepared by dispersing the required mass of freeze-dried protein fraction in ethanol/water (50/50 v/v). Chemicals, N-Ethylmaleimide (NEM) or β-mercaptoethanol (BME) were eventually added to the solvent before dispersing the proteins. The ageing time is defined 3 as the time elapsed since the sample has been prepared by dispersing the proteins in the solvent. Homogenization was ensured by a rotary shaking overnight at room temperature. Samples were then stored at 20°C for different ageing times.

Experimental methods

High-performance liquid chromatography. Protein size distribution was measured using size exclusion high-performance liquid chromatography (SE-HPLC) performed on an Alliance system equipped with a TSK G4000 SWXL column. Samples were diluted (at about 1mg/ml) in the elution buffer composed of 0.1 M sodium phosphate buffer at pH 6.8 and 0.1% sodium dodecyl sulfate (SDS). To investigate the role of hydrogen bonds, some tests were also performed using an elution buffer containing, in addition to SDS, urea at a concentration of 8M. Elution of the injected sample (20μl) was performed at 0.7 ml/min and the detection of the different species was recorded at a wavelength of 214 nm. The apparent molecular weight calibration of the column was obtained using a series of protein standards with molecular weight in the range 13 to 2 000 kDa according to [START_REF] Dahesh | Polymeric assembly of gluten proteins in an aqueous ethanol solvent[END_REF] Rheology. Rheological measurements were performed on an ARES strain controlled rheometer. A Couette geometry was used for samples concentrations below 300 mg/ml whereas higher concentrations were probed with a plate-plate geometry with rough surfaces to avoid slipping. Low viscosity silicon oil was used to avoid contact between air and the samples and thus prevent solvent evaporation. The following protocol was systematically used: after loading the sample in the shear cell, the less viscous samples were submitted to a shear rate of 1 s -1 during 60 s, before being allowed to rest for 30 minutes. The very viscous samples were only allowed to rest for 30 minutes after loading. Viscoelasticity measurements were run after the 30 min rest. We have checked that those protocols led to reproducible initial states. Frequency sweeps, strain sweep and stress relaxation measurements were performed. The extent of the linear regime was probed by running strain sweeps typically at a frequency of 1 rad/s. Frequency sweeps and stress relaxation were performed in the linear regime, with amplitude of the applied shear strain typically between 1 and 20%. Some additional tests were performed by loading a freshly prepared sample in the Couette cell of the rheometer and probing regularly the viscoelasticity of the sample over a period of 8 days. The results are shown in Supplementary data and demonstrate that the sample loading does not play any role on the gelation process.

Scattering experiments. Details concerning the experimental conditions for the scattering experiments are described in [START_REF] Dahesh | Polymeric assembly of gluten proteins in an aqueous ethanol solvent[END_REF] In brief, small-angle scattering experiments were conducted on different instruments using either X-ray or neutron beam. For the very low wave vector range (2.10 -3 -10 -1 nm -1 ), data were measured using neutrons on the KWS3 instrument operated by FRMII at the Heinz Maier-Leibnitz Zentrum, Garching, Germany. The ID2 beam line of the European Synchrotron radiation facilities (ESRF) at Grenoble, France, was used for the measurement of the intensity scattered in the intermediate q-range (0.04 -1.5 nm -1 ). Finally, an X-ray laboratory set-up was used to measure data at large wave vectors.

Infrared Spectroscopy. Fourier Transformed InfraRed (FTIR) spectroscopy experiments were performed on an Alpha FTIR Bruker apparatus equipped with the single reflection diamond Attenuated Total Reflection (ATR) module. Spectra were recorded by the co-addition of 24 scans at a resolution of 8 cm -1 . In order to isolate the protein signal, the background was measured with the solvent used to prepare the samples.

All measurements were performed at room temperature. Interestingly, all data acquired at different concentrations (in the range 105 to 528 mg/ml) and different ageing times (in the range 1 to 57 days) can be collapsed onto one of the two different master curves once the moduli (resp. the frequency) are rescaled by time-and concentration-dependent prefactors, as shown in Figure 2C-D, where bG'and bG" are plotted as a function of a . Note that few data sets where, in the whole experimentally accessible frequency range, G' and G" vary as a powerlaw, could not be discriminately associated with one of the two master curves corresponding to pre-gel and post-gel behaviors. Those data sets correspond to the gel point following the Winter and Chambon criterium [START_REF] Winter | Analysis of linear viscoelasticity of a cross-linking polymer at the gel point[END_REF].

The scalings evidenced by the building of the master curves nicely demonstrate the self-similarity of the sample viscoelasticity with concentration and ageing time. The pre-gel and post-gel master curves gathering all experiments are shown in Figure S2 (in SI). The two master curves are strikingly similar to those measured for cured synthetic polymers [START_REF] Adolf | Time-cure superposition during cross-linking[END_REF]. The pre-gel master curve (Fig. 2C) obtained at low concentrations and ageing times displays a terminal behavior at small frequency followed at large frequency by a regime where both moduli vary with the same power law with frequency, G'~G"~ with a critical exponent =0.8 [START_REF] Winter | Analysis of linear viscoelasticity of a cross-linking polymer at the gel point[END_REF]Winters & Mours, 1997). The same critical power law behavior is measured at large frequencies for the post-gel master curve obtained at larger concentrations and ageing times, and at lower frequency a cross-over toward an elastic regime with a frequency-independent storage modulus, G' 0 , larger than the loss modulus is measured. Note that stress relaxation measurements in the linear regime provide fully consistent results. As shown in Figure 3A, for short ageing time and for time larger than 0.1 s, the relaxation modulus G(t) monotonically decays as a power law with time with an exponent -. We found that the numerical value of the parameter is equal to the value measured at high frequency in a frequency sweep test (Fig. 2) as expected for a powerlaw gel [START_REF] Ng | Power law gels at finite strains[END_REF][START_REF] Winter | Analysis of linear viscoelasticity of a cross-linking polymer at the gel point[END_REF]. By contrast for long ageing times, G(t) decays in a more complex fashion and reaches a non-zero plateau at long times, G' p , which is the signature of a solid-like behavior, and is in quantitative agreement with the low-frequency plateau measured for the storage modulus in oscillatory measurements (G' 0 ). This is illustrated in Figure 3B for a sample of concentration C=317 mg/ml whose elastic plateau has been measured at different ageing times. We find that for all ageing times above gelation the elastic modulus measured by a stress relaxation test, G' p, is equal to that measured by a frequency sweep test, G' 0 , showing the consistency of our measurements and the reproducibility of our data.

Before gelation, we measure that the amplitude of the complex viscosity 𝜂 * = √ (

𝐺 ′ 𝜔 ) 2 + ( 𝐺 ′′ 𝜔 )
2 increases with ageing time (fig. 4A). * is found to decrease as a function of the frequency due to the shear thinning of the viscous suspensions. At short ageing time, a plateau at low frequency, 𝜂 0 * , is eventually reached, allowing to extract a zero-frequency complex viscosity. Master curves for a sample at different ageing times are built by normalizing the complex viscosity and the frequency by parameters that depend on the ageing times. This allows the evaluation of the zero-frequency complex viscosity, which is equivalent to the zero-shear viscosity following the so-called Cox-Merz rule, even

Comment citer ce document : Dahesh, M., Banc, A., Duri, A., Morel, M. H., Ramos, L. ( 2016). Spontaneous gelation of wheat gluten proteins in a food grade solvent. Food Hydrocolloids, 52, 1-10. DOI : 10.1016/j.foodhyd.2015.06.014 5 when it is not accessed experimentally and only the shear thinning regime at higher frequency is measured (Fig. 4B).

We show in Figure 5A that the zero-frequency complex viscosity increases both with ageing time and with concentration. This viscosity is expected to diverge at the gel point, which is not easily measured in the present study due to the very slow processes that take place over several days. We indeed evaluate, based on the transition from pre-gel to post-gel master curves, that the gel point decreases with concentration from ~ 57 days for C=185 mg/ml to ~1 day for C=238 mg/ml (Fig. 5B). Thanks to the post-gel master curve, one can evaluate the elastic plateau even when the rheological data do not allow to measure it directly as it occurs at too low frequency to be accessed experimentally. Its evolution with ageing time for different concentrations is reported in Figure 5C. The elastic plateau spans several orders of magnitude and displays a fast initial increase followed by a slower increase at larger time. This two-step kinetics is better emphasized in rescaled data (Fig. 5D), where both the elastic plateau is rescaled by the elastic plateau at long time, G' inf , and the ageing time by, t*, the cross-over time between the fast and the slow gelation regimes. We measure that t* continuously decreases with increasing concentration (inset Fig. 5D). A two-step kinetics, as also previously observed for instance for gelatin or chitosan hydrogels [START_REF] Normand | Gelation kinetics of gelatin: a master curve and network modeling[END_REF][START_REF] Hamdine | Viscoelastic properties of phosphoric and oxalic acid-based chitosan hydrogels[END_REF], reflects an initially fast cross-linking regime which subsequently slow down as further cross-linking or reinforcement of the existent cross-linking is hindered.

Critical gelation and link to the structure

Here, we analyze the rheological data in the framework of critical gelation. For a polymer undergoing a cross-linking reaction, 𝜀 = |𝑝 -𝑝 𝑐 | is defined as a measure of the distance from the gel point, where 𝑝 is the extent of the cross-linking reaction, and 𝑝 𝑐 is the critical extent for which an infinite cluster percolate over the whole sample [START_REF] Winter | Analysis of linear viscoelasticity of a cross-linking polymer at the gel point[END_REF]Winters & Mours, 1997[START_REF] Stauffer | Gelation and critical phenomena[END_REF]. A critical polymer gel occurs at 𝑝 𝑐 , for which 𝐺′ ∝ 𝐺" ∝ 𝜔 𝛥 . In the post-gel state, both the elastic plateau, G' 0 , and the characteristic time at which G' and G" cross, , are predicted to follow critical variations with the distance from the gel point [START_REF] Martin | Viscoelasticity of near-critical gels[END_REF][START_REF] Martin | Viscoelasticity near the sol-gel transition[END_REF]: 𝐺′ 0 ∝ 𝜀 𝑧 and 𝜏 ∝ 𝜀 -𝑦 , with critical exponents z and y such that = z/y. This leads to a power law relation between the two viscoelastic parameters, G' 0 and : 𝐺′ 0 ∝ 𝜏 -𝛥 , where an exponent that is the opposite to that of the scaling of the complex modulus at the critical point is expected. We have directly tested this prediction.

In Figure 6A, we demonstrate that, for more than 70 experimental conditions (spanning half a decade of concentration and almost two decades of ageing times) 𝐺′ 0 decreases as a power law with 𝜏 over 8 orders of magnitude yielding 𝐺′ 0 ∝ 𝜏 -𝑚 , with 𝑚 = 0.9 ± 0.2 , remarkably close to 𝛥 = 0.78 ± 0.07, the exponent for the evolution of the complex modulus with the frequency, as expected theoretically.

Hence our data present the hallmarks of viscoelasticity near critical gel. Those models, initially developed for polymer gels, have been found to satisfyingly account for the viscoelasticity of a large variety of synthetic polymers, including polydimethylsiloxane [START_REF] Chambon | Linear viscoelasticity at the gel point of a cross-linking PDMS with imbalanced stoichiometry[END_REF], polybutadienes (Mours & Winter, 1996), or polyacrylamide [START_REF] Larsen | Microrheology of the liquid-solid transition during gelation[END_REF] and also of biomacromolecules, including alginate [START_REF] Matsumoto | Viscoelastic and SAXS investigation of fractal structure near the gel point in alginate aqueous systems[END_REF], chitosan [START_REF] Hamdine | Viscoelastic properties of phosphoric and oxalic acid-based chitosan hydrogels[END_REF], gelatin [START_REF] Guo | Physical gelation of gelatin studied with rheo-optics[END_REF], and peptides [START_REF] Larsen | Microrheology of the liquid-solid transition during gelation[END_REF][START_REF] Cingil | Equivalent pathways in melting and gelation of well-defined biopolymer networks[END_REF].

A key ingredient of the critical gel model is the fractal nature of the gel. We have directly checked this assumption using scattering experiments, combining wide and small-angle X-ray scattering and ultraslow small-angle neutron scattering. Our data (fig. 6B) show that, over almost two decades, the scattered intensity decays as a power law with the scattering vector, q: 𝐼~𝑞 -𝑑 𝑓 , proving the fractal structure of the gluten gel with a fractal dimension 𝑑 𝑓 = 2. We find the same values for all gels with different concentrations. Note that this value is precisely the one theoretically expected for a branched polymer in good solvent as given by lattice-animal models [START_REF] Martin | Viscoelasticity near the sol-gel transition[END_REF]. This value is moreover fully consistent with our structural measurements in dilute regime [START_REF] Dahesh | Polymeric assembly of gluten proteins in an aqueous ethanol solvent[END_REF] that 6 demonstrate that the protein extract used here display in an ethanol/water 50/50 solvent the structural and dynamical hallmarks of polymer coils in good solvent conditions. Furthermore, models have been developed to relate the critical exponent of the viscoelasticity to structural properties of the polymer near the gelation point. In the case of unscreened excluded-volume interactions percolation theory predicts 𝛥 = 𝑑/(𝑑 𝑓 + 2), where d=3 is the space dimension and 𝑑 𝑓 is the fractal dimension of the polymer [START_REF] Muthukumar | Screening effect on viscoelasticity near the gel point[END_REF]. With 𝑑 𝑓 = 2, one predicts 𝛥 = 0.75, very close to our experimental values (𝛥 = 0.78). Note that if we assumed instead screened excluded-volume interactions we would expect 𝛥 = [𝑑(𝑑 + 2 -2𝑑 𝑓 )]/[2(𝑑 + 2 -𝑑 𝑓 ) = 0.5 for 𝑑 𝑓 = 2, hence very different from our experimental findings. This unambiguously confirms that the hydrodynamic interactions between the polymer chains are screened, as expected for polymer in good solvent conditions. Quantitative direct connections between structure and visco-elasticity for near-critical gels are very scarce in the literature [START_REF] Matsumoto | Viscoelastic and SAXS investigation of fractal structure near the gel point in alginate aqueous systems[END_REF][START_REF] Takenata | Comparison in fractal dimension between those obtained from structure factor and viscoelasticity of gel networks of 1,3:2,4-bis-O-(p-methylbenzylidene)-D-sorbitol in polystyrene melt at gel point[END_REF]. Here, our structural and rheological measurements in a concentrated regime provide consistent results in excellent agreement with structural measurements of the protein suspension in a dilute regime.

Origin of the spontaneous gelation process

To get some insight about the physical origin of the slow gelation, we first have measured over ageing time the molecular weight distribution of the proteins using high-performance liquid chromatography (HPLC) in a SDS-buffer (this buffer is expected to disrupt all weak bonds). Data (Fig. 7) clearly show a weak but continuous decrease of the peak corresponding to low molecular weight (MW) proteins (gliadin, MW around 30 kDa) and the region corresponding to (short) glutenin polymers (MW in the range 80-300 kDa). By contrast, the excluded peaks related to very large assemblies of proteins increases. New interchain disulfide bonds arising from disulfide /thiol exchange or thiol oxidation, as observed upon heating and/or mixing of gluten [START_REF] Morel | Mechanism of heat and shear mediated aggregation of wheat gluten protein upon mixing[END_REF][START_REF] Auvergne | Reactivity of wheat gluten protein during mechanical mixing: Radical and nucleophilic reactions for the addition of molecules on sulfur[END_REF], could be at the origin of our observations.

Accordingly, in the presence of a chemical (N-Ethylmaleimide, NEM) that blocks the free thiol groups and, thus, prevents any further disulfide-bonds to occur and hinders completely exchanges between existing disulfide bonds (Fig. 8A), we indeed measure a lower content of high molecular weight species. Moreover, we find that the HPLC profile of a 10 days old sample with NEM superimposes perfectly with the one of a 1-day old sample without NEM (Fig. 8B). Interestingly, we nevertheless find that the building-up of a gel still occurs. Indeed, for a sample containing an excess of NEM (1 mM, which corresponds to roughly 3.7 times more NEM than free thiol groups in the sample) an elastic modulus is measured. Moreover, the modulus is found to continuously increase with ageing time (fig. 8D). These data prove that an increase in disulfide interchain bonding is not essential for the gelation. The slightly lower elastic modulus, as compared to a reference sample without NEM, might nevertheless suggest a minor contribution of disulfide interchain bonding to the sample elasticity.

Experiments are also performed with β-mercaptoethanol (BME) which is a chemical commonly used to cleave disulfide bonds (Fig. 8A). BME shifts the thiol/disulfide balance of proteins, through the reduction of their inter-or intra-molecular disulfide bonds. In catalytic amount it can promote the reshuffling of protein disulfide bonds through thiol/disulfide exchange and therefore could assist protein unfolding. When used at a concentration of 3 mM in the solvent, which corresponds to about 13% of the total disulfide bonds, BME induces a shift of molecular weight distribution towards smaller protein species indicating a partial breakdown of inter-molecular disulphide bonds (Fig. 8B) but leads to a faster kinetics for the gel build-up (Fig. 8D), presumably thanks to a faster shuffling of the S-S bonds yielding a facilitated gelation. Notably however, the final elastic plateau is comparable to that of the reference sample, suggesting that BME does not modify the number and strength of the junctions in the polymer gel. By contrast, larger quantities (0.3 M), which corresponds to a 13 fold excess of BME compared to the total number of disulfide bonds, cleave all the disulfide bonds, allowing a totally different type of gel to form very rapidly (Fig. 8D), presumably through the 7 formation of intermolecular -sheets (Fig. 8C). Indeed, FTIR spectra of gels prepared with the different thiol modifiers show that the amide I of protein gels is nearly unmodified in presence of NEM or 3 mM BME whereas in presence of large quantities of BME the shape of the amide I band is totally different and dominated by a contribution at 1620 cm -1 assigned to -sheets (Fig. 8C).

Conclusive results are derived from experiments using urea, a chemical that destabilizes H-bonds. A gel sample readily dissolves in an ethanol/water solvent containing 8 M urea, whereas it keeps its integrity when put into contact with an ethanol/water solvent (Fig. 9A) demonstrating unambiguously that H-bonds formation is the motor for gelation, as also assumed for standard water hydrated gluten [START_REF] Ng | Large amplitude oscillatory shear flow of gluten dough: A model power-law gel[END_REF]. Size-exclusion chromatography confirms moreover that the MW size distribution profile is preserved by urea addition (Fig. 9B). The H-bonding could occur between the side amide groups of the glutamine aminoacids which are abundant in the repetitive domains of the gluten proteins (Fig. 1A) and are known to be prone to interact through H-bonds [START_REF] Rhys | The hydrogen-bonding ability of the amino acid glutamine revealed by neutron diffraction experiments[END_REF]. Note however that contrarily to what was observed with BME (0.3M), no modification of the infrared spectra could be detected with aging time. We believe that, in this case, the long polymer chains prevent the building of cooperative H-bonds as in interchain -sheets but instead a few isolated Hbonds forms, which are responsible to the slow gelation of the samples, but are not sufficient to lead to a significant change in the infrared signal with time.

Rheological properties of the novel gluten gels

We have quantified the rheological properties of the gels after long ageing times, once the mechanical properties do not significantly evolve any more. We measure that the elastic plateau at long time, G' inf , can be varied over a much extended range of numerical values, from 10 -2 Pa to 10 4 Pa (fig. 10A). As shown the elastic modulus grows very rapidly with concentration as it increases by almost 6 orders of magnitude while the concentration varies by a factor of 2. For concentration above 400 mg/ml, the elastic plateau, G' inf, increases smoothly with concentration, whereas below this concentration, the dependence of G' inf with concentration is very strong. Our data suggest that the plateau increases exponentially with the protein concentration.

Interestingly, an exponential increase of the elastic modulus with concentration has been recently observed with bicontinuous solid-stabilized emulsions [START_REF] Witt | Bijel reinforcement by droplet bridging: a route to bicontinuous materials with large domains[END_REF] and with protein gels formed by spinodal decomposition [START_REF] Gibaud | Unexpected decoupling of stretching and bending modes in protein gels[END_REF], where it has been interpreted using a model for a porous medium [START_REF] Pal | Porosity-dependence of effective mechanical properties of pore-solid composite materials[END_REF]. Given the structure of the gels investigated here, an analogy with a porous medium is not obvious. At the present time the physical origin of the very strong dependence of the elastic modulus with concentration remains to be clarified. On the other hand, our data can be compared with existing data for standard hydrated gluten. For a protein concentration of 400 mg/ml, the typical value for hydrated gluten, the elastic modulus of our novel gel is of the order of 4000 Pa, yielding a three to five-fold stronger gel than standard hydrated gluten (800-1500 Pa) [START_REF] Ng | Power law gels at finite strains[END_REF][START_REF] Pruska-Kedzior | Comparison of viscoelastic properties of gluten from spelt and common wheat[END_REF]. Hence, substitution of water by an ethanol/water mixture does not alter the viscoelastic characteristics of gluten proteins. However, in striking contrast with hydrated gluten, we are able to produce gels at unprecedented low gluten protein concentrations thus generating materials encompassing a broad range of lower elastic plateau values as compared to standard gluten. The novel gluten gels also exhibit a surprisingly large linear regime, as shown in Figure 10B where both the storage and loss modulus are found to be independent of the strain amplitude up to 𝛾 𝑐 ≈ 300%, again in sharp contrast with a wheat dough (𝛾 𝑐 < ~1%) [START_REF] Cornec | Characterisation of gluten subfractions by SE-HPLC and dynamical rheological analysis in shear[END_REF][START_REF] Hibberd | Dynamic viscoelastic behaviour of wheat flour doughs[END_REF][START_REF] Phan-Tien | Oscillatory and simple shear flows of a flourwater dough: a constitutive model[END_REF][START_REF] Uthayakumaran | Small and large strain rheology of wheat gluten[END_REF] and a standard hydrated gluten (𝛾 𝑐 is typically a few %) [START_REF] Cornec | Characterisation of gluten subfractions by SE-HPLC and dynamical rheological analysis in shear[END_REF][START_REF] Létang | Characterization of wheat flour-water doughs. Part I: Rheometry and microstructure[END_REF][START_REF] Uthayakumaran | Small and large strain rheology of wheat gluten[END_REF]. Moreover, the gels can be extended considerably before fracturing, pinpointing at a ductile behavior. In addition, they exhibit self-healing, highlighting the reversible/physical links building up the gel (fig. 10C). These features are in sharp contrast with other gels made with large amounts of BME, which are very brittle without self-healing properties, and for which all proteins have been cleaved (fig. 8B). Those

Comment citer ce document : Dahesh, M., Banc, A., Duri, A., Morel, M. H., Ramos, L. ( 2016). Spontaneous gelation of wheat gluten proteins in a food grade solvent. Food Hydrocolloids, 52, 1-10. DOI : 10.1016/j.foodhyd.2015.06.014 8 contrasting results highlight the crucial role of the native polymeric species in the remarkable rheological properties of the novel gluten gels.

Conclusions

We have shown that water/ethanol soluble blends of gliadin and glutenin (~ 50/50) extracted from gluten undergo a spontaneous gelation, which is driven by the formation of H-bonds. We have quantitatively rationalized the gelation using theoretical percolation models. The mechanical properties of the as-prepared gels can be tailored over an unprecedented range. They display shear elastic modulus spanning almost six orders of magnitude and exhibit an unexpectedly large linear elastic domain, in striking contrast with conventional hydrated gluten. We expect that those novel gels could serve as starting materials for the processing of plant-based protein food products. In the form of biodegradable glue or as electrospun fibers the protein blend could also find non-food applications. As such, the protein fraction investigated here constitutes a promising model to understand the molecular basis of the gluten elastic behavior. Contrarily to simple gluten/water mixture, which pre-exists as a gel, it offers the possibility to investigate in great details the interaction potential of the two main gluten protein classes, gliadin and glutenin. How screening of protein charges by salt and pH alter the gel formation kinetic and its elastic properties will be the subject of our future investigations.

Comment citer ce document : Dahesh, M., Banc, A., Duri, A., Morel, M. H., Ramos, L. ( 2016). Spontaneous gelation of wheat gluten proteins in a food grade solvent. Food Hydrocolloids, 52, 1-10. DOI : 10.1016/j.foodhyd.2015.06.014

11 Symbols correspond to data points evaluated from the scaling factors, a and b, used to fall the frequencydependent complex moduli onto the post-gel master-curve. The post-gel master curve gathering all experimental data is shown in SI (Fig. S1). The power law evolution (black line) is characteristic of near-critical gel viscoelasticity (B) Scattering profile of a sample (C=290 mg/ml) as obtained from combining several scattering techniques. The profile is typical of a polymer gel in good solvent conditions and evidences a hierarchical structure (as schematized in the cartoon). At large scattering vectors the wide peak corresponds to the liquid like order of the sample, the change of slope at q~3nm -1 gives the persistence length of polymeric chains (l 0 =0.7 nm) and the transition around q~5nm -1 is characteristic of the polymer blob size (ξ=1.8 nm). The power law decay at small wave vector indicates large scale heterogeneities characterized by a fractal dimension d f =2. More details are provided in [START_REF] Dahesh | Polymeric assembly of gluten proteins in an aqueous ethanol solvent[END_REF].

Supplementary data

Comparison between in-situ ageing and ex-situ ageing Because of the long ageing times, samples usually age in a hermetically closed container (ex-situ gelation). Aliquots of those samples are regularly loaded in the rheometer to probe their viscoelasticity, following a protocol described below in the Method section. To check for the possible role of the sample loading on the aging behavior, some additional tests were performed by loading a freshly prepared sample in the Couette cell of the rheometer and probing regularly the viscoelasticity of the sample over a period of 8 days. We quantitatively compare the viscoelasticity for a sample allowed to age in-situ to that of a sample that has aged ex-situ. We show in figure S1 comparable evolution with ageing time of the zerofrequency elastic plateaus for in-situ and ex-situ gelation of a sample with concentration C= 317 mg/ml. This proves that our protocol leads to results comparable to those obtained when a sample is allowed to age directly in the rheometer cell with a layer of silicon oil on the top to prevent evaporation of the solvent (in-situ gelation). Pre-gel and post-gel master curves

Figure 1 :Figure 2 :

 12 Figure 1: Gluten proteins and samples (A) Typical primary structures of both families of gluten proteins: gliadins and glutenins. Repetitive domains rich in proline (P), glycine (G) and glutamine (Q) residues are highlighted in blue. Cysteine residues involved either in intramolecular (inner lines) or intermolecular (outer lines) disulfides bonds are represented as short vertical rods (in red). Gliadins contain 0, 6 or 8 cysteines involved in intra-molecular disulphide bonds. Glutenins contain 4, 5 or 7 cysteines involved in intramolecular bonds and 1 or 3 cysteines involved in intermolecular disulphide bonds. (B) Comparison of protein dispersions: samples are prepared by dispersing (a) commercial gluten (the starting material used for protein fractionation) in water, (b) the gluten protein extract in water, and (c) the gluten protein extract in a 50/50 (v/v) water/ethanol solvent. In the three samples, the protein concentration is C=210 mg/ml. (C) Homogeneous gluten gels, with various protein concentrations as indicated on the picture, produced by dispersing the gluten protein extract in a 50/50 (v/v) water/ethanol solvent.

Figure 3 :

 3 Figure 3: Stress relaxation of the gluten suspensions and gels (A) Stress relaxation in the linear regime of a gel (C=317 mg/ml) at different ageing times. The amplitude of the sudden imposed shear strain was well in the linear regime (between 5% and 50%). (B) Comparison of the elastic moduli of samples at different ageing times measured by shear stress relaxation tests (G' p ) and by frequency sweep tests (G' 0 ). Symbols are experimental data and the line corresponds to G' p = G' 0 .

Figure 4 :

 4 Figure 4: Complex viscosity of the gluten suspensions (A) Complex viscosity * of samples with concentration C=211 mg/ml, for different ageing times (as indicated in the legend) as a function of the frequency. (B) Master curve obtained shifting data horizontally and vertically using scaling factors a' and b'. The zero shear complex viscosity is 0 *=1/b'.

Figure 5 :

 5 Figure 5: Kinetics and gel point (A) Zero shear complex viscosity 0 * of pre-gel samples of different concentrations as indicated in the legend as a function of ageing time. (B) Evolution of the gel point as a function of protein concentration. (C) Low frequency elastic modulus G' 0 of post-gel samples of different concentrations as indicated in the legend as a function of ageing time. Lines are guides for the eyes. (D) Master curve of post-gel kinetics, obtained from rescaling the data shown in (C). The insert displays the evolution of the characteristic time t* used to produce the main plot.

Figure 6 :

 6 Figure6: Critical gelation and link to the structure of the gel (A) Low frequency elastic modulus of gels as function of the relaxation time at the crossover between G' and G''. Symbols correspond to data points evaluated from the scaling factors, a and b, used to fall the frequencydependent complex moduli onto the post-gel master-curve. The post-gel master curve gathering all experimental data is shown in SI (Fig.S1). The power law evolution (black line) is characteristic of near-critical gel viscoelasticity (B) Scattering profile of a sample (C=290 mg/ml) as obtained from combining several scattering techniques. The profile is typical of a polymer gel in good solvent conditions and evidences a hierarchical structure (as schematized in the cartoon). At large scattering vectors the wide peak corresponds to the liquid like order of the sample, the change of slope at q~3nm -1 gives the persistence length of polymeric chains (l 0 =0.7 nm) and the transition around q~5nm -1 is characteristic of the polymer blob size (ξ=1.8 nm). The power law decay at small wave vector indicates large scale heterogeneities characterized by a fractal dimension d f =2. More details are provided in[START_REF] Dahesh | Polymeric assembly of gluten proteins in an aqueous ethanol solvent[END_REF]

Figure 7 :

 7 Figure 7: Ageing of the gluten gels High-performance liquid chromatography (HPLC) profiles of samples at different ageing times. Arrows indicate the evolutions with ageing time.

Figure 8 :Figure 9 :

 89 Figure 8: Role of di-sulfide bonds and thiol modifiers on the gluten gels (A) Chemical reactions of N-Ethylmaleimide (NEM) and β-mercaptoethanol (BME) with proteins containing free thiols and disulfide bond respectively. (B) HPLC profiles of fresh and 10 days old samples. Reference samples correspond to pristine samples (protein in water/ethanol) without additional chemicals. The other samples include a fresh sample containing 0.3 M BME and 10-days old samples containing 1 mM NEM or 3mM BME. Data are normalized by the protein concentration in the gel. (C) Amide I band of 10 days old samples aged with NEM or BME measured by infrared spectroscopy. (D) Comparison of the time-evolution of the low frequency elastic modulus of samples with concentration C=317mg/ml prepared without thiol modifiers (reference sample) and with different thiol modifiers: 0.3M of BME, 3mM of BME and 1 mM of NEM. The pictures illustrate the brittle structure of the gel prepared with 0.3M of BME compared to the ductility of the gels obtained in the other cases.

Figure 10 :

 10 Figure 10: Mechanical characteristics of the gluten gels (A) Low frequency elastic modulus of gels obtained at an infinite ageing time, G' inf , as a function of protein concentration. The diamond displays the characteristic value obtained for standard hydrated gluten. (B) Strain sweep of a 56-day old gel of concentration C=310 mg/ml illustrating the extremely large linear regime of the gels which can reach more than 300%. (C) Pictures illustrating the self-healing behavior of gels (here an 8-day old gel with C=537 mg/ml). Top left: pieces of gel that are used to form the healed material (bottom left). Right: stretching of the healed sample after a 10 minutes rest.

A

  Comment citer ce document : Dahesh, M., Banc, A., Duri, A.,Morel, M. H., Ramos, L. (2016). Spontaneous gelation of wheat gluten proteins in a food grade solvent. Food Hydrocolloids, 52, 1-10. DOI : 10.1016/j.foodhyd.2015.06.014 17

Figure S1 :

 S1 Figure S1:Comparison of G 0 ' measured for a sample with concentration C=317 mg/ml at different ageing times using different protocols. The sample has aged either in a flask and has been loaded in the rheological cell before each measurement (ex-situ), or has aged directly in the Couette cell of the rheometer with a silicon oil drop on the top to prevent solvent evaporation (in-situ).

Figure S2 :

 S2 Figure S2: (A) Pre-gel mastercurve plotted with data obtained with 20 different measurements. The concentration range of samples was (105-211) mg/ml and their ageing time was in the range (1-77) days. (B) Post-gel mastercurve plotted with data of 69 measurements. The concentration range of samples was (185-528) mg/ml and their ageing time was in the range (1-77) days. In A and B, the numbers x/y in the caption indicate the concentration in mg/ml (x) and the ageing time in days (y).

Results and discussion 3.1. Ageing time-and concentration-dependent viscoelasticity

	3. We investigate the rheological properties of the stable dispersions of the gluten protein blend in
	water/ethanol (50/50 v/v). Standard linear viscoelasticity measurements of the frequency-dependence
	of the storage modulus, G', and the loss modulus, G", are shown in Figure 2A-B for samples with
	different protein concentrations, C (105, 185, 317 and 370 mg/ml), and different ageing times (2, 7 and
	10 days). At low ageing time/concentration the terminal relaxation of viscoelastic materials is
	measured: the loss modulus, G", is larger than the storage modulus G', and G"~ and G'~ 2 , with
	the frequency. As ageing time and concentration increase, both moduli are found to increase and
	eventually a cross-over from a viscous fluid (G">G') to an elastic solid (G'>G") is measured.
	4
	Comment citer ce document :
	Dahesh, M., Banc, A., Duri, A., Morel, M. H., Ramos, L. (2016). Spontaneous gelation of wheat
	gluten proteins in a food grade solvent. Food Hydrocolloids, 52, 1-10. DOI :
	10.1016/j.foodhyd.2015.06.014

Acknowledgements

We thank J. Bonicel (IATE) for help in the SE-HPLC, P. Dieudonné (L2C) for the in-house SAXS measurements and C. Charbonneau (L2C) for help during the neutron scattering measurements. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities. We would like to thank Pawel Kwasniewski for assistance in using beamline ID02 at ESRF and Zhendong Fu for assistance during the neutron scattering experiments. This work was supported by the Laboratoire of Excellence NUMEV (ANR-10-LAB-20) and INRA (CEPIA department). Besides, this research project has been supported by the European Commission under the 7th Framework Program through the "Research Infrastructures" action of the Capacities Program, NMI3-II, Grant Agreement number 28388 to perform the neutron scattering measurements at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany.


Ana Mayela Ramos-De-La-Peña

Catherine M G C Renard

Louise Wicker

Julio C Montañez

Luis Alfonso García-Cerda

Juan Carlos Contreras-Esquivel

Environmental friendly cold-mechanical/sonic enzymatic assisted extraction of genipin from genipap (Genipa americana)

Keywords:

An efficient cold-mechanical/sonic-assisted extraction technique was developed for extraction of genipin from genipap (Genipa americana) peel. Ultrasound assisted extraction (285 W, 24 kHz) was performed at 5, 10 and 15 °C for 5, 10 and 15 min. After cold-extraction, genipin was separated from pectin and proteins by aid of fungal pectinesterase. The maximum yield of non-cross-linked genipin was 7.85 ± 0.33 mg/g, at 10 °C for 15 min by means of ultrasound extraction. The protein amount in extracts decreased in all samples. If mechanical process is combined with ultrasound assisted extraction the yield is increased by 8 times after the pectinesterase-assisted polyelectrolyte complex formation between pectic polysaccharides and proteins, avoiding the typical cross-linking of genipin. This novel process is viable to obtain non-cross-linked genipin, to be used as a natural colorant and cross-linker in the food and biotechnological industries.

Introduction

Genipa americana L. is a tree that is widely distributed from Mexico to the Caribbean region and South America [START_REF] Djerassi | Naturally ocurring oxygen heterocyclics. IX 0 . Isolation and characterization of genipin[END_REF]. It is also known as jagua, juito, huito and genipa (Spanish areas in Latin America), genipap and genipa (English), bois de fer (French) and genipapo (Portuguese) [START_REF]BTFP (BioTrade initiative/BioTrade facilitation programme), Market brief in the European Union for selected natural ingredients derived from native species. Genipa americana[END_REF]. G. americana and Gardenia jasminoides Ellis fruits contain genipin, an iridoid cross-linking compound [START_REF] Chiono | Genipincrosslinked chitosan/gelatin blends for biomedical applications[END_REF][START_REF] Ramos-Ponce | A simple colorimetric determination of the free amino groups in water soluble chitin derivatives using genipin[END_REF] that is able to react spontaneously with primary amino acids groups, peptides or proteins to form dark blue pigments. It is soluble in water, alcohol and propylene-glycol and it presents stability in the pH range from 4 to 9.

Genipin cytotoxicity is lower than other chemical cross-linking reagents. It has been reported that it is approximately 5000-10,000 times less cytotoxic than glutaraldehyde. Due to its healthy attributes and to its ability to develop blue color, genipin has been used as a natural food colorant in some regions of Asia [START_REF] Akao | Enzymic studies on the animal and intestinal bacterial metabolism of geniposide[END_REF]. Genipin also has been used to cross-link molecules such as chitosan or proteins [START_REF] Fraga | Kinetic study of chitosane/genipin system using DSC[END_REF]. In the cross-linking reaction, a bimolecular substitution occurs, that includes a substitution of an ester group in the genipin molecule by a secondary amide. The second reaction is a nuclephilic substitution of ester clustering. The cross-linking reactions allow the formation of blue pigments that include other reactions with a higher level of complexity [START_REF] Renhe | Obtention of blue colorant from jenipapo fruit[END_REF].

The blue color plays an important role in the food development industry. There is a need of natural blue colorants in the beverage, ready-meals, soups, breakfast cereal production, as well as in pet food, confectionery and baby food industries [START_REF]BTFP (BioTrade initiative/BioTrade facilitation programme), Market brief in the European Union for selected natural ingredients derived from native species. Genipa americana[END_REF]. The food industry has taken advantage of genipin characteristics in order to use it as a natural colorant in beverages [START_REF] Ueda | Production of anti-tumor-promoting iridoid glucosides in Genipa americana and its cell cultures[END_REF], juices, nectars, desserts and gels [START_REF] Barbosa-Da-Silva | Frutas Nativas da Regiaõ Centro-Oeste do Brasil[END_REF]. Moreover, genipin has also been used in the biomedical area as biomaterials cross-linking agent [START_REF] Muzzarelli | Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids[END_REF], in the forensic science as fingerprint developer [START_REF] Almog | Genipin -A novel fingerprint reagent with colorimetric and fluorogenic activity[END_REF], and in the textile industry, when cotton, wool and leathers are dyed [START_REF] Taylor | Potential application for genipin-modified gelatin in leather processing[END_REF][START_REF] Ding | Tanning effects of aluminum-genipin orvegetable tannin combinations[END_REF]. In addition, genipin has been included in analytical purposes, such as aminoacid quantification [START_REF] Lee | Colorimetric determination of amino acids using genipin from Gardenia jasminoides[END_REF], where studies have been carried out to elucidate the mechanism and kinetics of the cross-linking reaction between biopolymers containing primary amine groups and genipin [START_REF] Butler | Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin[END_REF]. Ramos-Ponce et al. ( 2010) have demonstrated that D-glucosamine is an amino sugar that can be cross-linked with genipin to determine the amount of genipin that is present in a sample [START_REF] Ramos-Ponce | A simple colorimetric determination of the free amino groups in water soluble chitin derivatives using genipin[END_REF]. Fig. 1 shows the chemical structure of genipin.

The currently methods used for genipin recovery involve the use of organic solvents such as chloroform and very specific and complex steps for purification (concentration under reduced pressure, chromatography, crystallization, etc.) [START_REF] Endo | The Constituents of Gardenia jasminoides geniposide and genipin-getiobioside[END_REF], that makes the final product very expensive and difficult to obtain. It is necessary to find new and feasible alternatives for genipin recovery promoting the environmental preservation using emerging technologies and moderate temperatures which avoid the blue color formation during the extraction process. Recovery of genipin is certainly a critical technological step because aqueous extracts release simultaneously pectic substances and proteins. Recently, an enzymatic approach for phytochemical separation after the extraction process has been reported, where exogenous pectinesterase and calcium promote precipitation of water soluble pectin into calcium pectate [START_REF] Contreras-Esquivel | Eco-enzymatic method for phytochemical recovery with pectinesterase and carbohydrate oxidases, Mexican Patent, under submission[END_REF]. Ultrasound assisted technology has gained great interest nowadays due to industries can be provided with practical and reliable ultrasound equipment. Its emergence as green novel technology has attracted the attention because it is concerned to the environmental sustainability [START_REF] Chemat | Applications of ultrasound in food technology: processing, preservation and extraction[END_REF]. This technology has demonstrated to enhance the mass transfer by acoustic-induced cavitation in la liquid medium and the mechanical effects induced by cavitation bubbles that include microjet impatcs and shockwaveinduced damage [START_REF] Esclapez | Ultrasound-assisted extraction of natural products[END_REF] in this case, of the plant cell wall of G. americana L. fruit. Ultrasound is an emerging technology that enhances the acquisition of clean products after extraction [START_REF] Mason | The uses of ultrasound in food technology[END_REF].

To date, however there has been no report on eco-friendly assisted-extraction of genipin from genipap under mild conditions by means of ultrasound. The aim of this work is to investigate the genipin recovery from genipap fruit after mechanical and mechanical/sonic enzymatic assisted extraction at cold temperatures in order to obtain non-cross-linked genipin extract free from pectin and proteins. This purified extract can be used as a natural colorant in the food industry, in dairies, savory/bakery, soft drinks, confectionary and beverages or as a raw material for preparation of highly purified genipin.

Materials and methods

Materials

Genipap fruit was obtained from Cintalapa city, Chiapas state, Mexico. Pectinesterase (9347.72 U/mL; Lot number KENOO510; Novoshape) from Aspergillus aculeatus was purchased from Novozymes Ò (Krogshøjvej, Bagsvaerd, Denmark. Isopropyl alcohol (98%) was procured from Coyotefoods Biopolymer and Biotechnology Co. (Saltillo City, Coahuila State, Mexico). D-glucosamine was obtained from Cargill (Indianapolis, Minnesota, USA). Genipin was procured from Chengdu King-tiger, Pharm-chem, Tech. Co., Ltd. (Dayi, Chengdu, China). All other reagents were analytical grade.

Methods

Cold-mechanical extraction and enzymatic assisted treatment

Genipap fruit was peeled and the flesh was frozen until use. Peel was weighted and 50 g were taken for each experiment. Peel was cut with a stainless steel knife into cubes. Water (200 mL) at different temperatures (5, 15 and 30 °C) was added to the genipap peel pieces. Temperature was selected in order to explore the genipin recovery between 5 and 30 °C, to promote the use of moderate temperatures to avoid the genipin cross-linking when temperature is higher than 30 °C. The mixture was blended for 1 min and the extract and solid phase were recovered separately through filtration with cheese cloth. The solid phase was weighed and dried at 60 °C for 12 h. The extract was incubated for 1 h after the addition of 500 U of PE and 50 mM CaCl2 in order to achieve the enzyme activation, avoiding the enzyme trapping by carboxyl groups on pectin. This enhances the interaction between salt ions with negatively charged groups, allowing the enzyme to interact with the ester bonds to be cleaved [START_REF] Banjongsinsiri | Effect of pectinmethylesterase and calcium infusion on texture and structure of fruits and vegetables[END_REF]. Extracts without PE enzyme were referred as blanks. After incubation, extracts were centrifuged at 8873g for 20 min in a Biofuge primo R Sorvall Ò (Hanau, Hesse, Germany) and the supernatant was removed from the pellet after filtration through cheese cloth. Pellet was obtained with 10 mL of isopropyl alcohol and weight was recorded in wet and dry basis before and after drying at 60 °C for 12 h. Centrifuged extracts were subjected to pH and chemical analysis.

Cold-mechanical/sonic enzymatic assisted treatment

This extraction process was similar to the mechanical procedure, except that a smaller scale was used, also temperatures and times of treatment were different. Genipap fruit peel was cut with a stainless steel knife into cubes of regular size (1 Â 1 Â 1 cm) and 25 g were mixed for 1 min in a blender which contained water (100 mL) at 5, 10 and 15 °C. After blending, 7 mL of solid-liquid sample were taken and placed into a glass test tube with screw cap. The rest of the sample was discarded. The sample was subjected to ultrasound treatment in a cup horn (Qsonica, LLC, Newton, Connecticut, USA) at 5, 10 and 15 °C for 5, 10 and 15 min (285 W, 24 kHz). Finished the ultrasound assisted extraction, 500 U of PE and 720 mM CaCl 2 were added to the samples and they were incubated for 1 h at the corresponding temperature. After incubation, samples were filtered through cheese cloth and through filter paper (Whatman TM, GE Healthcare, Little Chalfont, Buckinghamshire, UK). The liquid phase was stored at 9 °C until use. The solid phase was dried at 60 °C for 12 h, then weight was registered. Blanks were defined as samples treated mechanically but without being subjected to ultrasound assisted extraction. Fig. 3 shows the flow diagram of the cold-mechanical/sonic enzymatic assisted extraction.

Chemical and statistical analysis

Samples of 5 mL were taken from extracts to determine the cross-linked genipin [START_REF] Ramos-Ponce | A simple colorimetric determination of the free amino groups in water soluble chitin derivatives using genipin[END_REF]. A calibration curve (Fig. 2a) was prepared with genipin (0-500 mg L À1 ) and the spectra of cross-linked genipin is showed in Fig. 2b. Samples were mixed with D-glucosamine (2000 mg L À1 ) and heated for 1 h in a boiling water bath. After cooling, absorbance of samples was determined at 589 nm in a Shimadzu UV-vis Photodiode Array Spectrophotometer MultiSpec-1501 (Greater Toronto area, Ontario, Canada). Protein content was also determined [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF]. Pectinesterase activity of the commercial preparation was determined (pH 4.5/40 °C; Pectin: 1%, w/v) [START_REF] Kertesz | Pectic enzymes[END_REF]. Each experiment was done in quintuplicate. Analysis of variance (ANOVA) was done using the Statistica (StatSoft Inc., Tulsa, OK, USA) software.

Results and discussion

Cold-mechanical extraction and enzymatic assisted treatment

Fig. 4 shows the effect of temperature on extracts pH without PE and when enzyme was added. In both cases pH decreased from The pH decrease is associated with the liberation of water soluble pectin after the vegetable tissue disintegration [START_REF] Schols | Structural characterization of pectic hairy regions isolated from apple cell walls[END_REF] and with the formation of acetic acid and methanol upon the demethylation of pectin due to the pectinesterase activity [START_REF] Mccann | Plant cell wall architecture: the role of pectins[END_REF].

There was no statistical difference when temperature varied according to ANOVA. Cross-linked and non-cross-linked genipin liberation is reported in Table 1. The effect of temperature (5, 15 and 30 °C) on the genipin content can be seen in blanks and in extracts with PE. The cross-linked genipin content is higher than non-crosslinked due to genipin reacted with genipap proteins and also with D-glucosamine. In this case the total content of genipin (including free genipin) was cross-linked and quantified. The highest content of cross-linked genipin (2.41 ± 0.73) mg/g in blanks and with PE (2.02 ± 0.39) mg/g was obtained at 15 °C. The amount of cross-linked and non-cross-linked genipin was higher in blanks than in extracts with enzyme, this probably due to genipn could be trapped in the complex formed between pectin-proteins and calcium due to the intermolecular binding after the CaCl 2 addition [START_REF] Contreras-Esquivel | Eco-enzymatic method for phytochemical recovery with pectinesterase and carbohydrate oxidases, Mexican Patent, under submission[END_REF][START_REF] Yamasaki | Pectic enzymes in the clarification of apple juice part I. Study in the clarification reaction in a simplified model[END_REF][START_REF] Banjongsinsiri | Effect of pectinmethylesterase and calcium infusion on texture and structure of fruits and vegetables[END_REF]. Protein content and pellet weight in wet basis was estimated and Fig. 5 describes the behavior of protein liberation (Fig. 5a) and pellet yield (Fig. 5b) in blanks and in extract with PE when temperature was raised. The protein content increased as temperature was higher in blanks and when PE was added. This in agreement with solubilization of cell wall components when heat is increased. However, proteins in blanks were $2 times higher than in samples with enzyme. Yields of pellets were higher in the extracts with PE and they increased as temperature did. It has been reported that fungal PE has high specific activity at pH 3.5 [START_REF] Deuvetter | Mode of de-esterification of alkaline and acidic pectin methylesterases at different pH conditions[END_REF] which indicates that PE demethoxyled pectin and a complex was formed with CaCl 2, where proteins were also included in the complex [START_REF] Waldron | Cell wall polysaccharides: composition and structure[END_REF].

Absorbance at 589 nm of all samples (extracts with PE and without enzyme) was recorded in order to study the change in color of genipap extracts during processing according temperature changes. They are showed in Fig. 6. Blanks presented minimal values of absorbance at 30 °C (0.12 ± 0.02) probably due to genipap fruit used for 5 and 15 °C treatments had genipin previously cross-linked.

There was no statistical difference between temperatures in extracts with enzyme according to ANOVA. Absorbance values in both cases were lower than 0.3, which indicates that using moderate temperatures (between 5 and 30 °C) makes feasible the genipin recovery without developing the dark blue color which is typical after genipin cross-linking under oxygen presence [START_REF] Djerassi | Naturally ocurring oxygen heterocyclics. IX 0 . Isolation and characterization of genipin[END_REF].

Cold-mechanical/sonic enzymatic assisted treatment

Due to statistical difference was not found in the recovery of genipin when temperatures between 5 and 30 °C were used in the mechanical extraction, the interval of temperatures for Temperature (°C) Temperature (°C) ultrasonic treatment was reduced to 5-15 °C in order to keep avoiding the cross-linking of genipin and to find the adequate temperature for the environmental friendly recovery of genipin. Time of sonication was also determined in order to achieve the highest yield of genipin in the shortest period of time (5-15 min). Genipin was quantified after mechanical (blanks) and ultrasound assisted extraction, Fig. 7 shows the effect of temperature on genipin liberation in blanks. Genipin was measured after being cross-linked with D-glucosamine and after heating and without the addition of D-glucosamine. It can be seen that the genipin content in samples with D-glucosamine was 8 times higher than in samples which were only heated. There was not statistical difference between treatments of each response. The amount of cross-linked genipin with D-glucosamine also was higher ($4 times) than the reported in the previous mechanical process described above. This difference is attributed to the PE activity, that led to the formation of the polyelectrolyte complex, that included proteins in the matrix that avoided the cross-linking of genipin [START_REF] Banjongsinsiri | Effect of pectinmethylesterase and calcium infusion on texture and structure of fruits and vegetables[END_REF].

Fig. 8 indicates the amount of genipin cross-linked with D-glucosamine subjected to heat treatment and the detected only after heating when ultrasound assisted extraction took place at 5, 10 and 15 °C for 5, 10 and 15 min. According to ANOVA, there was not statistical difference between treatments when time was considered, instead of, different temperatures caused the statistical difference in this process. In this case, the amount of genipin cross-linked with D-glucosamine and heat was slightly higher than the genipin that only was heated. When the extraction was carried out at 10 °C, the highest genipin yield detected after only heating was 7.85 ± 0.33 mg/g, then it decreased when temperature was 15 °C to 6.00 ± 2.02 mg/g and the lowest yield was detected at 5 °C, being 4.15 ± 0.49 mg/g. Ultrasound assisted extraction of genipin led to the production of the major content of available genipin at 10 °C when treated for 15 min. This only heated genipin represents the genipin that reacted with the primary amino groups from endogenous proteins of Genipa americana fruit after heating. Before heating no blue coloration was observed for this available genipin extracted with ultrasound, meanwhile genipin obtained from mechanical extraction (blank) only, turned its color to blue even without heating. Fig. 9 shows this fact in blanks (b) and the Temperature (°C) ultrasound assisted extracted genipin (a) did not change their appearance after 65 days (stored at 9 °C) of extraction. This is an advantage of the ultrasound extraction process, due to higher yield of available genipin can be recovered, without being cross-linked and without heating.

The ultrasound assisted extraction showed to be a reliable technology for genipin extraction. [START_REF] Mason | The uses of ultrasound in food technology[END_REF] [START_REF] Mason | The uses of ultrasound in food technology[END_REF], indicate that mechanical effects of ultrasound enhances the penetration of solvent into cellular materials that promotes the mass transfer, and that this emerging technology cause plant cell wall disruption, which facilitates the liberation of contents. These liberated contents have been reported earlier after ultrasonic extraction of hemicellulose release from buckwheat hulls [START_REF] Hromádková | Ultrasonic extraction of plant materialsinvestigation of hemicellulose release form buckwheat hulls[END_REF]. The contents included proteins, pectic polysaccharides and hemicellulose, basically. Fernandes and Rodrigues (2012) [START_REF] Fernandes | Ultrasound as pre-treatment for drying of genipap (Genipa americana L.)[END_REF] studied the previous treatment to air drying process of Genipa americana fruit by means of ultrasound for 20 min. They reported 16.6% of reducing sugars loss during the process in ultrasonic bath, caused by the difference in sugar concentration between the fruit and the liquid medium. In our study, genipin was detected in the extract after being liberated from the cell wall. Fig. 10 shows the protein content liberated after the ultrasound assisted extraction of genipin at 5, 10 and 15 °C for 5, 10 and 15 min. No statistical difference could be observed between treatments, except when the extraction was carried out at 5 °C for 15 min. The total content of proteins detected in blanks was 2.41 ± 0.54 mg/g. The protein content obtained after ultrasound assisted extraction was lower than those obtained in the first mechanical stage. This difference is due to the ultrasound effect that promoted the liberation of polysaccharides, such as pectin [START_REF] Li | Optimization of the ultrasonically extraction of polysaccharides from Zizhyphus jujuba cv[END_REF] and also to the activity of PE added to the extract, that led to a more efficient demethylation of the pectin molecule, enhancing the formation of a polyelectrolyte complex between CaCl 2 and pectin, trapping proteins in the matrix formed. When periods of time of 15 min were used, the highest amount of cross-linked and non-cross-linked genipin was obtained. Short periods of time were used according to it has been reported that ultrasound requires short times to achieve the maximum yield of extraction [START_REF] Mason | The uses of ultrasound in food technology[END_REF].

Wang et al. (2011) [START_REF] Wang | Using orthogonal arrays to obtain efficient and reproducible extraction conditions of geniposide and genipin in gardenia fruit with liquid chromatography-mass spectrometry determinations[END_REF] obtained genipin from Gardenia fruit using methanol as solvent as well as sodium hydroxide, hydrochloric acid for solvent adjustment. They reported an average of 8.83 and 7.84 mg/g of genipin from G. jasminoides Ellis and G. jasminoides Ellis var. grandiflora Nakai fruits, respectively. These values are close to the obtained in our research and this fact indicates that average yield for genipin recovery can be achieved using emerging technologies and environmental friendly conditions.

Conclusions

Mechanical extraction of genipin from genipap fruit allows to recover cross-linked and non-cross-linked genipin. The presence of proteins in extracts was evidenced. PE increases the yield of pellet precipitated at 15 and 30 °C. Ultrasound enhances the liberation of genipin and proteins as well as the formation of a polyelectrolyte complex. The higest yield of genipin was found after ultrasound treatment at 10 °C for 15 min. Ultrasound assisted extraction improves the yield of non-cross-linked genipin by a factor of 8. Ultrasound assisted extraction represents a viable method for genipin recovery at cold temperatures.

Fig. 1 .Fig. 2 .

 12 Fig.1. Chemical structure of genipin[START_REF] Almog | Genipin -A novel fingerprint reagent with colorimetric and fluorogenic activity[END_REF].

Fig. 3 .

 3 Fig. 3. Cold-mechanical/sonic enzymatic assisted extraction.

Fig. 4 .

 4 Fig. 4. Effect of temperature on pH in extracts without PE and with enzyme.

Fig. 5 .

 5 Fig.5. Effect of temperature on protein content and pellet weight in extracts without PE and with enzyme.

Fig. 6 .Fig. 7 .Fig. 8 .

 678 Fig. 6. Effect of temperature on absorbance of extracts without PE and with enzyme.

Fig. 9 .Fig. 10 .

 910 Fig. 9. Extracts from peel of genipap fruit after ultrasound assisted extraction (a) and mechanical extraction (b) after 65 days of storage at 9 °C.

Table 1

 1 Cross-linked (with D-glucosamine) and non-cross-linked (only heated) genipin yield found in extracts + PE and in extracts where enzyme was not added (blanks) after mechanical extraction.

	Genipin	Cross-linked a			Only heated a
	Temperature (°C)	5	15	30	5	15	30
	Extracts + PE (mg/g)	1.62 ± 0.30	2.02 ± 0.39	1.66 ± 0.57	0.92 ± 0.16	1.15 ± 0.29	0.49 ± 0.24
	Blanks (mg/g)	1.70 ± 0.02	2.41 ± 0.73	1.81 ± 0.08	0.96 ± 0.13	0.97 ± 0.17	1.21 ± 0.01

a Average and standard deviation on: 5 samples.

Acknowledgements

This work was supported by R&D Center, Coyotefoods Biopolymer and Biotechnology Company. A.M. Ramos-de-la-Peña received the scholarship for her graduate degree from CONACYT. B. A. Puente-Urbina collaborated in the laboratory equipment supervision at Centro de Investigacion en Quimica Aplicada (CIQA), Saltillo city, Coahuila state, Mexico.