Thomas M Evans

David K Sing

Tiffany Kataria

Jayesh Goyal

Nikolay Nikolov

Hannah R Wakeford

Drake Deming

Mark S Marley

David S Amundsen

Gilda E Ballester

Joanna K Barstow

Lotfi Ben-Jaffel

Vincent Bourrier

Lars A Buchhave

Ofer Cohen

David Ehrenreich

Antonio García Muñoz

Gregory W Henry

Heather Knutson

Panayotis Lavvas

Alain Lecavelier Des Etangs

Nikole K Lewis

Mercedes López- Morales

Avi M Mandell

Jorge Sanz-Forcada

Pascal Tremblin

Roxana Lupu

An ultrahot gas-giant exoplanet with a stratosphere

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Infrared radiation emitted from a planet contains information about the chemical composition and vertical temperature profile of its atmosphere [START_REF] Madhusudhan | A temperature and abundance retrieval method for exoplanet atmospheres[END_REF][START_REF] Line | A systematic retrieval analysis of secondary eclipse spectra. I. A comparison of atmospheric retrieval techniques[END_REF][START_REF] Line | No thermal inversion and a solar water abundance for the hot Jupiter HD 209458b from HST/WFC3 spectroscopy[END_REF] . If upper layers are cooler than lower layers, molecular gases will produce absorption features in the planetary thermal spectrum [START_REF] Stevenson | Thermal structure of an exoplanet atmosphere from phase-resolved emission spectroscopy[END_REF][START_REF] Beatty | Evidence for atmospheric cold-trap process in the non-inverted emission spectrum of Kepler-13Ab using HST/WFC[END_REF] . Conversely, if there is a stratosphere-where temperature increases with altitude-these molecular features will be observed in emission [START_REF] Gillett | The 2.8-14-micron spectrum of Jupiter[END_REF][START_REF] Ridgway | Jupiter: identification of ethane and acetylene[END_REF][START_REF] Wallace | The thermal structure of the atmosphere of Jupiter[END_REF] . It has been suggested that stratospheres could form in highly irradiated exoplanets [START_REF] Fortney | A unified theory for the atmospheres of the hot and very hot Jupiters: two classes of irradiated atmospheres[END_REF][START_REF] Hubeny | A possible bifurcation in atmospheres of strongly irradiated stars and planets[END_REF] , but the extent to which this occurs is unresolved both theoretically [START_REF] Burrows | Theoretical spectra and light curves of close-in extrasolar giant planets and comparison with data[END_REF][START_REF] Spiegel | Can TiO explain thermal inversions in the upper atmospheres of irradiated giant planets?[END_REF] and observationally [START_REF] Line | No thermal inversion and a solar water abundance for the hot Jupiter HD 209458b from HST/WFC3 spectroscopy[END_REF][START_REF] Knutson | The 3.6-8.0μm broadband emission spectrum of HD209458b: evidence for an atmospheric temperature inversion[END_REF][START_REF] Haynes | Spectroscopic evidence for a temperature inversion in the dayside atmosphere of hot Jupiter WASP-33b[END_REF][START_REF] Wong | 3.6 and 4.5 μm Spitzer phase curves of the highly irradiated hot Jupiters WASP-19b and HAT-P-7b[END_REF] . A previous claim for the presence of a stratosphere 14 remains open to question, owing to the challenges posed by the highly variable host star and the low spectral resolution of the measurements [START_REF] Line | No thermal inversion and a solar water abundance for the hot Jupiter HD 209458b from HST/WFC3 spectroscopy[END_REF] . Here we report a near-infrared thermal spectrum for the ultrahot gas giant WASP-121b, which has an equilibrium temperature of approximately 2,500 kelvin. Water is resolved in emission, providing a detection of an exoplanet stratosphere at 5σ confidence. These observations imply that a substantial fraction of incident stellar radiation is retained at high altitudes in the atmosphere, possibly by absorbing chemical species such as gaseous vanadium oxide and titanium oxide.

We observed a secondary eclipse of WASP-121b on 10 November 2016 using the Hubble Space Telescope (HST) Wide Field Camera 3 (WFC3). Time series spectra were acquired using the G141 grism, which covers the 1.1-1.7 μm wavelength range. A secondary eclipse of WASP-121b was also observed with the Spitzer space telescope on 30 January 2017 in the 3.6 μm photometric channel of the Infrared Array Camera (IRAC). Further details of the observing set-up and reduction of these data sets are provided in Methods. For the WFC3 data, we produced a wavelength-integrated 'white' light curve by summing each spectrum along the dispersion axis (Fig. 1). The white light curve exhibits instrumental systematics, most notably a quasi-repeatable ramp within each HST orbit, which is a well-known systematic that affects HST/WFC3 data [START_REF] Stevenson | Thermal structure of an exoplanet atmosphere from phase-resolved emission spectroscopy[END_REF][START_REF] Beatty | Evidence for atmospheric cold-trap process in the non-inverted emission spectrum of Kepler-13Ab using HST/WFC[END_REF][START_REF] Cartier | Near-infrared emission spectrum of WASP-103b using Hubble Space Telescope/Wide Field Camera 3[END_REF][START_REF] Crouzet | Water vapor in the spectrum of the extrasolar planet HD 189733b. II. The eclipse[END_REF][START_REF] Wilkins | The emergent 1[END_REF][START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF] . We modelled the planet eclipse signal and instrumental systematics simultaneously by treating the data as a Gaussian process. The eclipse mid-time T mid and planet-to-star surface flux ratio F p /F s were allowed to vary in the fitting, while the planet-to-star radius ratio R p /R s , normalized semi-major axis a/R s , orbital inclination i, and orbital period P were held fixed to previously determined values [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF][START_REF] Delrez | WASP-121 b: a hot Jupiter close to tidal disruption transiting an active F star[END_REF] . Details of our light curve model and fitting procedure for both WFC3 and IRAC are provided in Methods. The best-fit model for the WFC3 light curve is shown in Fig. 1, and results are reported in Extended Data Table 1.

We produced spectroscopic light curves by summing each WFC3 spectrum within 28 channels across the 1.122-1.642 μm wavelength range. As described in Methods, we adopted a commonly used approach to remove wavelength-independent systematics as well as systematics arising from pointing shifts along the dispersion axis for each spectroscopic light curve [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF] . The resulting time series are well-behaved and exhibit minimal residual correlations (Extended Data Fig. 1). We fitted the spectroscopic light curves using the same Gaussian process methodology as described above and in Methods for the white light curve analysis, but only allowed F p /F s to vary while holding T mid fixed to the value determined for the white light curve. The best-fit models are shown in Extended Data Fig. 1 and the results are reported in Extended Data Table 2.

The wavelength-dependent eclipse depths measured with HST and Spitzer are shown in Fig. 2 and comprise the thermal emission spectrum for WASP-121b. A number of previous measurements of thermal emission from ultrahot gas giants have been consistent with radiation from isothermal blackbodies [START_REF] Cartier | Near-infrared emission spectrum of WASP-103b using Hubble Space Telescope/Wide Field Camera 3[END_REF][START_REF] Wilkins | The emergent 1[END_REF] . This may indicate that the atmospheric temperature remains constant over the pressures probed by the different wavelengths, or that the same pressure level is probed at all wavelengths, which could be the case if there is an optically thick cloud deck across the dayside hemisphere. Alternatively, the data precision may simply not be high enough to exclude an isothermal blackbody. When we fitted an isothermal blackbody to the WASP-121b thermal spectrum, we obtain a best-fit atmospheric temperature of 2,700 ± 10 K. However, this model, which is shown in Fig. 2 by the yellow line, provides a poor fit to the data with a χ 2 of 83.9 for 29 degrees of freedom. This allows us to exclude an isothermal blackbody spectrum for WASP-121b at 5σ confidence.

The inability of an isothermal blackbody to explain the data implies that the thermal spectrum probes a range of atmospheric layers with different temperatures. Given that the emission is strongest at 1.35-1.55 μm and 1.20-1.25 μm (Fig. 2), the temperature must be higher at pressures for which the atmosphere is optically thick at these wavelengths. For a decreasing temperaturepressure (T-P) profile (that is, where temperature decreases as pressure decreases), this would only be possible if the opacity of the atmosphere were relatively low within these bands compared to the surrounding wavelengths. However, no plausible gas-phase absorber-such as H 2 O, CO, VO, TiO, FeH or CrH-has this property [START_REF] Sharp | Atomic and molecular opacities for brown dwarf and giant planet atmospheres[END_REF] (see Methods and Extended Data Fig. 2), and refractory condensates (such as Al 2 O 3 , CaTiO 3 , FeO, MgSiO 3 and MgSiO 4 ) are spectrally featureless within the WFC3 bandpass [START_REF] Wakeford | Transmission spectral properties of clouds for hot Jupiter exoplanets[END_REF] . An increasing T-P profile is the only remaining possibility, with the strength of the thermal emission positively correlating with the atmospheric opacity.

To further interpret the measured spectrum, we performed a retrieval analysis to constrain the T-P profile and chemical species present in the atmosphere [START_REF] Madhusudhan | A temperature and abundance retrieval method for exoplanet atmospheres[END_REF][START_REF] Line | A systematic retrieval analysis of secondary eclipse spectra. I. A comparison of atmospheric retrieval techniques[END_REF][START_REF] Line | No thermal inversion and a solar water abundance for the hot Jupiter HD 209458b from HST/WFC3 spectroscopy[END_REF] . In our retrieval, we allowed the abundances of H 2 O, TiO, VO, FeH, CrH, CO, CH 4 and NH 3 to vary as free parameters, and assumed that these gases are well mixed vertically throughout the atmosphere. A one-dimensional analytic formulation was adopted for the T-P profile, which assumes radiative equilibrium and is flexible enough to describe atmospheres with and without stratospheres [START_REF] Line | A systematic retrieval analysis of secondary eclipse spectra. I. A comparison of atmospheric retrieval techniques[END_REF][START_REF] Line | No thermal inversion and a solar water abundance for the hot Jupiter HD 209458b from HST/WFC3 spectroscopy[END_REF][START_REF] Guillot | On the radiative equilibrium of irradiated planetary atmospheres[END_REF] . Further details of our retrieval methodology are provided in Methods.

Our best-fit thermal spectrum from the retrieval analysis is shown by the red line in Fig. 2, and gives a good fit to the data with a χ 2 value of 26.6 for 25 degrees of freedom. This model includes only H 2 O and VO opacity, as we found the inclusion of other molecules was not justified by the current data (see Methods). The retrieved T-P profiles are shown in Fig. 3, all of which exhibit a stratosphere between pressures of 10 -1 bar and 10 -5 bar, with an inferred temperature increase of Detection of the stratosphere is driven by the emission peaks at 1.35-1.55 μm and 1.20-1.25 μm, which coincide with H 2 O and VO bands (see Fig. 2 and Extended Data Figs 2 and3). Indeed, H 2 O has previously been detected in the transmission spectrum of WASP-121b [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF] , and high-altitude absorption of incident stellar radiation at optical wavelengths by VO, as well as by TiO, has been proposed as a possible mechanism for forming stratospheres in strongly irradiated exoplanets [START_REF] Fortney | A unified theory for the atmospheres of the hot and very hot Jupiters: two classes of irradiated atmospheres[END_REF][START_REF] Hubeny | A possible bifurcation in atmospheres of strongly irradiated stars and planets[END_REF] . However, although the inferred H 2 O abundance is broadly consistent with solar elemental abundances in chemical equilibrium, the 95% lower credible limit for the inferred VO abundance is about 1,000× solar. Such a high inferred VO abundance warrants scepticism. For instance, there is a well-known modelling degeneracy between the absolute pressure level of a stratosphere and the abundance of optical absorbers such as VO, with higher abundances giving stratospheres at lower pressures and vice versa [START_REF] Hubeny | A possible bifurcation in atmospheres of strongly irradiated stars and planets[END_REF] . It is also possible that the relatively simple T-P profile adopted in the retrieval analysis produces a biased VO abundance estimate, as it forces the atmosphere to be isothermal at low pressures (Fig. 3) and does not account for three-dimensional effects or nonequilibrium chemistry. Alternatively, the 1.20-1.25 μm feature could be emission from some other unknown source. Given these caveats, a robust detection or non-detection of VO must await further observations that are capable of spectrally resolving the emission bands at high signal-to-noise Figure 3 | Temperature-pressure profiles for WASP-121b. a, Grey lines show a random subset of T-P profiles sampled by the MCMC retrieval analysis. Red line shows the median temperature at each pressure level, and pink lines show ranges either side encompassing ±34% of the sampled profiles. Yellow line indicates the best-fit isothermal temperature of 2,700 K. Dashed line shows the approximate condensation curve for Ti and V compounds [START_REF] Lodders | Titanium and vanadium chemistry in low-mass dwarf stars[END_REF] . b, Histogram showing the temperature difference between pressures of 10 -1 bar and 10 -5 bar. The median increase across this pressure range is indicated by the red line, with pink lines showing the ranges either side encompassing ±34% of the samples. ratio; for example, with the upcoming James Webb Space Telescope. Nonetheless, the conclusion that WASP-121b has a stratosphere appears secure, as the data resolve H 2 O in emission while ruling out isothermal and decreasing T-P profiles at high confidence.

Using an approach described in previous studies [START_REF] Fortney | A unified theory for the atmospheres of the hot and very hot Jupiters: two classes of irradiated atmospheres[END_REF][START_REF] Marley | Thermal structure of Uranus' atmosphere[END_REF] , we calculate that a heat deposit equivalent to more than 20% of the incident stellar flux at pressures below 10 -3 bar is required to raise the stratosphere temperature to about 2,700 K (Fig. 3). Direct absorption of impinging stellar radiation by gaseous species with strong optical bands seems the most plausible means to achieve this. To estimate the required opacity of the putative absorber, we generated radiative-convective equilibrium models including a parameterized grey absorber across optical wavelengths [START_REF] Burrows | Theoretical spectra and light curves of close-in extrasolar giant planets and comparison with data[END_REF][START_REF] Spiegel | Can TiO explain thermal inversions in the upper atmospheres of irradiated giant planets?[END_REF] assuming 1× and 15× solar elemental abundances, with TiO and VO opacities removed. We find the predicted emission spectra and H 2 O emission features are in reasonable agreement with the data for opacities of 0.02 cm 2 g -1 for the 1× solar case and 0.06 cm 2 g -1 for the 15× solar case (Extended Data Fig. 4). Alternative heating mechanisms, such as the breaking of upwardpropagating gravity waves, are not expected to produce energy fluxes of the required magnitude [START_REF] Hickey | Gravity wave heating and cooling in Jupiter's thermosphere[END_REF][START_REF] Matcheva | Heating of Jupiter's thermosphere by dissipation of gravity waves due to molecular viscosity and heat conduction[END_REF] . Strong absorption within high-altitude clouds could also potentially produce a stratosphere [START_REF] Heng | On the e ects of clouds and hazes in the atmospheres of hot Jupiters: semianalytical temperature-pressure pro les[END_REF] . However, it is unclear whether a significant cloud deck could be maintained on the dayside hemisphere of WASP-121b, as the high temperatures are likely to inhibit the condensation of even the most refractory compounds of elements such as V, Ti, Ca and Fe (ref. 28).

WASP-121b is the first exoplanet with a stratosphere for which resolved spectral features have been observed in emission. Previous stratosphere detection claims have either since been revised [START_REF] Line | No thermal inversion and a solar water abundance for the hot Jupiter HD 209458b from HST/WFC3 spectroscopy[END_REF][START_REF] Knutson | The 3.6-8.0μm broadband emission spectrum of HD209458b: evidence for an atmospheric temperature inversion[END_REF] or are based on measurements of excess thermal emission without resolved spectral features [START_REF] Haynes | Spectroscopic evidence for a temperature inversion in the dayside atmosphere of hot Jupiter WASP-33b[END_REF][START_REF] Wong | 3.6 and 4.5 μm Spitzer phase curves of the highly irradiated hot Jupiters WASP-19b and HAT-P-7b[END_REF] . Published thermal spectra for other gas-giant exoplanets are either indistinguishable from isothermal emission otherwise consistent with decreasing temperature profiles [START_REF] Stevenson | Thermal structure of an exoplanet atmosphere from phase-resolved emission spectroscopy[END_REF][START_REF] Beatty | Evidence for atmospheric cold-trap process in the non-inverted emission spectrum of Kepler-13Ab using HST/WFC[END_REF][START_REF] Crouzet | Water vapor in the spectrum of the extrasolar planet HD 189733b. II. The eclipse[END_REF] . These results are in line with the overall finding that irradiated gas-giant atmospheres exhibit a wide range of properties [START_REF] Sing | A continuum from clear to cloudy hot-Jupiter exoplanets without primordial water depletion[END_REF] . Expanding the sample of ultrahot exoplanets with measured thermal emission spectra will be central to making sense of this diversity, particularly as we enter the era of the James Webb Space Telescope, which is anticipated to vastly improve the quality of exoplanet spectra across the near-to-mid-infrared wavelength range.

METHODS HST/WFC3 observations

A secondary eclipse of WASP-121b was observed using HST/WFC3 as part of Program (PIs D.K.S. and M.L.-M.). We observed the target for 6.9 h over five HST orbits, which covered the full planetary eclipse lasting 2.9 h. The first two HST orbits occurred before eclipse ingress, the third and fourth orbits occurred during the eclipse, and the fifth orbit occurred after the completion of eclipse egress (Fig. 1).

Observations were made in spectroscopic mode using forward-scanning with the G141 grism and a scan rate of 0.12 arcsec s -1 . Overheads were reduced by only reading out the 256 × 256 pixel subarray containing the target spectrum. We adopted the SPARS10 sampling sequence with non-destructive reads per exposure (NSAMP = 15) resulting in total integration times of 103 s and scans across 100-pixel rows of the cross-dispersion axis. With this configuration, we obtained science exposures in the first HST orbit and 16 exposures in each subsequent HST orbit. Typical count levels remained below 2.5 × 10 4 electrons per pixel.

HST/WFC3 data reduction

The target flux was extracted from the IMA files produced by the CALWF3 pipeline (v3.4) by taking the difference between successive non-destructive reads. The background was removed by subtracting the median count in a box of pixels spanning 110 columns along the dispersion axis and 20 rows along the cross-dispersion axis well away from the target spectrum. For each readdifference, we determined the flux-weighted centre of the scanned spectrum along the crossdispersion axis and set to zero all pixel values located more than 35 pixels above and below this pixel row. This removed flux contributions from nearby contaminant stars and cosmic ray strikes from the masked region. Final reconstructed frames were produced by adding together the readdifferences.

The target spectrum was then extracted from each frame by summing the flux within a rectangular aperture spanning the full dispersion axis and positioned on the central cross-dispersion row of the scan. We did this for a range of different cross-dispersion aperture widths spanning 100 to pixels in ten-pixel increments. The wavelength solution was determined by cross-correlating each target spectrum against a Phoenix stellar model 31 with properties similar to the WASP-121 host star (effective temperature T eff = 6,400 K, log[g (cgs)] = 4.0, Fe/H = 0) modulated by the throughput of the G141 grism.

HST/WFC3 white light curve analysis

White light curves were generated for each trial aperture by summing the spectra along the dispersion axis (Fig. 1). Before fitting the light curves, we discarded the first HST orbit because of the particularly strong ramp systematic it exhibited, which is a well-known feature of WFC3 data sets [START_REF] Stevenson | Thermal structure of an exoplanet atmosphere from phase-resolved emission spectroscopy[END_REF][START_REF] Beatty | Evidence for atmospheric cold-trap process in the non-inverted emission spectrum of Kepler-13Ab using HST/WFC[END_REF][START_REF] Cartier | Near-infrared emission spectrum of WASP-103b using Hubble Space Telescope/Wide Field Camera 3[END_REF][START_REF] Crouzet | Water vapor in the spectrum of the extrasolar planet HD 189733b. II. The eclipse[END_REF][START_REF] Wilkins | The emergent 1[END_REF][START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF]32,33 . We also discarded the first exposure of each remaining HST orbit as these had significantly lower counts than subsequent exposures in the same orbit.

To model the white light curves we adopted a Gaussian process methodology similar to that outlined in our previous work [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF] . We defined our data likelihood as a multivariate normal distribution with a deterministic mean function μ to model the planetary eclipse signal and covariance matrix K to capture additional correlations in the data, such that:

p(d | u, w) = N(μ, K+)
where p denotes a probability density function, d is a vector containing the flux measurements, u is a vector containing the mean function parameters, w is a vector containing the covariance parameters, N denotes a multivariate normal distribution, and  is a diagonal matrix containing the squared photon-noise uncertainties for each data point. For the mean function μ, we used:

μ(t, t ; c 0 , c 1 , F p /F s , T mid ) = [c 0 + c 1 t ] E(t ; F p /F s , T mid )
where t is a vector containing the observation timestamps, t is standardized time (that is, time t minus the mean divided by the standard deviation), c 0 and c 1 are parameters defining a linear trend, E is the analytic function for a planetary eclipse taken from ref. 34, F p /F s is the planet-to-star flux ratio, and T mid is the eclipse mid-time. For the eclipse signal E we ignored limb darkening and fixed the remaining parameters to the previously determined values listed in Extended Data Table 1. For the covariance function, we adopted a Matérn ν = 3/2 kernel with HST orbital phase φ, dispersion drift x, and cross-dispersion drift y as the input variables. As with time t for the linear trend in the mean function μ, each of these input variables was standardized before being provided as input to the Gaussian process (that is, mean-subtracted and divided by the standard deviation). Thus, the entries of the covariance matrix were given by:

K ij = A 2 (1+D ij 3 1/2 ) exp[-D ij 3 1/2 ]
where A defines the characteristic correlation amplitude and:

D ij = [η 2 φ (φ i -φ j ) 2 + η 2 x (x i -x j ) 2 + η 2 y (y i -y j ) 2 ] 1/2
where η φ , η x and η y are the inverse correlation length scales and the primed variables are standardized. The free parameters of our white light curve model were therefore u = [c 0 , c 1 , F p /F s , T mid ] for the mean function and w = [A, η φ , η x , η y ] for the covariance function. Our prior distribution over the model parameters took the form p(u,w) = p(c 0 ) p(c 1 ) p(F p /F s ) p(T mid ) p(A) p(η φ ) p(η x ) p(η y ). Uniform distributions were adopted for p(c 0 ), p(c 1 ), p(F p /F s ), and p(T mid ). A gamma distribution was adopted for p(A) with the form Gamma(α=1, β=100) = 100 exp[-100A]. We found that priors less strongly favouring small values of A (for example, a uniform or log-uniform distribution) resulted in the Gaussian process model being biased by a subset of outliers in the data to infer implausibly short correlation length scales with large correlation amplitudes. Effectively, this resulted in a model that was over-fitted to the data, justifying the adoption of the stronger gamma distribution prior. Log-uniform distributions were adopted for p(η φ ), p(η x ), and p(η y ) (that is, we fitted for log[η φ ], log[η x ] and log[η y ] with uniform distribution priors).

The posterior distribution p(u, w | d) ∝ p(d | u, w) p(u, w) was marginalized using affine-invariant Markov chain Monte Carlo (MCMC) as implemented by the emcee Python software package 35 .

We randomly initialized five groups of 150 walkers close to the maximum likelihood solution, which was located by minimizing the negative logarithm of the posterior distribution using nonlinear optimization as implemented by the fmin routine of the scipy.optimize Python software package 36 . Each of the five walker groups was run for 500 steps before discarding the first 250 steps as burn-in. The resulting five chains displayed good mixing and convergence, with Gelman-Rubin statistic values well within 1% for each model parameter 37 . The five individual chains were then combined to produce the final posterior samples.

This analysis was repeated for each of the trial apertures used to extract the target spectra (see above). All inferred values for F p /F s agreed to within 1σ regardless of the aperture used. We adopt the 160-pixel aperture reduction for the remainder of this study and report the MCMC results in Extended Data Table 1. The standard deviation of the best-fit model residuals for this analysis was 64 p.p.m., which is within 4% of the theoretical photon-noise floor (Fig. 1), while the other apertures gave either larger uncertainties for F p /F s or larger residual scatter relative to photon noise.

HST/WFC3 spectroscopic light curve analysis

Spectroscopic light curves were produced using the same method as adopted in our previous work [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF]32,33 . We started by identifying the out-of-eclipse spectra based on the best-fit white light curve model and taking the median of these to form a master stellar spectrum. For each individual spectrum, we determined the lateral shift along the dispersion axis and a wavelength-independent rescaling of the flux that minimized the residuals with the master spectrum. The residuals obtained in this way were binned into 28 spectroscopic channels across the 1.122-1.642 μm wavelength range, which avoided the steep edges of the G141 grism response. Each channel spanned 4 pixel columns along the dispersion axis, equivalent to 0.019 μm in wavelength. The time series of binned residuals were then added to the eclipse signal of the best-fit white light curve model to produce the spectroscopic light curves shown in Extended Data Fig. 1. This process removed systematics due to pointing drifts along the dispersion axis and the wavelength-independent ('common-mode') component of the flux time series. The latter included the ramp systematic, but also the wavelength-integrated eclipse signal, hence manually adding the eclipse signal from the white light curve best-fit model back in the final step.

To fit the spectroscopic light curves, we adopted the same data likelihood and prior distributions as were used for the white light curve analysis. The only exception was that we did not fit for T mid as a free parameter but instead held it fixed to the best-fit value determined for the white light curve. We also adopted the same fitting methodology that was used for the white light curve analysis, first identifying the maximum likelihood solution by nonlinear least squares and using it as a starting point for affine-invariant MCMC to marginalize over the posterior distribution. The results are reported in Extended Data Table 2 and best-fit models are shown in Extended Data Fig. 1. Median scatter in the best-fit model residuals across the 28 channels was equal to photon noise, with a 1σ range about the median of 90%-110% times photon noise.

As an aside, we note that the apparent size of WASP-121b when viewed from the zenith geometry at secondary eclipse could vary as a function of wavelength due to the wavelength-dependent opacity of the atmosphere. In transmission, this effect produces a variation in R p /R s of up to ∼0.4% about the mean across the WFC3 G141 bandpass [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF] . Given that the eclipse depth scales as (R p /R s ) [START_REF] Line | A systematic retrieval analysis of secondary eclipse spectra. I. A comparison of atmospheric retrieval techniques[END_REF] , this translates to a potential bias in the inferred planetary flux of up to ∼0.8% in any given wavelength channel, which is well within the 1σ uncertainties obtained from our light curve fitting (Extended Data Table 2).

Spitzer/IRAC observations, data reduction and light curve analysis

A single secondary eclipse of WASP-121b was observed using Spitzer/IRAC as part of Program 13044 (PI D.D.). Observations were made in the 3.6 μm photometric channel using a 32 × 32 pixel subarray and an exposure time of 2.0 s per image. We observed the target for 8.5 h, which included the full eclipse, as well as a pre-eclipse baseline of 3.2 h and a post-eclipse baseline of 2.4 h. Data reduction was performed using the methodology described in our previous work 38,39 . We analysed the light curve by fitting for an eclipse signal with F p /F s and T mid allowed to vary, while treating instrumental systematics caused by the intrapixel sensitivity variations of the IRAC detector with pixel level decorrelation (PLD) as described in ref.

39. An 800,000 step MCMC was used to derive estimates for the model parameters, giving an eclipse depth of (R p /R s ) 2 F p /F s = 3,670 ± 130 p.p.m. and mid-time of T mid = 2,45 7,783.77740 ± 0.00068 (BJD TDB ).

1D atmosphere modelling of WASP-121b

We used the 1D atmosphere ATMO model [40][41][42][43][44] to perform forward and retrieval modelling of the measured WASP-121b thermal emission spectrum. ATMO computes the 1D temperaturepressure (T-P) structure of an atmosphere in plane-parallel geometry. It can calculate forward models assuming radiative, convective and chemical equilibrium. It can also be used as a retrieval tool to compute the emission and transmission spectra from an input T-P profile and arbitrary chemical abundances 45 .

For the retrieval analysis, we used a model planetary atmosphere with 50 pressure levels evenly spaced in log pressure between 10 -8 bar and 500 bar. We adopted the parameterized T-P profile of ref. 23, fitting the data assuming either one or two optical channels. This gave three to five parameters for the T-P profile: the Planck mean thermal infrared opacity, κ IR ; the ratios of the optical to infrared opacities in the two channels, γ 1 and γ 2 ; a partition of the flux between the two optical channels, α; and an irradiation efficiency factor, β. We set the planetary radius equal to 1.694 Jupiter radii (R J ) which corresponds to the lowest-altitude probed by the near-infrared transmission spectrum [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF] . An internal planetary temperature of 100 K was assumed and the same Phoenix stellar model as described above was used for the input flux from the WASP-121 host star.

We adopted uniform priors for all free parameters in our model with the following ranges: 10 -5 to 10 -0.5 for κ IR ; 10 -4 to 10 1.5 for γ 1 and γ 2 ; 0 to 1 for α; and 0 to 2 for β. Uniform priors between 0 and 0.05 were also adopted for the mixing ratios of chemical species other than H and He. The upper limit placed on the metal abundances is justified by the fact that WASP-121b is known to be a gas giant. We ran a suite of retrievals including various combinations of H 2 O, TiO, VO, FeH, CrH, CO, CH 4 and NH 3 , using nonlinear least-squares optimization to locate the minimum χ 2 solution for each model. However, we found that the inclusion of molecules other than H 2 O and VO did not offer a significant improvement in the fit to the data. This is quantified in Extended Data Table 3, where we provide the full list of retrievals performed with accompanying Bayesian information criterion (BIC) values. We also found that using two optical channels rather than one for the T-P profile did not improve the quality of the fit nor did it substantially alter the inferred stratosphere profile. Thus, our final model had five free parameters: volume mixing ratios for H 2 O and VO plus κ IR , γ 1 , and β. For this model, we marginalized over the posterior distribution using differential-evolution MCMC 46 . We ran 10 chains for 30,000 steps each, at which point the Gelman-Rubin statistic for each free parameter was within 1% of unity, indicating that the chains were well-mixed and had reached a steady state. Following ref. 46, we then discarded a burn-in phase from all chains corresponding to the step at which all chains had found a χ 2 below the median χ 2 value of the chain. The remaining samples were then combined into a single chain to estimate the posterior distribution. The results are reported in Extended Data Table 4 and plotted in Extended Data Fig. 5.

A limitation of the retrieval analysis is that the inferred T-P profile is not derived self-consistently with the inferred chemical abundances. The latter determine the opacity of the atmosphere, which in turn controls the radiative transfer and thus the T-P profile. As a check, we used the chemical abundances obtained from the retrieval analysis to generate self-consistent T-P profiles in radiative-convective equilibrium. In one test, the VO abundance was set to the median value from the retrieval, and in a second test it was set to the lower value of the 1σ credible range. In both cases, the H 2 O abundance was set to its median value from the retrieval. The results are shown in Extended Data Fig. 4. Between pressures of about 10 -3 bar to 10 -5 bar, the temperatures of the radiative-convective equilibrium models range from approximately 2,400 K to 3,000 K, in agreement with the retrieved T-P profile shown in Fig. 3 at the uncertainty levels. The thermal spectra show H 2 O and VO emission features, with overall flux levels broadly matching those measured in the WFC3 G141 bandpass but giving a poorer agreement for the 3.6 μm IRAC data point (Extended Data Fig. 4). The approximate nature of the parameterized T-P profile is most prominently seen at the lowest pressures below ∼10 -5 bar where it is forced to be isothermal at about 2,900 K (Fig. 3), whereas the radiative-convective models rise to temperatures exceeding 3,000 K. This results in isothermal blackbody emission at optical wavelengths (Fig. 2), in contrast to the self-consistent model which shows prominent optical VO emission features. Furthermore, the retrieved VO abundances are probably biased towards large values, as the isothermal approximation at low pressures limits the emission contributions from those pressure levels. However, the H 2 O retrieved abundance and emergent spectra are expected to be less affected, as the contribution functions at these wavelengths are deeper in the atmosphere, where the parameterized retrieval T-P profile used for the retrieval is more accurate.

To help quantify the corresponding optical opacities needed to produce the observed stratosphere, we implemented the approach of refs 11 and 12, in which an arbitrary absorber was added throughout the atmosphere with grey opacity across the 0.43-1 μm optical wavelength range. We assumed 1× and 15× solar elemental abundances, with gaseous TiO and VO opacities removed. With this set-up, a grid of models in radiative-convective equilibrium was generated for a range of heat recirculation factors and assuming the grey absorber had opacity κ of 0.002, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2 and 2 cm 2 g -1 . All models exhibited stratospheres between pressures of 10 -2 bar and 10 -5 bar. Two examples with zero heat recirculation are plotted in Extended Data Fig. 4, one for κ = 0.02 cm 2 g -1 and solar metallicity, and the other with κ = 0.06 cm 2 g -1 and 15× solar metallicity. Both show H 2 O in emission and reproduce the overall level of thermal emission measured in the WFC3 G141 bandpass, while over-predicting the flux measured in the 3.6 μm IRAC bandpass.

Validating the ATMO retrieval code

We benchmarked our ATMO retrieval and T-P profile parameterization code by applying it to the published thermal spectrum of WASP-43b [START_REF] Stevenson | Thermal structure of an exoplanet atmosphere from phase-resolved emission spectroscopy[END_REF]47,48 . Retrieval analyses performed on these data with the CHIMERA code 2,3 have previously indicated near-solar abundances of H 2 O along with a noninverted T-P profile for the planetary atmosphere. We performed a retrieval with the same data and fit parameters as the CHIMERA study published in ref. 48. The latter consisted of five parameters for the T-P profile and abundances for H 2 O, CO, CO 2 , CH 4 , NH 3 and HCN. For consistency, we turned off scattering in our retrieval analysis, as this was not included in the CHIMERA study. We also note that CHIMERA uses the HITRAN database 49 for many of the molecular opacities, including H 2 O, whereas ATMO primarily uses the ExoMol database 50 . We find good agreement with the retrieved T-P profiles and H 2 O abundance reported in ref. 48 (Extended Data Fig. 6). Text labels in the key give the rescaling factors that have been applied to each cross-section to fit them on a single vertical axis, with the exception of the H 2 O cross-section which has not been rescaled. Note that the VO cross-section has been rescaled by a factor of 0.03, which is consistent with the abundance of VO relative to H 2 O inferred for the best-fit model (Extended Data Table 4).

Extended

Extended Data Figure 3 | A comparison of models with and without stratospheres. a, A sequence of T-P profiles with and without stratospheres computed using the analytic parameterization of ref. 23.The red line corresponds to a model with a strong stratosphere obtained from the MCMC retrieval analysis and the green line shows a model with a weaker stratosphere. The light blue and dark blue lines show, respectively, models with a decreasing temperature profile and a strongly-decreasing temperature profile. The yellow line indicates the bestfit isothermal model with a temperature of 2,700 K. b, Corresponding contribution functions averaged over the WFC3 G141 bandpass, indicating the approximate pressures probed at these wavelengths. c, Corresponding thermal spectra, with H 2 O and VO bands seen in emission for the models with stratospheres (red and green lines) and in absorption for the models with decreasing temperature profiles (light blue and dark blue lines). The isothermal model has a featureless blackbody spectrum (yellow line).

Extended Data Figure 4 | Self-consistent models in radiative-convective equilibrium. a, Similar to Fig. 2, but showing self-consistent models in radiative equilibrium: median H 2 O and VO abundances obtained from the retrieval analysis (light blue line); H 2 O abundance set to median value and VO abundance set to the lower value of the 68% credible range (green line); solar metallicity, but without VO and TiO, and with an arbitrary absorber with absorption cross-section κ = 0.02 cm 2 g -1 (pink line); and 15× solar metallicity, but without VO and TiO, and with an arbitrary absorber with κ = 0.06 cm 2 g -1 (dark blue line). For the last two models, the arbitrary absorber has grey opacity across the 0.43-1 μm wavelength range and is assumed to be distributed uniformly throughout the atmosphere. The best-fit isothermal model with temperature 2,700 K is also shown (yellow line). Brown circles with error bars, HST data with 1σ uncertainties: inset, HST data on a magnified scale. b, Corresponding T-P profiles, all of which have a stratosphere across the 10 -2 bar to 10 -5 bar pressure range.

Thick lines indicate the main pressure levels probed across the WFC3 G141 bandpass. and Spitzer/IRAC observations. Vertical error bars give 1σ uncertainties and horizontal error bars give photometric bandpasses. The red line shows the best-fit spectrum obtained from an ATMO retrieval analysis, with shading indicating the regions encompassing 68%, 95% and 99.7% of the MCMC samples. The inset shows a magnified view of the HST data. The retrieved volume mixing ratio (VMR) for H 2 O is in good agreement with the published value obtained using the CHIMERA retrieval code 2,3 and the ATMO retrieval achieves a better fit to the data as measured by the minimum χ 2 . b, Best-fit T-P profiles retrieved with ATMO (solid blue line) and CHIMERA (solid green line). The two analyses agree to within the 68% credible ranges (blue and green dashed lines). Free parameters are those that were allowed to vary in the model fitting, and fixed values were obtained from previous analyses of primary transit light curves [START_REF] Evans | Detection of H 2 O and evidence for TiO/VO in an ultra-hot exoplanet atmosphere[END_REF][START_REF] Delrez | WASP-121 b: a hot Jupiter close to tidal disruption transiting an active F star[END_REF] . For the free parameters, quoted values give the median of the MCMC samples with ranges either side encompassing ±34% of the samples. Note that the eclipse depth is equal to (R p /R s ) 2 (F p /F s ). Values for c 1 , log[η φ ], log[η x ] and log[η y ] have arbitrary normalizations owing to standardization of their associated variables before fitting (see Methods). The eclipse mid-time T mid is quoted as a Barycentric Julian Date in the Barycentric Dynamical Time standard (BJD TDB ), which was determined by converting from JD UTC using the online tools of ref. 51. To do so, we assumed a geocentric reference frame for HST, which introduces an error of up to ∼0.02 s. However, this is insignificant compared to the ∼1 s uncertainty of the HST timestamps 51 and the ∼30 s uncertainty in the measurement of T mid itself. Summary of the different models for which retrieval analyses were performed, where n is the number of degrees of freedom. The quality of the obtained fits to the measured thermal spectrum are indicated by the χ 2 and Bayesian information criterion (BIC) values. Models are arranged in order of increasing BIC value. Note that the best-fit isothermal model has χ 2 = 83.9 for n = 29 (see main text), corresponding to a BIC value of 85.9.

Extended

Extended

Extended Data

Figure 1 |

 1 Figure 1 | Wavelength-integrated white light curve for WASP-121b.a, Raw normalized flux with photon-noise 1σ error bars (pink circles) and best-fit Gaussian process model (grey line). Gaps in the time series are due to the target being occulted by the Earth during each HST orbit. Quasirepeatable systematics are evident for each HST orbit as well as a visit-long drift in the baseline flux level. b, Relative change in the system flux in parts per million (p.p.m.) with photon-noise 1σ error bars (pink circles) after correcting for the instrumental systematics, with best-fit eclipse model (grey line). c, Best-fit model residuals in p.p.m. with photon-noise 1σ error bars (pink circles).

Figure 2 |

 2 Figure 2 | Emission spectrum for WASP-121b. a, Eclipse depths measured with HST (brown circles) and Spitzer (black circle, top right), and a ground-based photometric measurement 20 (green circle). Vertical error bars give 1σ uncertainties and horizontal error bars give photometric bandpasses. Yellow line shows the best-fit isothermal blackbody spectrum with a temperature of 2,700 K. Red line shows the bestfit model fromthe retrieval analysis. Inset shows the HST data on a magnified scale.b, Planetary flux with stellar contribution removed. Measured spectra for M8 and L1 dwarfs 30 are shown for comparison (blue and purple lines), exhibiting H 2 O absorption bands (bracketed) characteristic of these spectral types. For WASP-121b the H 2 O band at 1.35-1.55 μm appears in emission. Spectra for 2,400 K and 2,900 K blackbodies (grey lines) indicate the approximate temperature range probed by the data. c, Similar to b, but showing only the WFC3 bandpass and with a possible emission feature due to VO also indicated at 1.20-1.25 μm.

Data Figure 1 |Extended Data Figure 2 |

 12 Spectroscopic light curves for WASP-121b. a-d, Raw normalized light curves for each of the spectroscopic channels with photon-noise 1σ error bars (orange and green circles), and best-fit eclipse signals multiplied by linear time trends (grey lines). Vertical offsets have been applied for visual clarity. Labels indicate central wavelengths for each channel. e-h, Model residuals after removing the best-fit eclipse signal and linear time trend for each of the spectroscopic light curves in a-d, respectively, with photon-noise 1σ error bars (orange and green circles) and stochastic components of the best-fit Gaussian process models (black lines). Note that for most channels, the correlations remaining in the residuals after accounting for a linear time trend are minimal. Model thermal spectrum for WASP-121b broken down by emission source and absorption cross-sections of important molecules. a, Similar to Fig.2, showing the HST measurements of the WASP-121b thermal spectrum (brown circles; error bars, 1σ uncertainties). Red line shows the best-fit model (H 2 O and VO) obtained from the retrieval analysis. Other coloured lines show model thermal spectra generated using the same T-P profile as for the best-fit model but with the opacity due to each molecule turned off oneby-one: VO off (blue line) and H 2 O off (green line). b, Absorption cross-sections for plausible gas-phase absorbers (colour coded, key at top right) across the WFC3 bandpass.

Data Figure 5 |Extended Data Figure 6 |

 56 Posterior distributions from MCMC retrieval analysis for WASP-121b. Panels along the diagonal show the marginalized posterior distributions for each parameter of the thermal emission model (κ IR , γ 1 , β, H 2 O abundance, VO abundance), shown left to right in the columns, and top to bottom in the rows. Solid orange lines show the posterior medians and dashed orange lines show the ranges either side encompassing ±34% of the samples. Panels below the diagonal show the posterior distribution as a function of each parameter pair with increasing probability from light to dark shades. As γ 1 controls the optical opacity, there is a strong anti-correlation with the radiation efficiency factor β, where lower radiation efficiency values are compensated by higher optical opacities and vice versa. There is also a strong degeneracy between the abundances of each molecule (H 2 O, VO) and the parameter κ IR which controls the overall atmospheric opacity. Higher abundances and higher values of κ IR result in stratospheres at lower pressures and vice versa. ATMO retrieval code applied to WASP-43b thermal emission data. a, Black data points show the measured thermal emission data for WASP-43b taken from ref. 48, composed of HST/WFC3

Data Table 1 | Parameter values for the WASP-121b white light curve fit

	Free parameters	Value	Units
	eclipse depth	1132 -35 +37	p.p.m.
	Tmid	2457703.45879 -0.00039 +0.00039	BJDTDB
	c0	0.9996 -0.0021 +0.0009	
	c1	-0.000217 -0.000061 +0.000046	arbitrary normalization
	A	1431 -748 +3305	ppm
	log[]	-0.35 -1.57 +1.14	arbitrary normalization
	log[x]	-2.87 -1.83 +1.81	arbitrary normalization
	log[y]	-2.82 -2.62 +2.78	arbitrary normalization
	Fixed parameters	Value	Units
	P	1.2749255	day
	Rp	1.694	RJ
	Rs	1.458	R⨀
	a/Rs	3.825	
	i	88.91	degree
	b = a cos(i)/Rs	0.073	
	e	0	

Table 3 | Comparison of different retrieval analyses for WASP-121b

 3

		Molecules	n	 2	B.I.C.		Molecules	n	 2	B.I.C.
	1	H2O, VO	25 26.6	43.6	19	VO, FeH, CrH	24 30.3	50.7
	2	H2O, VO, FeH	24 23.7	44.1	20	VO, TiO	25 34.2	51.2
	3	H2O, VO, CrH	24 25.1	45.5	21	VO, TiO, FeH	24 31.9	52.3
	4	H2O, VO, TiO	24 25.5	45.9	22	VO	26 39.1	52.7
	5	H2O, VO, TiO, FeH	23 22.4	46.2	23	VO, CH4	25 38.7	55.7
	6	H2O, VO, CH4	24 26.3	46.7	24	VO, FeH	25 39.1	56.1
	7	H2O, VO, CO	24 26.5	46.9	25	H2O, CO	25 44.6	61.6
	8	H2O, VO, NH3	24 26.5	46.9	26	CrH	26 49.0	62.6
	9	VO, CrH	25 30.3	47.3	27	H2O	26 49.3	62.9
	10	H2O, VO, FeH, CH4	23 23.6	47.4	28	H2O, CrH	25 48.0	65.0
	11	H2O, VO, FeH, NH3	23 23.7	47.5	29	TiO, FeH	25 48.6	65.6
	12	H2O, VO, FeH, CO	23 23.8	47.6	30	H2O, TiO	25 50.0	67.0
	13	VO, TiO, CrH	24 28.1	48.5	31	TiO	26 54.8	68.4
	14	H2O, VO, TiO, CO	23 25.3	49.2	32 H2O, TiO, FeH	24 48.1	68.5
	15	H2O, VO, TiO, FeH, CO	22 22.5	49.7	33	H2O, TiO, CO	24 50.0	70.4
	16	H2O, VO, CH4, NH3	23 26.3	50.1	34	H2O, TiO, FeH, CO	23 47.5	71.3
	17	VO, NH3	25 33.3	50.3	35	H2O, FeH	25 60.7	77.7
	18	H2O, VO, FeH, CH4, NH3	22 23.5	50.7	36	H2O, FeH, CO	24 63.9	84.3

Acknowledgements This work is based on observations with the NASA/ESA HST, obtained at the Space Telescope Science Institute (STScI) operated by AURA, Inc. This work is also based in part on observations made with the Spitzer Space Telescope, which is operated by the Jet Propulsion Laboratory, California Institute of Technology under a contract with NASA. The research leading to these results has received funding from the European Research Council under

Code availability. Custom code used to extract the HST spectra from the raw data frames and custom code used to extract and analyse the Spitzer data are available upon request. Publicly available custom codes were used for the Gaussian process modelling (http://github.com/tomevans/gps) and eclipse signal modelling (http://github.com/tomevans/planetc), both of which are publicly available but not maintained in a user-friendly state. The HST light curve MCMC fitting was performed using the open source emcee code (http://github.com/ dfm/emcee). The MCMC retrieval analyses were performed using the publicly available package exofast (http://astroutils.astronomy.ohio-state.edu/exofast). The ATMO code used to compute the atmosphere models is currently proprietary.

Data availability. Raw HST data frames are publicly available online at the Mikulski Archive for Space Telescopes (MAST; https://archive.stsci.edu). Raw Spitzer data frames are publicly available online at the NASA/IPAC Infrared Science Archive (IRSA; http://irsa.ipac.caltech.edu/Missions/spitzer. html). Reduced data products and models used in this study are available in Supplementary Information.

Extended Data


A Nesterenko

I Alric

F Silvestre

V Durrieu

Comparative study of encapsulation of vitamins with native and modified soy protein

Keywords:

Microencapsulation of hydrophobie (oc-tocopherol) and hydrophilic (ascorbic acid) vitamins by native (non-modified) and modified soy protein isolate (SPI) was carried out using a spray-drying technique. Proteins' functional properties were modified by acylation and cationization reactions in aqueous alkaline media. The results obtained demonstrated that SPI modification resulted in decreased emulsion droplet size and viscosity. Ali preparations with ascorbic acid (M) had lower viscosity and microparticle size than those with oc-tocopherol (T). Moreover, grafting of fatty acid chains ta SPI by acylation improved its amphiphilic character and affinity with hydrophobie substances. Thus, the microencapsulation effi ciency of T was increased from 79.7% ta 94.8% and the microencapsulation efficiency of M was reduced from 91.8% ta 57.3% compared ta native SPI. Conversely, attachment of quaternary ammonium cationic groups ta proteinic chains by cationization, increased SPI solubility and favored the M microencapsu lation. This study illustrated that an appropriate modification of SPI can improve the microencapsulation efficiency of suitable active cores.

Introduction

Over the past decades, environmental requirements have become of great importance. In order to replace synthetic polymers and animal derived products, there is an increasing interest in the industrial use of renewable resources and development of naturally occurring materials for new applications. Natural polymers such as vegetable proteins have attracted considerable research activities because of their availability, biodegradability, renewable character and various interesting functional properties. Among them, pro teins extracted from vegetable seeds (soybean, pea, barley, wheat, rice, oat, sunflower) have been reported as having good emulsifying and foaming capacities, water solubility, amphiphilic and film forming properties [START_REF] Nunes | Vegetable proteins and milk puddings[END_REF].

Due to their good physico-chemical properties, vegetable pro teins represent a highly suitable wall-forming material for micro encapsulation of active components to use in the food industry, pharmaceutics and cosmetics (Nesterenko, Alric, Silvestre, & Durrieu, 2013). Microencapsulation allows the isolation of the material in microencapsulation by spray-drying had been reported by various authors. This natural polymer showed a high efficiency for coating different active substances: orange oil [START_REF] Kim | Microencapsulation properties of gum arabic and several food proteins: spray-dried orange oil emulsion particles[END_REF], fish oil [START_REF] Augustin | Maillard reaction products as encapsulants for fish oil powders[END_REF], stea rin/palm oil [START_REF] Rusli | Stabilization of oils by microencapsulation with heated protein-glucose syrup mixtures[END_REF], phospholipid [START_REF] Yu | Preparation of phospholipid microcap sules by spray drying[END_REF], flavors [START_REF] Charve | Encapsulation performance of proteins and traditional materials for spray dried flavors[END_REF], casein hydrolysate [START_REF] Favaro-Trindade | The use of spray drying technology ta reduce bitter taste of casein hydrolysate[END_REF][START_REF] Ortiz | Pro duction and properties of casein hydrolysate microencapsulated by spray dry ing with soybean protein isolate[END_REF], paprika oleoresin [START_REF] Rascon | Carotenoid retention and storage stability of spray-dried paprika oleoresin using gum arabic and soy protein isolate as wall materials[END_REF] and soy oil [START_REF] Tang | Microencapsulation properties of soy protein isola te and storage stability of the correspondingly spray-dried emulsions[END_REF].

It is widely accepted that the antioxidant properties of r,. tocopherol (vitamin E) and ascorbic acid (vitamin C) are responsible in part for their biological activity [START_REF] Packer | Direct observation of a free radical interaction between vitamin E and vitamin C[END_REF]. Nevertheless, environmental factors, such as oxygen, temperature, moisture and UV affect the stability of these compounds and involve their deterioration. Microencapsulation could be an effi cient method for the protection and stabilization of r,.-tocopherol (T) and ascorbic acid (M). However, the nature of the wall matrix particularly affects the degree of protection of the active core, the microparticles' stability and the retention efficiency.

Modifiable character is one of the important advantages of proteins. Modification of proteinic chains leads to changes in the properties and behavior of this natural polymer and diversification of protein functionalities. In microencapsulation, functionalization of proteinic chains makes it possible to obtain microparticles with new properties, different from those obtained with other wall materials.

Grafting of fatty acid chains to proteins by acylation is well known in order to enhance their hydrophobicity, surface-active functionality and emulsifying capacity [START_REF] Matemu | Improved emulsifying properties of soy proteins by acylation with saturated fatty acids[END_REF][START_REF] Wong | Functional and biological activities of casein glycomacropeptide as influenced by lipophilization with medium and long chain fatty acid[END_REF]. In fact, the incorporation of hydrocarbon chains (hy drophobie part) into the protein macromolecules (hydrophilic part) allows the creation of amphiphilic structures with improved surface activity [START_REF] Rondel | Synthesis and properties of lipoamino acid-fatty acid mixtures: influence of the amphiphilic structure[END_REF]. On the other hand, the introduction of quaternary ammo nium groups to polysaccharides (Channasanon, Graisuwan, Kiatkamjornwong, & Hoven, 2007;[START_REF] Wang | Cationization of Ganodenna luddum polysaccharides in concentrated alkaline solutions as gene carriers[END_REF] or to ani mal derived proteins [START_REF] Kiick-Fischer | Controlling absorbency in gelatin net works: preparation and characterization of alkylated, crosslinked gelatin[END_REF][START_REF] Zohuriaan-Mehr | Protein and homo poly(amino acid)-based hydrogels with super-swelling properties[END_REF] by cationization is used to enhance their solubility, antibacterial properties as well as their hydrophilic properties (water absorption and swelling ca pacity). Nevertheless, there is no data in the literature dealing with cationization of vegetable proteins. Both acylation and cationiza tion reactions could be suggested as an effective way to obtain SPI with defined characteristics.

Therefore, the objective of this work was to study the influ ence of SPI modification by acylation and cationization, on the microencapsulation of hydrophobie (T) and hydrophilic (M) vi tamins by the spray-drying technique. In the context of "green" chemistry [START_REF] Galuszkaa | The 12 principles of green analytical chemistry and the SIGNIFICANCE mnemonic of green analytical practices[END_REF], modi fication reactions were carried out without any use of organic solvents and chemical catalysts. The effect of SPI modifications on both solution/emulsion and microparticle properties was also investigated.

Materials and methods

Materials

Soy protein isolate (SPI), 90% pure, was purchased from Lustre! Laboratoires SAS (Saint Jean de Vedas, France). The term 'native SPI' was used in this study for ail samples prepared with non-modified commercial soy protein isolate. Ali other chemicals were of analytical grade. r,.-Tocopherol, L-ascorbic acid, sodium hydroxide, dodecanoyl chloride, glycidyltrimethylammonium chloride, cyclo hexane (HPLC grade), iodine and sodium thiosulfate were pur chased from Sigma (Saint-Quentin Fallavier, France).

SPI modifications

The acylation reaction was carried out on SPI using dodecanoyl chloride (DDC) following the Schotten-Baumann reaction as described previously [START_REF] Nesterenko | Influence of soy protein's structural modifications on their microencapsulation properties: a-tocopherol microparticles preparation[END_REF]. The molar ratio DDC/NH 2 of protein used for the reaction was 0.5/1 and the sample obtained was named SPI-A.

The SPI cationization reaction was carried out in aqueous solu tion (5% w/w) at 40 °C or 70 °C using glycidyltrimethylammonium chloride (GTMAC). When the SPI solution reached reaction tem perature, pH was adjusted to 10.0 with 4 M NaOH and GTMAC was added (molar ratios GTMAC/NH 2 were 1, 2 or 4). The pH of the solution was maintained at 10.0 during the 1 h reaction period. The reaction was ended by adjusting the pH of the solution to 7.0 using 4 M HCI. The mixture of cationized SPI was cooled, freeze-dried at 20 Pa (Cryo-Rivoire equipment, Cryonext, Saint Gely du Fesc, France) and stored at 4 °C. Samples obtained were named SPI-C. The degree of cationization (DC) was evaluated using the o-phtal dialdehyde method (OPA) [START_REF] Church | Spectropho tometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins[END_REF][START_REF] Goodno | A fluorimetric assay for available lysine in proteins[END_REF] and defined as follows:

OC(%) = ( no -nm) x 100 no (1)
where n 0 is the molar quantity of amino groups per gram of native SPI, and nm the molar quantity of amino groups per gram of cat ionized SPI.

SPI solubility profiles

Native SPI solubility was compared to the cationized sample (SPI-C) and the blank sample (SPI-Cbtank treated under cationization conditions without GTMAC). Solubility profiles of SPI were deter mined as described in a previous study [START_REF] Nesterenko | Influence of soy protein's structural modifications on their microencapsulation properties: a-tocopherol microparticles preparation[END_REF]. Briefly, protein mixtures in deionized water (5% w/w) were pre pared at different pH values and stirred at 70 °C for 1 h. Suspensions were centrifuged at 10,000 x g for 15 min (Sigma Laborzentrifugen, Osterode, Germany). The soluble protein fraction in the superna tant was analyzed using the Kjeldahl method and solubility (S%, w/ w) was calculated from the following equation: S(%) = protein con � ent in the � upern � tant x 100 total protem content m solution

Microencapsulation by spray-drying

(2)

Protein based microparticles were prepared using a two-step procedure. An aqueous solution of protein (native or modified) was mixed with active core material. Then a liquid preparation (solution or emulsion) was spray-dried to obtain a microparticle powder.

Solution/emulsion preparation

The wall material (SPI) was dissolved in deionized water (8% w/ w) at 70 °C for 1 h under constant mechanical stirring (1000 rpm). In order to allow maximum protein solubilization, the pH of the solution was fixed at 10.5. Active material (T or M) was then mixed with SPI solution to obtain the preparation in which the protein/ active core ratio was 2/1 ( 11.5% of total solids ). Liquid preparations were homogenized with a high-pressure homogenizer (APV Sys tems, Albertslund, Denmark) at 50 MPa. The intense mechanical force developed during homogenization, contributed to protein structure modifications, such as unfolding of proteinic chains. Polar and non-polar regions of the protein were exposed to new envi ronments, which made them more surface-active [START_REF] Rampon | Evidence that homogenization of BSA-stabilized hexadecane-in-water emulsion induces structure modification of the nonadsorbed protein[END_REF]. Preparations with T were named SPI/T, SPI-A/T and SPI-C/T for native, acylated and cationized soy protein respectively. Preparations with M were named SPI/M, SPI-A/M and SPI-C/M for native, acylated and cationized soy protein respectively.

Solution/emulsion characterization

Light scattering and optical microscopy were used to check good dispersion of T in the proteinic solution and droplet size uniformity. Emulsions were analyzed after high-pressure homogenization for 1 h. The oil droplet size distribution of homogenized emulsions (SPI/T, SPI-A/T and SPI-C/T) and zeta-potential of protein solutions were measured using Zetasizer Nano-ZS equipment (Malvern In struments, Worcestershire, UK). To avoid multiple scattering ef fects, liquid preparations were diluted 100 times with deionized water before measurements. A relative refractive index 1Jo n / 1Jwater = 1.12 (1Joil = 1.49, 1Jwater = 1.33) was used to analyze the data, assuming that ail droplets were spherical in shape. The volume particle diameter (D43 or D v ) was taken and used as an indicator of the emulsion size. Additionally, emulsions were visualized using an Eclipse E600 optical microscope (Nikon, Sendai, Japan), linked to a digital video camera (DXM1200, Nikon, Sendai, Japan) at a magni fication of lO00x.

Apparent viscosity of all liquid preparations after high-pressure homogenization was determined at 20 °C and shear stress variation between 0 and 1 N/m 2 for 3 min, using a Rheometer CSLlO0 (Carri Med LTD, Dorking, UK) with a 6 cm diameter plate-cone geometry and 0.035 rad angle. Liquid preparations were characterized as Newtonian fluids.

Microparticle preparation

Freshly homogenized liquid preparations were spray-dried using a Mini Spray Dryer B-290 (Büchi, Flawil, Switzerland) un der stable process conditions as follows: inlet air temperature at 124 ± 4 °C and outlet at 74 ± 4 °C, drying air flow rate of 35 m 3 , spray flow rate of 0.47 m 3 /h, liquid feed flow rate of 0.33 x 10-3 m 3 /h and aspiration of 100%. Microparticles were collected from the cyclone collector, shut hermetically in opaque packaging and stored at 4 °C. The spray-drying yield was defined as follows:

Spray -drying yield(%) __ m _ p __ X 100 msPI+Core

(3)

where msPI+Core the initial mass of solids added in liquid prepara tion including SPI and active core (T or M) and m p is the mass of collected powder (dry matter). The moisture of obtained micro particles was determined with an infrared moisture balance (Sartorius, Goettingen, Germany) by drying the sample at 105 °C to constant weight during 5 min.

Microparticle characterization

The amount of T retained in microparticles during drying was determined using UV/VIS spectroscopy [START_REF] Faria | Characterization and singlet oxygen quenching capacity of spray-dried microcapsules of edible biopolymers containing antioxidant molecules[END_REF]. Briefly, about 5 mg of microspheres containing the T to be determined were dissolved in 10 mL of cyclohexane. The solution was stirred for 10 min and filtered through a 0.2 µm PIFE membrane filter. The absorbance of the solution was measured using a UV Spectrometer (UV-1800, Shimadzu, Kyoto, Japan) at 298 nm. To determine M content of the dry microparticles, the AOAC (Association of Official Analytical Chemists) standard methodology with an iodometric titration procedure was used [START_REF] Aoac | Iodine in iodized salts[END_REF]. Each sample of microparticle was analyzed at least three times.

The microencapsulation process was monitored for both retention efficiency (RE) and Joad efficiency (LE). RE was defined as the percentage of estimated active core content in particles ob tained ( Coreex p ) over theoretical core content ( Core th eo) in initial liquid preparation.

RE(%)

= C oreex p x 100 C or etheo

(4)
The difference between experimental and theoretical values was caused by active core Joss during spray-drying. LE, corre sponding to active core content per 100 g of powder, was calculated as:

LE(%) = mcexp x 100 mm ( 5 )
where mc,xp is the estimated mass of core in microparticles, and m m the mass of the analyzed sample of microparticles ( dry matter).

Particle size distribution was determined by the scattering pattern of a transverse laser light using the Scirocco 2000 equip ment (Malvern Instruments, Worcestershire, UK). The volume particle diameter (D43 or D v ) was calculated as the mean of three measurements per sample.

The morphology of the microparticles was observed with a scanning electron microscope LEO435VP (LEO Electron microscopy Ltd., Cambridge, UK) operated at a voltage of 8 kV. In order to examine the inner structure of prepared microparticles, the pow der was first frozen in liquid nitrogen and broken up in a mortar. Samples were deposited on conductive double-sided adhesive tape and sputter-coated with silver.

Statistical analysis

The experimental data was statistically analyzed using Minitab 16 software (State College, USA). A one-way analysis of variance (ANOVA) was performed to determine significant differences (P < 0.05) between the samples. Tukey's test was adopted as the multiple comparison procedure.

Results and discussions

SPI modifications

SPI cationization

During cationization in alkaline aqueous media, the GTMAC reacted with nucleophilic sites of the protein i.e. primary amino groups (N-terminal and lysine residues), which were the most reactive ones. The reagent used (GTMAC) is toxic because of the presence of an epoxy group. However, during cationization at pH 10.0 in water, the GTMAC unreacted with protein was hydrolyzed to (2,3-dihydroxypropyl)trimethylammonium chloride, and this sec ondary product did not have any toxicity. Indeed, it was used as an additive for care products to promote the retention of moisture [START_REF] Baldaro | Procedure for the prepa ration of puridied cationic guar[END_REF]. The total hydrolysis of GTMAC under the experimental conditions employed for cationi zation of SPI, was verified by nuclear magnetic resonance (NMR) of hydrogen. Thus, cationized SPI was used for microencapsulation experiments without previous purification. From the results presented on Fig. 1A, the degree of cationiza tion (DC) of SPI increased with the quantity of GTMAC in the re action media, and the temperature. Similar phenomena had been reported in a study dealing with cationization of starch using GTMAC [START_REF] Kavaliauskaite | Interaction of starch with glycidyltrimethylammonium chloride and properties of derived products[END_REF].

The highest modification rate (DC = 91.6%) was obtained at a molar ratio GTMAC/NH 2 of 4/1, after 1 h reaction time at 70 °c. To confirm the presence of positively charged groups on SPI, zeta potential measurements were made. The zeta potential of native SPI at pH 10.0 was -44.4 mV, and as shown on Fig. 18, the zeta potential of SPI after cationization had increased. However, values obtained were negative because of the high amount of negative net charges (Cao-) on the proteinic chains in alkaline media (pH 10.0). After cationization, the majority of primary amino groups (NH 2 ) were replaced by positively charged functions. Thus, the augmen tation in zeta-potential with increase of DC confirmed qualitatively the presence of positively charged trimethylammonium groups on proteinic chains.

The effect of SPI cationization on its functional properties, especially the solubility profiles, was studied (Fig. 2). The solubility of SPI and SPI-C was compared to a blank sample (SPI-CbJank treated for 1 h at 70 °C and pH 10.0 without GTMAC). The blank sample was more soluble than native SPI for ail pH values. This observation could be related to the partial unfolding of proteinic chains during treatment at alkaline pH and high temperature. On the other hand, for native SPI and SPI-CbJank samples, the isoelectric point was in a pH range between 4.5 and 5.0. However, the attachment of posi tively charged groups to SPI by cationization resulted in an obvious ----,------,----,--------.---,------ shift of the protein isoelectric point into the alkaline range (pH of 6.0-6.5). After cationization, the number of net positive charges of the protein was increased and the number of basic NH 2 functions reduced. This affected the overall balance of acid to basic groups in the protein and resulted in the shift phenomenon.

The solubility of SPI-C was higher than the solubility of SPI at pH 1-10, which confirmed a significant contribution of polar cationic groups on protein affinity with water. At acidic pH, positive charges fixed to SPI by cationization increased the repulsion between pro teinic chains. This favored protein-solvent interactions and explained the increase in protein solubility. Conversely, in alkaline solution (pH> 10), the attraction between cationic ((CH 3 ) 3 N + ) and carboxylic (Cao-) groups resulted in the decrease of protein-sol vent interactions. Thus the lowest protein solubility could be explained by the formation of more compact structures.

The cationized sample SPI-C (DC of 91.6%) obtained at 70 °C and molar ratio GTMAC/NH 2 of 4/1, was used as wall material for microencapsulation experiments.

SPI acylation

In agreement with our previous report [START_REF] Nesterenko | Influence of soy protein's structural modifications on their microencapsulation properties: a-tocopherol microparticles preparation[END_REF], soy protein ( degree of acylation of 33.2%) modified with dodeca noyl chloride (DDC) was the more efficient wall material for T microencapsulation compared to other modified proteins (hydro lyzed, hydrolyzed and acylated, acylated with octanoyl chloride and hexadecanoyl chloride). Therefore, SPI acylated with DDC (molar ratio DDC/NH 2 of 0.5/1) was selected for the present study.

Microencapsulation with native and modified SPI

Effect of SPI modifications on solution/emulsion properties

The preparation of a feed liquid containing wall material and an active core, was the first step involved in the process of microen capsulation by spray-drying. In the case of T encapsulation, the liquid preparation was an oil-in-water emulsion, whereas it was a solution in the case of M coating. The proteinic matrix material used for T and M microencapsulation was native (SPI), acylated (SPI-A) and cationized (SPI-C) soy protein.

The different characterizations of solutions/emulsions and mi croparticles are summarized in Table 1. Structural modifications of proteinic chains affected the viscosity of liquid preparations and the mean droplet size in emulsions.

Fatty acid hydrophobie chains attached to water-soluble pro tein by acylation favored the improvement of the amphiphilic character of SPI. Moreover, the DDC unreacted with SPI was hy drolyzed to sodium dodecanoate having surfactant properties. This enhancement in surface-active properties of SPI resulted in lower emulsion viscosity for sample SPI-A/T compared to sample SPI/T. A similar result was reported earlier [START_REF] Derkatch | Rheological properties of concen trated emulsions stabilized by globular protein in the presence of nonionic surfactant[END_REF], and the g SPI, SPI-A and SPI-C: native, acylated ( degree of acylation -33.2%) and cationized ( degree of cationization -91.6%) soy protein isola te respectively; T: a-tocopherol; M: ascorbic acid.

h RE: retention efficiency of a-tocopherol determined by UV spectroscopy and retention efficiency of ascorbic acid determined by iodometric titration. i LE: load efficiency or active core content per 100 g of powder.

phenomenon of viscosity fall was attributed to the changes in the composition of interface layers in the emulsion. The SPI-A/T emulsion (with acylated protein) had a reduced oil droplet size compared to SPI/T emulsion (0.7 µm and 1.1 µm respectively). The uniformity of droplet size in emulsions obtained was observed by optical microscopy (Fig. 3A). The intense mechanical forces applied to the pre-emulsion during high-pressure homogenization resulted in partial unfolding of proteinic chains and oil droplet dispersion. The emulsion was stabilized by an adsorbed layer of surface-active proteins, forming a protective barrier around the dispersed oil droplets. This protein layer provided immediate and effective protection of the fine droplets against coalescence and gave a good stability to the emulsion. The decrease in droplet diameter observed for SPI-A/T emulsion compared to SPI/T emul sion, could be due to the increased surface activity of SPI after acylation, and thus better oil droplet dispersion. The same behavior was observed for sunflower protein/a.-tocopherol emul sion in our previous study [START_REF] Nesterenko | New way of biomaterials valorization: use of sunflower protein for a-tocopherol microen capsulation[END_REF].

On the other hand, grafting of cationic quaternary ammonium groups to native SPI also involved a decrease in viscosity for both T and AA Iiquid preparations. After cationization, proteinic matrix chains became more hydrophilic [START_REF] Kiick-Fischer | Controlling absorbency in gelatin net works: preparation and characterization of alkylated, crosslinked gelatin[END_REF][START_REF] Zohuriaan-Mehr | Protein and homo poly(amino acid)-based hydrogels with super-swelling properties[END_REF]. This increase in hydrophilic properties of SPI favored the mobility of proteinic chains in aqueous media and, presumably, could explain the decreased viscosity for samples SPI-C/T and SPI-C/AA. A decrease in the mean droplet size in the case of T based emulsions was observed. However, the optical micrograph of the SPI-C/T emulsion (Fig. 3A) showed the formation of coalesced T droplets with diameters from 5 to 10 µm. The improved hydrophilic character of SPI chains could reduce their affinity with hydrophobie T resulting in Jess efficient protein adsorption on the oil droplet surface and thus reduced protection against coalescence and emulsion stability.

Volume droplet size distributions of oil-in-water emulsions are shown in Fig. 3B. Ali the emulsions had droplet size distributions with explicit bimodal behavior. A second minor population with a size of about 5 ± 2 µm could be attributed to some coalesced droplets. The droplet size dispersion of SPI/T was narrower than that of SPI-A/T, which confirmed that the droplet size distribution was more uniform in the case of native protein based emulsion. It is important to note that this population of coalesced droplets was larger in the SPI-C/T emulsion, which confirmed the decrease in emulsion stability.

Effect of SPI modification on the microencapsulation process

A significant difference in spray-drying yield for emulsions with T (61-68%) compared to preparations with AA (77-87%) was observed. This could be attributed to the hydrophobie and viscous character of a.-tocopherol that involved a higher microparticle accumulation inside the drying chamber. In addition, an increase in microparticle size for spray-dried T based emulsions (7.6-9.3 µm) compared to AA based solutions (4.8-5.2 µm), was also due to a microparticle agglomeration effect induced by the presence of surface oil. Nevertheless, these results demonstrated that native soy proteins could efficiently encapsulate hydrophobie (T) and hydrophilic (AA) core material with retention efficiencies of 79.7% and 91.8% respectively. Higher retention efficiency of AA compared to T could be attributed to the hydrophilic character of SPI and its better affinity to the hydrophilic core. A wide range ofbiopolymers has been reported in the literature as efficient wall materials for ascorbic acid encapsulation by spray drying (retention efficiency varying from 85% to 101%). These include: maltodextrin and cashew tree gum [START_REF] Moreira | Ascorbic acid and anthocyanin retention during spray drying of acerola pomace extract[END_REF], gum Arabie and rice starch [START_REF] Trindade | The stability of ascorbic acid micro encapsulated in granules of rice starch and in gum arabic[END_REF], pea protein and carboxymethylcellulose [START_REF] Pierucci | New microencapsulation system for ascorbic acid using pea protein concentrate as coat protector[END_REF]. The microencapsulation of a.-tocopherol by spray-drying has been studied with different natural wall materials such as maltodextrin and gum Arabie [START_REF] Faria | Characterization and singlet oxygen quenching capacity of spray-dried microcapsules of edible biopolymers containing antioxidant molecules[END_REF], pea protein and carboxymeth ylcellulose [START_REF] Pierucci | Comparison of a-tocopherol microparticles produced with different wall materials: pea protein a new interesting alternative[END_REF], and showed retention efficiency ranging from 73% to 87%. Compared to literature values, the retention efficiencies obtained in this study are highly satisfactory.

The results reported in Table 1 demonstrated that SPI acylation enhanced the RE of the hydrophobie active substance (T) from 79. 7% to 94.8% but reduced the RE of the hydrophilic active substance (AA) from 91.8% to 57.3%. During drying, the surface composition of microparticles was significantly influenced by the nature of feed liquid ingredients. In our case, the proteins were the most surface active components in the liquid preparation and they would adsorb to the air-liquid interface of the drying droplets. This pref erential adsorption combined with protein film forming properties, was responsible for the formation of a smooth protein skin on the microparticle surface Uayasundera, [START_REF] Jayasundera | Surface modification of spray dried food and emulsion powders with surface-active proteins: a review[END_REF]. SPI acylation increased hydrophobie and surface-active character of proteinic chains. This modification was successful with regard toT encapsulation, because of hydrophobie interactions occurring between wall material and active core [START_REF] Nesterenko | Influence of soy protein's structural modifications on their microencapsulation properties: a-tocopherol microparticles preparation[END_REF][START_REF] Nesterenko | New way of biomaterials valorization: use of sunflower protein for a-tocopherol microen capsulation[END_REF]. After SPI acylation, the hydrophobie microdomaines were formed in water because of hydrophobie association between fatty acid moities (Lee, Jo, Kwon, Kim Y.H., &Jeong, 1998). Thus, the formation ofprotein film on the air-liquid interface was more effective, the exposure of oil to the microparticle surface was minimized, and T was better protected. Conversely, acylated SPI showed much Jess effectiveness in micro encapsulation of a hydrophilic compound (AA), because of the reduced affinity between core and wall material.

In contrast to the positive effect of SPI acylation on T retention efficiency, the attachment of polar trimethylammonium functions to SPI by cationization resulted in the decrease of RE values for this hydrophobie substance. This was due to the enhanced hydrophilic character of cationized SPI, which led to lower emulsion stability. More oil could be expected at the microparticle surface and the Joss of active core during spray-drying was increased. On the other hand, the enhanced spray-drying yield for SPI-C/AA preparation was observed because of better affinity between active core and modified wall material.

Effect of SPI modification on microparticle size and morphology

As can be clearly seen in Fig. 4A, the morphology of spray-dried microparticles was characterized by spherical shape with di ameters ranging from 1 to 10 µm. The surface of SPI microparticles was smooth and compact without presence of fissures. For the SPI A/T sample, the inner structure of partiel es showed the presence of small pores indicating that oil droplets were well distributed in the protein matrix. In the case of AA based microparticles, the disper sion of active core crystal powder in the protein matrix, involved the formation of a dense non-porous wall.

The microparticle size distributions displayed in Fig. 4B and results presented in Table 1, show that AA based microparticles (5.2 µm for SPI/AA) had lower average size compared to T based microparticles (9.3 µm for SPI/T). This variation in the particle size was most likely due to the agglomeration ofT based microparticles because of the presence of surface oil. Moreover, the difference in morphology of spray-dried droplets (homogeneous solution SPI/AA or heterogeneous emulsion SPI/T) and in density of active core materials affected the size of microparticles produced. As reported in the literature, the expected size of microparticles with vegetable protein produced by spray-drying, ranged from 2-3 µm [START_REF] Pierucci | New microencapsulation system for ascorbic acid using pea protein concentrate as coat protector[END_REF][START_REF] Pierucci | Comparison of a-tocopherol microparticles produced with different wall materials: pea protein a new interesting alternative[END_REF]) to 9-18 µm (Favaro-Trindade et al., 2010;[START_REF] Ortiz | Pro duction and properties of casein hydrolysate microencapsulated by spray dry ing with soybean protein isolate[END_REF], which was comparable and within the particle size range obtained in this work.

Conclusions

This paper has demonstrated that some suitable modifications of proteinic matrix material can increase the microencapsulation efficiency of different active compounds. For this, soy protein isolate (SPI) was modified by acylation and cationization and used as wall material for a-tocopherol (T) and ascorbic acid (AA) microencapsulation using the spray-drying technique.

The attachment of a polar trimethylammonium function to SPI by cationization affected its hydrophilic properties, solubility and isoelectric point. Protein cationization resulted in reduced T retention and in enhanced spray-drying yield for AA based prep arations. SPI acylation led to lower emulsion droplet size and viscosity because of enhanced surface-active properties. Due to the better affinity between hydrophobie core material and acyl ated SPI, the retention efficiency was enhanced from 79.7% to 94.8%. Microparticles obtained had spherical shape and an internai morphology depending on the nature of the active core (porous for liquid core and dense for crystal core). Thus, native and modified SPI appear to be promising carriers for different delivery systems, such as nutraceutical, cosmetic or pharmaceutical products.

  The effect of SPI cationization conditions (temperature and molar ratio GTMAC/NH2) on (A) DC and (B) zeta potential at pH 10.0.

  Fi g . 2. Effect of cationization on the solubility profile of SPI.

Fig

  Fig. 3. (A) Optical micrographs (scale bar -10 µm) and (B) droplet size distributions of T based emulsions with native (SPI/Tl, acylated (SPI-A/Tl and cationized (SPI-C/Tl soy protein.

Fig

  Fig. 4. (A) Scanning electron micrographs ( external and internai structures. scale bar -2 µm) and (B) particle size distributions of SPI-A/T and SPI/M microparticles.

Table 1

 1 Properties of liquid preparations and spray-dried microparticles based on native and modified SPI. Different Ietters in the same line indicate a statistical difference between the mean values (P < 0.05 ).

	Measured property	Wall/core materials g
		SPI/T	SPI/M	SPI-A/T	SPI-A/M	SPI-C/T	SPI-C/M
	Emulsion droplet size, Dv (µm) Liquid preparation viscosity (mPa s)	1.1 ± 0.02" 15.0 ± 0.02"	ND 8.9 ± 0.03 c	0.7 ± 0.04 c 8.o ± o.02 ct	ND 9.8 ± 0.04 b	0.9 ± 0.03 b 4.1 ± 0.03 f	ND 5.8 ± 0.04•
	Spray-drying yield (%) RE h (%) LE i (%) Mean particle size (µm)	68 79.7 ± 1.0 b 26.3 ± 0.6 b 9.3 ± 0.2"	81 91.8 ± 0.7" 30.3 ± 0.2" 5.2 ± 0.2 c	66 94.8 ± 2.2" 31.3 ± 0.7" 7.7 ± 0.2 b	77 57.3±4.l c 18.9 ± 1.4 c 4.8 ±0.S C	61 38.3 ± 1.0 d 12.6 ± 0.3 d 7.6 ± 0.3 b	87 92.3 ± 2.8" 30.5 ± 0.9" 4.9 ± o.z c
	a-f