Maria Isabel

Landim Neves

Sylvie Desobry-Banon

Italo Tuler Perrone
email: italotulerperrone@gmail.com

Stephane Desobry
email: stephane.desobry@univ-lorraine.fr

Jérémy Petit

Maria Isabel Landin

Stéphane Desobry

Jeremy Petit
email: jeremy.petit@univ-lorraine.fr

Encapsulation of curcumin in milk powders by spray-drying: Physicochemistry, rehydration properties, and stability during storage

Keywords: curcumin, antioxidant activity, encapsulation, skim milk, storage stability, color

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

Introduction

Curcumin is a bioactive polyphenol extracted from turmeric rhizome (Curcuma longa) [START_REF] Sharma | Curcumin: the story so far[END_REF] that has anti-inflammatory, antimicrobial, anti-amyloid, and antitumor properties. Polyphenols present antioxidant activity, hence they are prone to induce beneficial effects on human health [START_REF] Fang | Effect of spray drying and storage on the stability of bayberry polyphenols[END_REF]. Curcumin is known to present a very low solubility in water (2.99 × 10 -8 M), a characteristic that limits its use in food products, especially in aqueous solutions [START_REF] Sharma | Curcumin: the story so far[END_REF][START_REF] Joe | Biological properties of curcumin -cellular and molecular mechanisms of action[END_REF]. Besides, its sensitivity to environmental conditions, including elevated temperatures, light, extreme pH, moisture, and oxygen, makes curcumin susceptible to degradation during processing and storage [START_REF] Fang | Effect of spray drying and storage on the stability of bayberry polyphenols[END_REF]. Thus, several studies have been carried out aiming at developing techniques for its protection in food products [START_REF] Fang | Effect of spray drying and storage on the stability of bayberry polyphenols[END_REF][START_REF] Sahu | Fluorescence study of the curcumin -casein micelle complexation and its application as a drug nanocarrier to cancer cells[END_REF][START_REF] Donsì | Encapsulation of bioactive compounds in nanoemulsion-based delivery systems[END_REF][START_REF] Mazzarino | Elaboration of chitosan-coated nanoparticles loaded with curcumin for mucoadhesive applications[END_REF][START_REF] Livney | Milk proteins as vehicles for bioactives[END_REF][START_REF] Tsali | Valorization of grape pomace: Encapsulation and storage stability of its phenolic extract[END_REF][START_REF] Hasan | Liposome encapsulation of curcumin: physico-chemical characterizations and effects on MCF7 cancer cell proliferation[END_REF]. Microencapsulation is a commonly used technique to stabilize and protect bioactive compounds in microscopic particles. With this technique, a micro/nano environment is created, incorporating unstable ingredients within a carrier matrix and developing a protection to the external environment [START_REF] Fang | Encapsulation of polyphenols-a review[END_REF].

Encapsulation provides protection against degradation reactions and prevents the loss of flavor, nutrients, and/or bioactive compounds. In addition, it can be used to limit the release of components during food processing and storage, while controlling the release rate in physiological (gastric, intestinal) conditions [START_REF] Livney | Milk proteins as vehicles for bioactives[END_REF][START_REF] Soottitantawat | Microencapsulation by spray drying: Influence of emulsion size on the retention of volatile compounds[END_REF][START_REF] Madene | Flavour encapsulation and controlled release -a review[END_REF][START_REF] Yazdi | Heating of milk alters the binding of curcumin to casein micelles: A fluorescence spectroscopy study[END_REF][START_REF] Corgneau | Recent advances on lactose intolerance: Tolerance thresholds and currently available answers[END_REF][START_REF] Corgneau | Développement de formulations alimentaires diététiques adaptées aux intolérants au lactose et présentant une meilleure digestibilité[END_REF][START_REF] Nikolova | Développement de matrices permettant de protéger des substances actives lors d'un traitement thermique[END_REF]. Numerous methods of microencapsulation exist: spray-drying, spray chilling/cooling, coacervation, extrusion, fluidized bed coating, liposome entrapment, and molecular inclusion [START_REF] Fang | Effect of spray drying and storage on the stability of bayberry polyphenols[END_REF]. Spray-drying surely represents the most commonly used encapsulation technique, owing to its many advantages:

-it is economical: around ten times cheaper than the lyophilization process; -versatile: various configurations of air and spray flows exist (co-current, counter-current, or combined flows), and the spray can either be produced by a centrifugal atomizer or an atomizing nozzle; -and it is suitable for meeting the quality and efficiency constrains of various industrial fields such as pharmaceuticals, cosmetics, and food.

Generally, spray-dried powders have good techno functional properties like good rehydration ability, high flowability and long shelf-life [START_REF] Nikolova | Impact of spray-drying process parameters on dairy powder surface composition and properties[END_REF][START_REF] Schuck | Analytical methods for food and dairy powders[END_REF]. Spray-drying is especially useful for encapsulating various heat-sensitive food ingredients such as polyphenols, flavonoids, anthocyanins, prebiotics, phytosterols and polysaccharides, because the temperature increase in the matrix remains limited throughout the drying process [START_REF] Fang | Effect of spray drying and storage on the stability of bayberry polyphenols[END_REF][START_REF] Fang | Encapsulation of polyphenols-a review[END_REF][START_REF] Ray | An overview of encapsulation of active compounds used in food products by drying technology[END_REF]. Indeed, during most of the drying time, the surface of the particle remains wet and the local temperature of the surrounding air does not exceed its wet temperature that is around 50 °C in classical spray-drying conditions [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF].

Consequently, the alteration of heat-sensitive compounds remains limited [START_REF] Filková | Industrial Spray Drying Systems. Chapter 9[END_REF].

Milk is an interesting matrix candidate for encapsulation by spray-drying [START_REF] Livney | Milk proteins as vehicles for bioactives[END_REF], owing to its structural and physicochemical properties: binding of ions and small molecules, surface-active and self-assembly properties, possibility of gelation, pHdependent gel-swelling behavior, possibility to control matrix rehydration kinetics and thus bioactive release, interactions with macromolecules to form complexes and conjugates with synergistic combinations of properties, various shielding capabilities that are essential for protecting sensitive encapsulated compounds, biocompatibility and biodegradability, possibility to control the bioaccessibility of the bioactive and promote its bioavailability [START_REF] Livney | Milk proteins as vehicles for bioactives[END_REF][START_REF] Tsali | Valorization of grape pomace: Encapsulation and storage stability of its phenolic extract[END_REF].

The protection efficiency of milk powders towards encapsulated bioactive compounds is mastered by their main physicochemical properties, such as chemical composition, particle size distribution, and particle shape factors. In particular, granulometric characteristics (that can be deduced from particle size distribution) of a powder is an important parameter because it influences many functional properties such as flowability and rehydration capacity [START_REF] Nikolova | Impact of spray-drying process parameters on dairy powder surface composition and properties[END_REF][START_REF] Schuck | Analytical methods for food and dairy powders[END_REF][START_REF] Toro-Sierra | Impact of spray-drying conditions on the particle size of microparticulated whey protein fractions[END_REF][START_REF] Fu | Effect of particle shape and size on flow properties of lactose powders[END_REF][START_REF] Juliano | Food powders flowability characterization: theory, methods, and applications[END_REF].

The ability to reconstitute milk from powder is an essential property for industrials and consumers using powdered dairy ingredients or products. The International Dairy Federation has defined three reconstitution indices, namely solubility, dispersibility, and wettability indices [START_REF]GEA[END_REF][START_REF] Fil | Détermination de la dispersibilité et de la mouillabilité[END_REF], that describe different steps of powder rehydration and depend on powder composition and affinity between water and its components, but also on particle size, shape, and structure (porosity) [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF][START_REF] Gaiani | Morphological descriptors and color as a tool to better understand rehydration properties of dairy powders[END_REF][START_REF] Murrieta-Pazos | Food powders: Surface and form characterization revisited[END_REF].

During the storage of dairy powders, several changes can be observed mainly depending on storage temperature and powder water content. Dairy powders are susceptible to undergo agglomeration and caking, which are known to alter various powder characteristics like particle size distribution, rehydration ability, and color [START_REF] Gaiani | Morphological descriptors and color as a tool to better understand rehydration properties of dairy powders[END_REF][START_REF] Murrieta-Pazos | Food powders: Surface and form characterization revisited[END_REF][START_REF]Colour in food: improving quality[END_REF]. When employed as an encapsulation matrix, physicochemical and techno functional properties of powdered milk must be kept as much as possible in order to ensure and maintain maximum protection of the active material [START_REF] Fang | Effect of spray drying and storage on the stability of bayberry polyphenols[END_REF][START_REF]Colour in food: improving quality[END_REF][START_REF] Ray | An overview of encapsulation of active compounds used in food products by drying technology[END_REF].

The objective of the current work was to produce a milk powder encapsulating curcumin and study the evolution of degradation of curcumin power during storage at different temperatures. Then, spray-drying was performed to produce milk powders in which curcumin is encapsulated. Physicochemical characteristics (particle size distribution, moisture content, colorimetric properties) and rehydration behavior (accessed by the wettability, dispersibility, and solubility indices) were evaluated for the fresh powder. Encapsulation efficiency was also determined just after spray-drying.

Last, curcumin-loaded milk powder was stored at different temperatures [START_REF] Fang | Encapsulation of polyphenols-a review[END_REF][START_REF] Fil | Détermination de la dispersibilité et de la mouillabilité[END_REF], and 50 °C) to determine the stability of curcumin upon aging.

Material and methods

Material

Skim milk powder "Lait spray 0 %", used to prepare concentrates to be spraydried, was purchased from Ingredia (Arras, France); its moisture content was 3.5 % (w/w). Curcumin powder (≥ 94 % purity) was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). All other chemicals, of analytical grade, were provided by Carlo Erba (Milan, Italy).

Production of milk powders by spray-drying

The production of milk with and without curcumin was conducted by spraydrying 810 g skim milk concentrates at 30 % dry extract. Curcumin was solubilized in ethanol at maximum concentration (1 mg.mL -1 ). The curcumin concentration in skim milk concentrates was set at 0.083 mg/g milk powder (dry basis, d.b.) as the concentration of ethanol in spray-dried concentrates should not exceed 2 % (w/w) according to the spray-drying pilot specifications. The employed spray-dryer was a Micra Spray MS150 (Anhydro, Soeborg, Danemark) with a co-current flow single tower equipped with a bi-fluid nozzle (60 100 + Buffer Air Fluid 120, Spraying System). The operating parameters were defined according to Perrone et al. [START_REF] Perrone | Influence of pilot spray dryer settings on moisture and water activity of whole milk powder[END_REF] that have studied the influence of drying parameters on water activity. The objective is to obtain powders with a water activity lower than 0.2, which makes them less sensitive to biochemical alterations during storage [START_REF] Perrone | Influence of pilot spray dryer settings on moisture and water activity of whole milk powder[END_REF]. In this work, spray-dried skim milk powders with curcumin and without curcumin will be noted LC and LS respectively.

Immediately after spray-drying, powders were stored at 10 °C in sealed polypropylene bags. The spray-drying yield (%) was calculated by dividing the dry mass of produced powder by the dry mass of spray-dried milk (810 g).

In order to regulate the outlet air temperature at 90 °C, the inlet air temperature and the product flow rate needed to be adjusted, depending on weather conditions, as inlet air humidity was not regulated (Table 1). A fan power of 70 % and a spray pressure of 1 bar were maintained for all drying experiments.

According to Table 1, even if spray-drying conditions were close, significant differences were denoted between the two spray-drying assays. Globally, the inlet air temperature and the concentrate flow-rate of the second assay (LS2 and LC2) were a little higher. In general, when the inlet flow is higher, the outlet temperature tends to be lower, which could be seen in the second assay.

Curcumin extraction and dosage

First, it was found in preliminary studies (data not shown) that ethanol is the best solvent for curcumin extraction; moreover, it is readily available and less dangerous than other organic solvents. A wavelength of 425 nm was chosen for performing curcumin quantification by UV-visible spectrophotometry.

Curcumin extraction was achieved according to the following procedure: 0.1 g curcumin-loaded skim milk powder was solubilized in 5 mL distilled water using a Stuart SB3 rotator for 50 min at 40 rpm at 25 ± 1 °C. Then, 10 mL diethyl ether was added, and the mixture was allowed to stir in the same conditions. The mixture was then centrifuged at 5 088 x g for 5 min with a Hereaus Megafuge 8R centrifuge (Thermo Fisher Scientific) and the supernatant was recovered and placed under a dinitrogen atmosphere until complete evaporation of diethyl ether. Finally, 1 mL ethanol was added. The curcumin solution was diluted hundredfold before analysis by UV-Visible spectrophotometry at 425 nm. Curcumin ethanolic solutions up to 0.1 mg/mL were analyzed in the same conditions for calibration purposes.

The results are given in milligrams of curcumin per gram of skim milk powder (d.b.) (mg/g), according to the following equation:

= Curc

(Equation 1)

And the proportion of spray-dried curcumin found in analyzed powder was calculated according to: % = . 100 (Equation 2)

Where mp designates the mass of skim milk powder (d.b.) (g), mc the mass of detected curcumin (mg), [Curc]conc the curcumin concentration in the feed concentrate (0.083 mg/g dry mass of skim milk concentrate), and [Curc]powder the curcumin concentration found in powders (in mg/g dry mass of skim milk powder).

Physicochemical properties of powders 2.4.1. Particle size distribution

The particle size distribution was measured using a Mastersizer 3000 laser diffraction granulometer (Malvern Instruments), supplied with the Aero S dry dispersion unit. To obtain a correct obscuration (1 %), samples were dispersed at 1.8 bar air pressure, 100 % air pressure, 40 % feed rate, and 2.7 mm hopper length. The particle size estimator was chosen as the volume mean diameter and the mean particle size d50 was defined as the diameter for which 50 % of analyzed particles have lower size [START_REF] Petit | A dimensional analysis approach for modelling the size of droplets formed by bi-fluid atomization[END_REF].

Moisture content and water activity

The moisture content was determined by the AOAC method 925.10 [START_REF]Official methods of analysis[END_REF]. The water activity was determined with the water activity analyzer HygroPalm apparatus (HP23-AW-A, Rotronic, Croissy-Beaubourg, France). value represents the color varying from yellow (+) to blue (-) [START_REF]Colour in food: improving quality[END_REF]. The chrorma, that defines the degree of color saturation, was calculated with the following formula: C* = √(a*²+b*²). Also, the hue angle (H*), derived from the two coordinates a* and b*, was determined as arctan(b*/a*). All measurements were taken as the average of at least three points on the center and the periphery of the Petri dish filled with powder samples.

Powder functionalities

The rehydration properties of milk powders are essential techno-functional properties for application purposes. The rehydration behavior and kinetics of spraydried skim milk powders were characterized.

Rehydration behavior

The water solubility index was determined by the methodology described in [START_REF] Fil | Détermination de la dispersibilité et de la mouillabilité[END_REF][START_REF] Schuck | Analytical methods for food and dairy powders[END_REF]. The wettability and dispersibility indices were measured according to [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF]. The solubility was calculated from the volume of sediment (mL) remaining after two centrifugations. The procedure consists in adding 10 g powder in 100 mL distilled water, stirring the mixture during 90 s at 3 800 rpm, letting it at rest for 15 min, then stirring it briefly with a spatula and pouring it into the centrifuge for 5 min at 160 x g.

Fill the tube with water up to the 50 ml mark. Disperse the sediment in the water and centrifuge again at 160g for 5 min. Note the quantity of sediment (ml). The insolubility index (II) is equal to the volume of sediment (ml) remaining after the second centrifugation from 50 ml of reconstituted liquid. The solubility index (SI), expressed as a %, is equal to:

! = 100 -2 . !! (Equation 3)
Wettability analysis consists in measuring the time required for a powder sample to cross water surface in given conditions: 100 mL distilled water was added into a 400-mL beaker and then 10 g skim milk powder was gently poured at the water surface.

The dispersibility index is the percentage of powder mass which can pass through 200-µm mesh size sieve. 10 g skim milk powder was added to 100 g distilled water, then the mixture was vigorously stirred with a spatula for 15 s. After that, the mixture was sieved, and the dry extract of the filtrate was determined after drying at 105 °C for 7 h in an oven. The dispersibility index (DI), expressed as a %, is equal to:

$! = %&''()*.+ ,-
).

.//01 2-.// (Equation 4)

Where w is the weight of the powder used, XRW is the residual free water content of the powder (% w/w), XDM is the dry matter of the filtrate after sieving (% w/w). Express the result to one decimal place.

Powder aging during storage

The stability of encapsulated curcumin during storage was also evaluated.

Powder samples were stored at 10, 25, and 50 °C for 2 months in hermetically-sealed plastic pots at 0.43 water activity by using a saturated solution of potassium carbonate [START_REF] Mandato | Liquids' atomization with two different nozzles: Modeling of the effects of some processing and formulation conditions by dimensional analysis[END_REF]. Qualitative analysis of curcumin by UV/visible spectrophotometry at 425 nm was performed on aged powders after the extraction step.

Statistical analysis

Spray-dried powders were produced in duplicate and all the analyses were triplicated. Statistical analysis (ANOVA to check the occurrence of significant differences among samples and Tukey's HSD test to identify what samples were significantly different) was carried out with STATISTICA 7.0 software (StatSoft) at p < 0.05.

Results and discussion

Spray-drying

Spray-dried powder amounts and yields obtained in the first and second spraydrying assays were represented in table 2.

Powder yields were higher in the first spray-drying assay. In the second spraydrying, environmental conditions led to a little reduction of the outlet air temperature, which favored powder adhesion to spray-dryer walls [START_REF] Filková | Industrial Spray Drying Systems. Chapter 9[END_REF][START_REF] Murrieta-Pazos | Food powders: Surface and form characterization revisited[END_REF][START_REF] Gaiani | Etude des mécanismes de réhydratation des poudres laitières: influence de la structure et de la composition des poudres[END_REF]. Most losses during spray-drying were due to particles adhering to the spray-dryer walls, but there was also a proportion of very small particles that were not retained by the cyclone. In fact, there is a relationship between the size of the particles and losses in the exhaust air: the smaller the particles, the harder they can be recovered with a cyclone [START_REF] Gaiani | Etude des mécanismes de réhydratation des poudres laitières: influence de la structure et de la composition des poudres[END_REF][START_REF] Cuq | Advances in food powder agglomeration engineering[END_REF].

Physicochemical properties

Physicochemical characteristics mean particle size, moisture content, water activity, and colorimetric parameters (lightness L*, chroma C* and hue angle h*) of skim milk powders with and without curcumin were determined (Table 3).

Mean particle diameters of spray-dried powders were smaller than those found in the literature for milk powders, even on the same pilot-scale plant [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF][START_REF] Toro-Sierra | Impact of spray-drying conditions on the particle size of microparticulated whey protein fractions[END_REF][START_REF] Fil | Détermination de la dispersibilité et de la mouillabilité[END_REF][START_REF] Petit | A dimensional analysis approach for modelling the size of droplets formed by bi-fluid atomization[END_REF].

This could result from the presence of ethanol in skim milk concentrates, which was used to solubilize curcumin. Indeed, viscosity of concentrates containing a small proportion of ethanol is expected to be lowered, which is known to enhance the liquid jet breakup, leading to smaller droplets and thus smaller particles [START_REF] Petit | A dimensional analysis approach for modelling the size of droplets formed by bi-fluid atomization[END_REF][START_REF] Mandato | Liquids' atomization with two different nozzles: Modeling of the effects of some processing and formulation conditions by dimensional analysis[END_REF]. All spraydried powders presented very close mean diameters, showing that the presence of curcumin did not impact the size of spray-dried particles.

All produced powders presented water contents close to 5 % (w/w), which is a sign of a good storage ability of powders [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF]. No significant difference was denoted among powders with or without curcumin, whatever the spray-drying assay. Fang [START_REF] Fang | Effect of spray drying and storage on the stability of bayberry polyphenols[END_REF] found that spray-dried powders present lower moisture content at higher temperatures, which was also observed in the present study. The atmospheric conditions during the second atomization were warmer and wetter, which also contributed to produce a wet and adherent powder.

The spray-drying conditions significantly influenced the water activity of the samples, as the powders produced during the first spray-drying assay had a slightly lower water activity (0.12) than those of the second spray-drying assay (0.14). This difference can be attributed to the higher feeding flow-rate of concentrate of the second spray-drying assay, which led to lower temperature and probably a higher relative humidity of outlet drying air [START_REF] Gaiani | Etude des mécanismes de réhydratation des poudres laitières: influence de la structure et de la composition des poudres[END_REF][START_REF] Cuq | Advances in food powder agglomeration engineering[END_REF].

The introduction of curcumin in food powders is expected to change their color by increasing their yellowness [START_REF] Rajasekaran | A lucid build-up of nanostructured curcumin, quercetin and their interaction with DNA[END_REF]. This was checked by colorimetric analysis (Table 3). Powder luminosity was significantly different for skim milk with and without curcumin and for the two spray-drying assays. The presence of curcumin increased powder lightness but also its chroma (representing the degree of color saturation),

showing that powders with curcumin were brighter and more colored. The main color of 13 all powders was greenish-yellow, even in the absence of curcumin. For skim milk powders free from curcumin, this can be explained by the presence of riboflavin, a yellowish-green colored milk compound.

Powder functionalities

Most food additives and ingredients are presented in powder form and must be rehydrated before use; hence, water-powder interactions and more generally the ability to reconstitute a powder in water are essential properties for the formulation of many food products [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF][START_REF] Gaiani | Etude des mécanismes de réhydratation des poudres laitières: influence de la structure et de la composition des poudres[END_REF]. Rehydration indices of produced skim milk with and without curcumin are displayed in Table 4.

Skim milk powder in the absence of curcumin was poorly wettable (wettability times ranging between 30 and 35 min), but upon curcumin addition, powder became not wettable (wettability times superior to 45 min). Wettability was the only factor that showed significant difference between skim milk powders with and without curcumin whatever the spray-drying assay. Dispersibility and solubility of LS and LC differed in the first spray-drying assay but were similar in the second one. This shows that a small change in spray-drying conditions may have a tremendous impact on powder functionalities. Indeed, it can be guessed that modifying the evaporation rate (mainly by changing concentrate feed rate, drying air temperature or flow-rate) may have an impact on the location of curcumin within skim milk particles, as more curcumin at the surface of particles may affect their rehydration capacity, owing to the hydrophobicity of this bioactive compound. This explanation is in accordance with the results of the first spray-drying assay, where skim milk powder without curcumin exhibited lower wettability and solubility but a better dispersibility. Moreover, it can be guessed that curcumin addition modified powder surface structure. According to Murrieta-Pazos et al. [START_REF] Murrieta-Pazos | Food powders: Surface and form characterization revisited[END_REF], the surface of skim milk powders is mainly covered by proteins. Due to the interaction of curcumin with casein, modification of casein micelle conformation probably occurred [START_REF] Cuq | Advances in food powder agglomeration engineering[END_REF][START_REF] Rajasekaran | A lucid build-up of nanostructured curcumin, quercetin and their interaction with DNA[END_REF][START_REF] Khanji | Structure, gelation, and antioxidant properties of curcumin-doped casein micelle powder produced by spray-drying[END_REF], which induced a change of the interaction sites of caseins at the surface of particles and affected interactions of powder with water.

It is considered that a powder is dispersible when dispersibility index exceeds 85 % [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF]. Therefore, spray-dried powders of the current study had low dispersibility.

Generally, a soluble powder is not very dispersible because it dissolves easily when being in contact with water, so it does not separate into small particles but solubilizes quickly in water [START_REF] Schuck | Analytical methods for food and dairy powders[END_REF][START_REF] Gaiani | Etude des mécanismes de réhydratation des poudres laitières: influence de la structure et de la composition des poudres[END_REF]. The high solubility of spray-dried powders is thus consistent with their poor dispersibility. Also, producing powders at pilot scale results in low mean particles size (see the d50 values around 15 µm, which is relatively small for food powders) [START_REF] Gnagne | Characterisation of flow properties of foutou and foufou flours, staple foods in West Africa, using the FT4 powder rheometer[END_REF], and powders constituted by small particles have a tendency to be cohesive [START_REF] Chever | Agglomeration during spray drying: Physical and rehydration properties of whole milk/sugar mixture powders[END_REF] and thus hard to disperse.

Storage study

Sample color was followed during storage (Table 5 and Figure 1) with a view to correlate colorimetric changes to the evolution of curcumin content in powders (Figure 2). Indeed, curcumin is yellow, and its alteration leads to color loss [START_REF] Kharat | Physical and chemical stability of curcumin in aqueous solutions and emulsions: Impact of pH, temperature, and molecular environment[END_REF] or the formation of degradation products having a more brownish/reddish color [START_REF] Priyadarsini | The chemistry of curcumin: from extraction to therapeutic agent[END_REF][START_REF] Tennesen | Studies on curcumin and curcuminoids. V. Alkaline degradation of curcumin Z. Lebensm. Unters[END_REF].

As previously discussed from table 1 results, for all spray-dried powders, the lightness at time zero was high. Furthermore, color saturation was higher in skim milk powders containing curcumin, due the yellow color by curcumin. Also, the main color of all powders was similar: greenish-yellow.

During storage, a decrease in powder lightness was observed for all samples and this darkening phenomenon was more intense at 50 °C. At 10 and 25°C, the color saturation remained unaffected in the course of storage, but at 50 °C color saturation increased for powders without curcumin, while it slightly decreased for powders with curcumin. Moreover, the hue angle showed a decrease at 50 °C, indicating a shift of powders without curcumin to orange-yellow, while it remained similar all along the storage for powders with curcumin. It can be guessed that at 50 °C, the decrease in luminosity, increase in saturation and increase in angle hue corresponds to the formation of brown pigments, due to the occurrence of the Maillard reaction. In fact, Maillard reaction corresponds to a group of chemical reactions initiated by the condensation of an amino group with a reducing sugar and resulting in the formation of brown compounds [START_REF] Gaiani | Etude des mécanismes de réhydratation des poudres laitières: influence de la structure et de la composition des poudres[END_REF].

The formation of brown pigments is evidenced by photographs displayed in Skim milk powders with curcumin tended to remain a little more yellow than powders without curcumin [START_REF] Wang | Spray-drying of curcumin-loaded octenylsuccinated corn dextrin micelles stabilized with maltodextrin[END_REF], due that a significant proportion of curcumin was still present after 60 days storage at 50 °C, as evidenced by curcumin quantification (Figure 2).

During storage, curcumin was more altered when stored at high temperature, as expected for this thermolabile compound: about 83 %, 72 %, and 58 % residual curcumin were found after 60 days storage at 10, 25, and 50 °C, respectively (Figure 2).

This demonstrates the importance of adequate conditioning and storage conditions to avoid changes in powder characteristics and the amount of residual bioactive material [START_REF] Gaiani | Morphological descriptors and color as a tool to better understand rehydration properties of dairy powders[END_REF][START_REF] Murrieta-Pazos | Food powders: Surface and form characterization revisited[END_REF][START_REF]Colour in food: improving quality[END_REF][START_REF] Chever | Agglomeration during spray drying: Physical and rehydration properties of whole milk/sugar mixture powders[END_REF][START_REF] Kharat | Physical and chemical stability of curcumin in aqueous solutions and emulsions: Impact of pH, temperature, and molecular environment[END_REF]. Encapsulation in dairy matrices may be a way to limit its alteration, owing to the interaction with casein micelles [START_REF] Khanji | Structure, gelation, and antioxidant properties of curcumin-doped casein micelle powder produced by spray-drying[END_REF] that can improve curcumin stability [START_REF] Naksuriya | A kinetic degradation study of curcumin in its free form and loaded in polymeric micelles[END_REF].

The decrease in curcumin content was correlated to the decrease in yellow color:

indeed, the Pearson correlation coefficients between residual curcumin content and color saturation were 0.64, 0.79, 0.82 for powders stored at 10, 25, and 50 °C, respectively, showing a marked positive correlation whatever the storage temperature.

Thus, the variation in concentration of curcumin explains a large part of the evolution of powder color during storage. The stronger correlation for powders stored at 50 °C may be derived from the increase in curcumin degradation rate at this higher temperature [START_REF] Penyimpanan | Stability of curcumin in turmeric oleoresin-β-cyclodextrin inclusion complex during storage[END_REF].

Figure 3 illustrates the link between chroma (C*) and curcumin content. It can be noted that, whatever the storage temperature, both chroma and curcumin concentration tended to decrease with storage time. Besides, storage temperature had a negative effect on curcumin-loaded skim milk powder chroma and this can be put in relation with the fact that low temperatures help maintain curcumin color [START_REF] Naksuriya | A kinetic degradation study of curcumin in its free form and loaded in polymeric micelles[END_REF][START_REF] Penyimpanan | Stability of curcumin in turmeric oleoresin-β-cyclodextrin inclusion complex during storage[END_REF]. For all temperatures, from 14 storage days, the decrease in curcumin content was accompanied by a decrease in powder chroma and this phenomenon was more evident at 10 °C. For all temperatures, between 28 and 42 days, the decrease in powder chroma was more significant compared to the other storage times. It may be guessed that curcumin alteration during storage begins with curcumin located at the powder surface, which strongly affects powder color. Then, at longer storage times, curcumin hidden in the powder bulk starts to be degraded, but without much affecting powder surface appearance. Hence, the link between powder surface color saturation and curcumin content may be more relevant at relatively short storage times. At 25 °C and especially at 50 °C, for the higher storage times (approximately from 42 days), the positive correlation between chroma and curcumin content became less evident, as at these temperatures the powders were expected to undergo Maillard reaction, which results to the formation of brown pigments, thus affecting powder color independently from the degree of curcumin alteration. It can then be concluded that the relation between chroma and curcumin content was not linear due the existence of other factors affecting powder color.

This correlation between powder color and curcumin content is very interesting for industrials, as they can use powder color as an indicator to easily and quickly evaluate the degree of curcumin alteration upon storage.

This study permitted to show that the presence of curcumin in spray-dried skim milk powders did not significantly affect their main physicochemical characteristics, except color, which was obviously more yellow in curcumin-loaded powders, and color stability, which was improved by curcumin. Differences found among investigated powders were more related to small changes in spray-drying conditions. It was also suggested that Maillard reaction could be limited during storage (even at temperature as high as 50 °C) by addition of a small curcumin proportion in dairy powder formulation.

Also, it was noted that curcumin alteration upon storage at high temperature remained limited and this can be explained by the protective role of the dairy matrix against curcumin oxidation. Previous studies with milk proteins have shown that milk proteins (especially casein micelles) can substantially contribute to the protection of bioactive compounds like curcumin by forming complexes with these molecules through hydrophilic and/or hydrophobic interactions [START_REF] Livney | Milk proteins as vehicles for bioactives[END_REF][START_REF] Yazdi | Heating of milk alters the binding of curcumin to casein micelles: A fluorescence spectroscopy study[END_REF][START_REF] Priyadarsini | The chemistry of curcumin: from extraction to therapeutic agent[END_REF][START_REF] Naksuriya | A kinetic degradation study of curcumin in its free form and loaded in polymeric micelles[END_REF].

Conclusion

This study dealing with encapsulation of curcumin in skim milk powder by spray-drying permitted to evidence the impact of small changes in spray-drying conditions, as well as the presence of curcumin on physicochemical and functional properties of resulting powders. Spray-drying yield was slightly affected by minor variations of spray-drying conditions. In the second spray-drying assay, the air outlet temperature was a little lower, which increased particle adhesion to the spray-dryer walls and slightly decreased process yield. This also impacted powder water activity: powders of the second assay had a slightly higher water activity, consistently with the lower air outlet temperature. Variations in spray-drying conditions between the two assays were so close that other physicochemical (mean particle size, span, moisture content) and functional (wettability, dispersibility, solubility) properties were similar.

The presence of curcumin in milk did not affect the spray-drying yield.

Physicochemical characteristics (mean particle size, span, moisture content, and water activity) were also unchanged due to the presence of curcumin with the notable exception of powder color. Indeed, curcumin-loaded powders were more bright (higher luminance L*) and had a higher saturation of yellow color (higher chroma C* for a hue angle h* corresponding to yellow). As regards powder functional properties related to reconstitution properties, solubility and dispersibility were unaltered by the presence of curcumin, contrarily to wettability which was markedly lowered in the presence of curcumin. Indeed, this hydrophobic compound may be found at powder surface, decreasing interactions between powder surface and water, thus making it less wettable.

During storage, changes in color were observed: color saturation decreased, indicating a decrease in powder yellowness. The color loss was enhanced when storage temperature was increased. Color loss was correlated with the decrease in curcumin concentration, hence powder color may constitute an indicator for following the degree of alteration of curcumin encapsulated in dairy powders. For all studied temperatures, the decrease in curcumin content during storage was low (28 % loss after 6 months storage at 25 °C), showing the suitability of skim milk as an encapsulation matrix for protecting curcumin.

This study allowed verifying the possibility of relating, during storage, the evolutions of curcumin content and powder color saturation (chroma). It permitted to highlight the negative effect of storage temperature on curcumin content. As a perspective of this study, it would be interesting to evaluate the evolution of antioxidant activity of curcumin-loaded milk powders upon storage in order to investigate if curcumin keeps its antioxidant power when encapsulated in milk powders and stored.

2. 4

 4 .3. Colorimetry Powder color was characterized with a Microflash 200D apparatus (Data color International, Lawrenceville, USA). For each sample, 10 g powder was poured in a Petri dish (leading to a powder layer of approximately 1 cm high) and color measurement was performed according to the three colors coordinates L, a*, b* of CIEL*a*b* system. The L* value represents sample luminosity, varying from black (0) to white (100), the a* value represents the color varying from red (+) to green (-) and the b*

Figure 1 :

 1 Figure 1: after 60 days storage at 50 °C, the color change was more pronounced for

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Skim milk powders with (LC) and without curcumin (LS), at fresh state and after 60 days storage at 50 °C.LS LC

Table 1 .

 1 Spray-drying conditions for the production of skim milk powders with (LC1 and LC2) or without curcumin (LS1 and LS2). Te = inlet temperature; Ts = outlet temperature; QL = skim milk concentrate flow rate.Values in the same row followed by the same superscripted letters did not significantly differ according to Tukey's HSD test (p < 0.05).

	Te (°C)	Ts (°C)	QL (mL/min)
	LS1 161.6 ± 2.1ª	91.6 ± 1.2 b	35.0 ± 2.

Table 2 :

 2 Mass of spray-dried powders and yields for the two spray-drying assays.

		Powder mass (g)	Yield (%)
	LS1	598,25
	LC1	685,06
	LS2	548,84
	LC2	562,27

Acknowledgements

The authors acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) in Brazil for financial support and the LIBio laboratory in France for scholarship and technical support.

Table 3. Physicochemical characteristics of skim milk powders with and without curcumin obtained by spray-drying.

Values in the same column followed by the same superscripted letters did not significantly differ according to Tukey's HSD test (p < 0.05). d50 (µm) Moisture (% (w/w)) Water activity (-) L* (-) C* (-) h* (-) LS1 14.10 ± 0.01 a 4.80 ± 0.17 a 0.12 ± 0.00 a 94.12 ± 0.03 b 7.23 ± 0.04 a 100.22 ± 0.24 a LC1 15.20 ± 0.01 c 5.58 ± 0.17 a 0.