Fanny Lazzaro

Arnaud Saint-Jalmes

Frédéric Violleau

Christelle Lopez

Mireille Gaucher-Delmas

Marie-Noëlle Madec

Eric Beaucher

Frédéric Gaucheron
email: frederic.gaucheron@rennes.inra.fr

Gradual disaggregation of the casein micelle improves its emulsifying capacity and decreases the stability of dairy emulsions

Introduction

The casein micelle consists of a highly aggregated particle of 150 to 200 nm diameter constituted of proteins (i.e. the four casein molecules s 1 , s 2 , , ), and minerals (mainly calcium phosphate) that ensure its colloidal stability (Dalgleish & Corredig, 2012;Holt & Horne, 1996;Holt, Carver, Ecroyd, & Thorn, 2013;[START_REF] Marchin | Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle x-ray scattering/ultrasmall-angle x-ray scattering[END_REF][START_REF] Schmidt | Micellar aspects of casein[END_REF][START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | Cryo-transmission electron tomography of native casein micelles from bovine milk[END_REF][START_REF] Walstra | On the stability of casein micelles[END_REF]. The casein micelle has a key role in food products, especially dairy products, as it often contributes to their functional properties (i.e. the ability to form and/or stabilize networks such as gels, foams and emulsions, etc) (Foegeding & Davis, 2011).

The colloidal properties of the casein micelle (structure, composition, charge, hydration, etc) can be modified by controlling environmental factors such as pH, salt and chelating agent addition, temperature, etc (de Kort, Minor, Snoeren, van Hooijdonk, & van der Linden, 2011;Gaucheron, 2004;[START_REF] Silva | pH-induced demineralization of casein micelles modifies their physicochemical and foaming properties[END_REF]. However, only a few studies have described the link between the colloidal organization and the functional properties of the modified casein micelle [START_REF] Broyard | Modifications of structures and functions of caseins: a scientific and technological challenge[END_REF]. Of all their functional properties, the capacity of the casein micelle to emulsify and stabilize oil in water emulsions is of great interest for the food industry, especially for the dairy industry. Indeed, many dairy products are edible emulsions (e.g. cream and ice-cream, infant formulae, etc) (Barbosa-Cánovas, [START_REF] Barbosa-Cánovas | The Rheology of Semiliquid Foods[END_REF]Guzey & McClements, 2006).

Emulsions consist of mixtures of two immiscible liquids (such as oil and water), one of the liquids being dispersed as droplets in the other [START_REF] Mcclements | Food emulsions principles, practices, and techniques[END_REF]. These systems are thermodynamically unstable. The two phases will separate as a result of creaming, flocculation (agglomeration) and/or coarsening (fusion by coalescence or Oswald ripening) of the droplets. It is crucial to control both their formation and their stability during manufacture and storage to ensure the final quality of food emulsions,.

One way to improve the formation and the stability of emulsions is to use emulsifying agents that adsorb at the oil-water interface and lower its tension. This results in the formation of

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smaller droplets that are less prone to creaming. The adsorbed layer formed by the emulsifying agents at the droplet surface can also protect the emulsion against flocculation and coalescence. Emulsifying agents can be assessed according to two main characteristics: their ability to facilitate the blending of the emulsion phases (i.e. emulsifying capacity) and their ability to stabilize the emulsion (i.e. emulsion-stabilizing capacity). Caseins are known to adsorb at the interface, either in individual or aggregated form (Dickinson, 1999), and are therefore able to fulfill the role of emulsifying agent.

The emulsifying and stabilizing capacity of caseins is associated with their chemical nature and conformation at the interface and also depend on their aggregation state. Poorly aggregated casein systems such as sodium caseinate (30 to 50 nm diameter -formed by extreme acid demineralization of native casein micelle) (Pitkowski, Durand, & Nicolaï, 2008) have enhanced emulsifying properties but are less effective for the stabilization of emulsions than highly aggregated casein micelles [START_REF] Courthaudon | Surface active and emulsifying properties of casein micelles compared to those of sodium caseinate[END_REF][START_REF] Mulvihill | Surface active and emulsifying properties of caseins/caseinates as influenced by state of aggregation[END_REF].

However, little information is available on the emulsifying properties of the intermediate aggregation states of casein micelles. [START_REF] Ye | Functional properties of milk protein concentrates: emulsifying properties, adsorption and stability of emulsions[END_REF] contributed to this information by studying different milk protein concentrates (MPCs) containing both casein and whey proteins as well as lactose in soya oil-based emulsions. Demineralization of the MPCs was induced by cation exchange but did not control the diffusible phase.

The aim of our study was to investigate the effects of the gradual disaggregation of pure casein micelles on their colloidal properties and on their emulsifying and stabilizing capacity in model dairy emulsions. Tri sodium citrate (TSC), a calcium chelating salt, was used to remove calcium and inorganic phosphate from the casein micelle and to produce four suspensions of differently demineralized casein aggregates (CAs). Dialysis was performed on each suspension to control their diffusible phases. The CAs in these suspensions were characterized physico-chemically and used to form two types of emulsion to study their emulsifying and emulsion-stabilizing capacity separately. In addition, emulsions containing were of analytical grade.

Materials

Purified casein micelles were used to monitor our system. They were supplied by Gillot SAS (Saint Hilaire de Briouze, France) and obtained by microfiltration (0.1 μm pore size membrane) of raw skimmed milk followed by diafiltration against milli-Q water and spray dried according to Pierre, Fauquant, Le Graët, & Maubois (1992) and [START_REF] Schuck | Déshydratation par atomisation de phosphocaséinate natif obtenu par microfiltration sur membrane[END_REF] on Bionov facilities (Rennes, France). The powder comprised 96% (w/w) proteins -especially caseins (97%) (w/w). Residual whey proteins (3%) (w/w), lactose and diffusible calcium were present in the powder.

Anhydrous milkfat (AMF, melting point 32°C) was sup plied by Corman (Limboug, Belgium).

Preparation of different CA suspensions

Casein micelle powder was suspended in milli-Q water at a concentration of 28 g kg -1 and NaN 3 (1.6 g kg -1 ) was added for conservation (Fig. 1A). To ensure good resuspension of the powder, the suspension was stirred at 900 rpm for 6 h at 40°C in a water bath and then for 16 hours at room temperature. The rehydration of the casein micelle powder was checked by laser light diffraction as defined by [START_REF] Matijevic | Analytical methods for food and dairy powders[END_REF]. The results expressed in volume showed that more than 90 % of the particles were of size of casein micelles (150

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT nm diameter)
. This suspension was used to prepare four CA suspensions (S1, S2, S3 and S4). In S2, S3 and S4 varying amounts of a stock solution of TSC (0.85 mol kg -1 in milli-Q water, pH 7.0) were added to reach final concentrations of 4, 13 and 34 mmol kg -1 , respectively. S1 was kept as a control suspension (without addition of TSC). These suspensions were stirred for 30 min and then diluted with milli-Q water to reach an intermediate casein concentration of 25 g kg -1 . The pH was then adjusted to 7.0 with HCl 1M. S1, S2, S3 and S4 were left overnight at room temperature and the pH of each was readjusted if necessary. S1, S2, S3 and S4 were then dialyzed against an aqueous solution saturated in calcium and phosphate (5 mmol kg -1 NaH 2 PO 4 .2H 2 O and 5 mmol kg -1 of CaCl 2 .2H 2 O, pH was adjusted to 7.0 using 1 M NaOH). The aim of the dialysis was to remove the added citrate and the ions solubilized from the casein micelle. This provided an identical ionic environment for all the CAs in the four different suspensions. Using a solution saturated in calcium and phosphate also provided the advantage of limiting any further demineralization that might have been induced by classical dialysis against pure water. This was performed in two steps: first the suspensions were individually dialyzed (in separate baths) for 27.5 h at room temperature against a total volume of 44 times each suspension volume, and the baths were changed four times. The second step was combined dialysis (in the same bath) of the four CA suspensions for 15 h at room temperature against a volume 11 times the total suspension volume. The molecular weight cut-off of the dialysis membrane was between 12 and 14 kDa (Spectra/Por, Rancho Dominguez, Canada). The last dialysis bath was then filtered on a 2.5 μm filter paper and used to dilute the suspensions to reach a final casein concentration of 19.7 ± 0.6 g kg -1 . The final pH was 6.98 ± 0.04. The dialyzed CA suspensions, named S1 d , S2 d , S3 d and S4 d , were prepared in duplicate.

Recovery of the diffusible phases of the CA suspensions

The diffusible phases of each CA suspension were obtained by ultrafiltration for 30 min at 20°C on Vivaspin 20 concentrators (molecular weight cut-off 10 kg mol -1 , Vivascience,

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Palaiseau, France). They were used for the determination of diffusible cation and anion concentrations in the CA suspensions, as well as for the dilution of the CA suspensions and the emulsions for determination of the zeta potentials and sizes.

Preparation of the two types of emulsion

Two types of emulsion (E ec for "emulsifying capacity" and E st for "stability") were prepared with each of the four CA suspensions in order to evaluate the emulsifying and emulsionstabilizing capacity of the CAs (Fig. 1B).

Emulsions E ec were prepared with the CA suspensions diluted at a protein concentration of 1.2 g kg -1 with milli-Q water and then added to the 60°C melt ed AMF at a 30:70 (v/v) ratio.

The mixture was emulsified at 50°C in a water bath using a Polytron PT 3100 (Kinamatica AG, Littau, Switzerland) at 29,000 rpm for 5 min. Working at a limited protein concentration (1.20 g/kg compared to our emulsification system) highlighted the differences between the CAs by producing emulsions with different droplet sizes. E st emulsions were prepared following the same procedure except that the CA suspensions were kept at a protein concentration of about 20 g kg -1 . In this case, the choice of an excess protein concentration produced emulsions with similar droplet sizes, necessary for the study of the stabilizing capacity of the CAs. E st emulsions were divided into several samples and stored in transparent, cylindrical, hermetically sealed tubes at 50°C for 3 weeks. The temperature of 50°C was chosen in order to prevent the formation of fat crystals in the emulsion that could affect their physical stability [START_REF] Lopez | Coupling of time-resolved synchrotron X-ray diffraction and DSC to elucidate the crystallization properties and polymorphism of triglycerides in milk fat globules[END_REF].

Each week, one sample was analyzed by laser light diffraction, electrophoretic light scattering, multiple light scattering and confocal microscopy to follow the evolution of the emulsion. Two replicate emulsions were made for each type of emulsion.

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Analysis

Mineral composition and distribution

Total cations (calcium, magnesium, sodium and potassium) and diffusible cations and anions (inorganic phosphate, citrate and chloride) were determined in the CA suspensions and in their diffusible phases, respectively. Total anions were determined in the diffusible phases CA suspensions previously acidified at pH 4.6 with a 10% (v/v) acetic acid solution. Cation concentrations were measured by atomic absorption spectrometry (Varian 220FS spectrometer, Les Ulis, France) as described by [START_REF] Brulé | Etude de la teneur en éléments minéraux des produits obtenus lors de l'ultrafiltration du lait sur membrane[END_REF]. Anion concentrations were determined by ion chromatography (Dionex ICS 3000, Dionex, Voisin le-Bretonneux, France) as described by Gaucheron, Le Graët, Piot & Boyaval (1996). Colloidal concentrations were deduced by subtracting diffusible from total ion concentrations. The calcium demineralization rates corresponded to the percentage of solubilized calcium compared to total calcium initially present in the suspensions prior to dialysis.

Protein content

Protein content was determined in the CA suspensions and in their respective ultracentrifuged supernatants to deduce the non-sedimentable casein concentrations. The Kjeldahl method (IDF standard 20-1,2014) was used to determine the total nitrogen concentration in the samples, and a conversion factor of 6.38 was used to convert nitrogen to protein concentration. Measurements were performed in duplicate.

Pellet hydration and sedimentable protein concentrations

Twenty grams of CA suspension were ultracentrifuged at 20°C for 1 h at 100,000 g (Sorvall Discovery 90 SE, Hitachi, Courtaboeuf, France) and the ultracentrifuged pellets were recovered. Hydration was deduced according to the weight loss after drying the ultracentrifuged pellets of each sample mixed with Fontainebleau sand in an oven at 105°C for 8 h (FIL-IDF Standard 26A, 1993).

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Sedimentable protein concentrations were deduced from the proportion of pellets and hydration data by considering that ultracentrifuged pellets consisted mainly of proteins and water (mineral weights were disregarded). Measurements were performed in duplicate. was filtered through 0.1 μm filter paper. This filtered solution was used as the eluent for the AsFlFFF separation, and for the ten-fold dilution of the samples.

During the AsFlFFF run, the laminar flow was fixed at 1 mL min -1 and only the cross flow varied. The first focusing-injection step (10 min) consisted of setting up the cross flow at 1.5 mL min -1 for 1 minute. Then 30 μL of sample were injected while the cross flow was maintained at 1.5 mL min -1 for 9 min. This allowed the analytes to diffuse away from the membrane according to their R h . The elution step then started with a 5 min plateau at a cross flow rate of 1 mL min -1 followed by a linear decrease of 5 min to reach 0.15 mL min -1 for 25 min. The cross flow was finally stopped to eliminate all the particles that might have remained in the AsFlFFF channel.

Under our operating conditions, the AsFlFFF worked in normal mode, which means that larger particles were retained in the channel for longer times than smaller ones, providing that all particles had similar density.

The AsFlFFF was connected to an 18 angle DAWN-DSP MALLS detector (Wyatt Technology, Santa Barbara, CA, USA) ( = 633 nm), an Optilab Rex Refractometer (Wyatt Technology, Santa Barbara, CA, USA) ( = 685 nm), and an Agilent 1100 UV detector ( =

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concentrations and a calculated extinction coefficient of 9.009 L g -1 cm -1 was determined and used (bovine serum albumin at 280 nm in the eluent). Astra software version 6.0 was used to analyze the UV and Rayleigh ratio data and determine the MW and R h values. In this study, it was assumed that the different CAs were spherical and homogenous in composition. R h were determined between 20 and 28 min (population A) via Berry formalism of a Debye plot. R h cannot be calculated directly between 14 to 20 min (populations B and C) because of the low Rayleigh ratio signal in this time range. For suspensions S1 d and S2 d , the R h values between 20 and 24 min were therefore fitted with a first order exponential model that was extrapolated between 14 and 20 min (populations B and C). This treatment was not possible on S4 d because of the low Rayleigh ratio signal, and the values of R h determined for S4 d calculated between 20 and 28 min were not accurate enough for their extrapolation (see section 3.1.2).

Similar tests were performed to determine MW values, with slight modifications. The MW values of S1 d and S2 d were fitted with exponential models of first order between 18 to 20 min and the models were extrapolated between 14 to 18 min. In contrast to R h , the S4 d Rayleigh ratio was high enough to determine MW. For this suspension, the MW values were fitted between 15.5 and 16.5 min (population B) and the model was extrapolated between 14 and 15.5 min (population C).

Zeta potential ( )

The electrophoretic mobility of CAs (in CA suspensions) and milkfat droplets (in emulsions)

were measured by electrophoretic light scattering using a Zetasizer 3000 HS (Malvern Instruments, Worcestershire, UK). CA suspensions and emulsions were diluted in their corresponding diffusible phases. Diluted CA suspensions were filtered through a 0.45 μm pore size membrane to eliminate possible dust particles prior to analysis.

Henry's equation:

= ( 3 μ / 2 f(Ka) ) (1) M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
where is the viscosity and the dielectric constant of the solution, was applied to determine the apparent zeta potential ( ) of the particles from their electrophoretic mobility μ. F(Ka) = 1.5 was used according to the Smoluchowski approximation. The measurements for the CA suspensions were performed at 20°C and the viscosit y and the dielectric constant of the dissociating medium (water) were 1.00 cp and 80.4, respectively. Measurements for the emulsions were performed at 50°C with a viscosity o f 0.55 cp and a dielectric constant of 70.2. Measurements were performed in triplicate.

Droplet size distribution in emulsions

The milkfat droplet size distributions were determined by laser light diffraction immediately after the preparation of the emulsions and after 7, 14 and 21 days of storage at 50°C, using a

Mastersizer 2000 (Malvern Instruments, Worcestershire, UK) equipped with a He/Ne laser ( = 633 nm) and an electroluminescent diode ( = 466 nm). The refractive indices were set at 1.46 (at 466 nm) and 1.458 (at 633 nm) for milkfat and 1.33 for water. Before measurements, samples were dispersed in milli-Q water as was, or were previously diluted ten times in a solution of 1% (w/w) SDS to separate aggregated milkfat droplets and estimate the extent of droplet flocculation. All distributions and/or their corresponding mode values (i.e. the maxima of the size distribution) were used to compare the emulsions. Specific surface areas (area per unit mass) were used for the determination of the protein surface concentrations.

Measurements were performed in triplicate.

Confocal microscopy of the emulsions

The microscopy observations were carried out with a Nikon Eclipse-TE2000-C1si confocal microscope (Nikon, Champigny sur Marne, France) equipped with argon and He-Ne lasers operating at 488 and 543 nm excitation wavelengths, respectively (emissions were detected between 500 and 530 nm and between 565 and 615 nm, respectively). One milliliter of emulsion was stained using 100 μL of a milkfat soluble Nile Red fluorescent dye solution (0.1% w/w in propane diol) and 50 μL of a Fast green FCF solution (1% w/w in water) to stain the proteins. The samples were left for 15 min at 50°C prior to observation. Microscopy

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observations were performed at 50°C using a thermal PE100-NI System plate warmer (Linkam Scientific Instruments Ltd., Tadworth Surrey, England). Images were collected with an oil immersion objective with a magnification of x 60. Characteristic images were selected from the 9 images taken for each sample.

Interfacial tension and dilational rheology

An oscillatory drop tensiometer (Tracker, Teclis, France) was used to measure the interfacial tension ( ) and the interfacial dilatational moduli (E * , E' and E'') at the milkfat/CA suspension interfaces, at 50°C. The CA suspensions and the las t dialysis bath (control) were used to form a pendant drop of 10 μL at the tip of a syringe that was suspended in an 8 mL cuvette containing melted milkfat (50°C). Two opposite forc es, gravity and the force related to , were exerted on the drop to induce its shape. Analysis of the shape of the drop 5 min after its formation (equilibrium state) made it possible to calculate the value by solving the Laplace equation [START_REF] Ravera | Interfacial dilational rheology by oscillating bubble/drop methods[END_REF].

Dilatational rheology was performed on our system by applying the conditions used by Silva, Saint-Jalmes, de Carvalho, & Gaucheron (2014) with slight modifications. Briefly, a sinusoidal oscillation of the drop volume of 10% at a frequency of 0.2 Hz was applied to a 2 min old 10μL CA suspension drop in the melted milkfat at 50°C. The volume variation engendered a controlled oscillatory compression/dilation of the droplet interfacial area A and resulted in the surface tension oscillation as a function of time (t). Monitoring of (t) and determination of its phase shift ( ) compared to A(t) made it possible to calculate the complex (E*), elastic (E') and viscous (E'') moduli of the adsorbed interfacial layer. Purely elastic and solid-like interfacial layers had E E and tended to 0, whereas viscous and fluid-like interfacial layers had E > E and a large .

Creaming stability ratio

A transparent, cylindrical, hermetically sealed glass tube was filled with 20 mL of fresh E st emulsion and placed in the measurement chamber of a Turbiscan MA2000 multiple light

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scattering optical analyser Turbiscan MA2000 (Formulaction, France). The tube was scanned at 50°C from top to bottom by a 850 nm ligh t source and the back scattered light was recorded every 40 μm. Analysis of the back scattered signal as a function of the height of the tube determined the total height of the emulsion (H) and the thickness of the creamed layer (h). The creaming ratio (r c ) was defined as r c = H/h. The measurements were performed on each E st emulsion after 0, 7, 14 and 21 days of storage at 50°C.

Surface protein concentration

The method of separation of the non-adsorbed proteins from the emulsion droplets was derived from [START_REF] Patton | A method for isolation of milk fat globules[END_REF]. Forty-four milliliters of E st emulsion were gently mixed with 5 g saccharose in 50 mL centrifuge tubes and maintained at 50°C in a water bath. The tubes were centrifuged at 200 g for 20 min at 50°C and frozen at -20°C. The frozen tubes were cut at the interface to separate the creamed milkfat droplets at the top of the tube and the aqueous phase containing saccharose and non-adsorbed caseins at the bottom. The milkfat droplet phases were transferred to other centrifuged tubes, melted at 50°C and redispersed in 15 mL of 4% (w/w) SDS solution. The tubes were centrifuged at 1 500 g for 20 min at 50°C, frozen at -20°C and cut to separate t he top milkfat phase from the bottom aqueous SDS phase containing the adsorbed caseins. The first bottom saccharose aqueous phase (containing the non-adsorbed caseins) and the second bottom aqueous SDS phase (containing the adsorbed proteins) were both analyzed in terms of protein concentration using Kjeldahl and micro-Kjeldahl methods, respectively. The amounts of casein adsorbed at the interfaces were related to the specific surface areas of the droplets (previously determined by laser light diffraction) to calculate the interfacial casein concentrations and the percentages of adsorbed caseins.

Statistics

Measurements were carried out on each of the replicates of suspensions S1 d , S2 d , S3 d and S4 d , emulsions E1 ec , E2 ec , E3 ec and E4 ec and emulsions E1 st , E2 st , E3 st and E4 st , except for

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AsFlFFF, and dilatational rheology measurements. The standard deviations were calculated for each determination.

Results

The results are presented in two steps with first a focus on the physico-chemical and colloidal characteristics of the CAs in suspensions only. Their functional properties are then described when used as emulsifying agents in our model dairy emulsions.

Physicochemical characterization of casein aggregate suspensions

Mineral characteristics

Colloidal calcium and inorganic phosphate concentrations (Table 1) decreased simultaneously and in a correlated fashion (Fig. 2) in the order: S1 On the other hand, the concentration of colloidal sodium (Table 1) increased in the suspensions with the increase in added TSC. This increase was correlated with the decrease in colloidal calcium concentration (Fig. 2), and therefore with the decrease in inorganic phosphate concentration. The chloride ions were only diffusible in the CA suspensions (Table 1). The sodium and chloride present in the saturated dialysis baths (counter ions of phosphate and calcium) mainly contributed to the high colloidal and diffusible concentrations observed in the dialyzed CA suspensions.

Magnesium and potassium were not present in the CA suspensions because these ions were not present in the purified casein micelles. Diffusible ion concentrations (Table 1) were similar in the suspensions and no diffusible or colloidal citrate was found after the dialysis step. Diffusible calcium was close to zero for all suspensions.

Colloid characterization

Hydration of the ultracentrifugation pellets was constant for S1 d , S2 d and S3 d and slightly lower for S4 d (Table 2). The concentration of sedimentable proteins decreased with the increase in the amount of TSC added and a reduction of 82% was found when comparing 2). The non-sedimentable casein content thus increased from 8 to 18 g kg -1 with the addition of TSC to the CA suspensions (Table 2).

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT suspension S1 d with S4 d (Table
Similar zeta potentials (22.7 ± 1.3 mV) were measured for each CA suspension (Table 2).

The UV, Rayleigh ratio and the calculated MW and R h of the CAs in the suspensions obtained by AsFlFFF are represented as a function of elution time, respectively (Fig. 3). For each suspension, three peaks that corresponded to three different populations of particles

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The proportions of the different populations of particles depended on the amount of added TSC: the largest particles (A) disappeared when the TSC concentration increased, permitting the appearance of the two smallest populations (B and C). Nevertheless, the loss in surface area under the A peak was not equal to the gain in surface area under the B and C peaks due to the fact that the largest particles not only absorbed but also diffused the UV signal compared to small particles that only absorbed the UV signal.

Functional characterization of casein aggregate suspensions

Emulsifying capacity of casein aggregate suspensions

The particle size distribution profiles of E1 ec , E2 ec , E3 ec and E4 ec emulsions are presented in Microstructural observations confirmed the decrease in the size of the emulsion droplets as a function of the increase in TSC concentration in the CA suspensions. Moreover, flocculation of the emulsion droplets was characterized in each emulsion, in agreement with particle size measurements (Fig. 4).

The interfacial tension ( ) at the melted milk fat/CA suspension interface was measured to evaluate the activity of the CAs at the milkfat droplet surface. Blank interfacial tension determined on the last dialysis bath of the CA suspensions was 10 mN m -1 . The presence of CAs decreased to around 5 -6 mN m -1 whatever the added TSC concentration.

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Emulsion-stabilizing capacity of the casein aggregate suspensions

The evolution of the creaming ratios (r c ) of E st emulsions over time are shown in Figure 6.

None of the emulsions were stable against creaming. Phase separation was easily observable after 7 days of storage and did not vary during the following 14 days. The determination of r c indicated that the thickness of the creamed layers decreased with the increase in added TSC in the CA suspensions.

Laser light scattering measurements and confocal microscopy observations were performed on each emulsion throughout storage at 50°C (Fig. 7 ). Given that the particle size distribution profiles were monomodal (data not shown), the evolution of the mode value of each emulsion as a function of time is represented. The light scattering measurements were carried out in the presence and absence of SDS. Indeed, this small surfactant is able to dissociate flocculated droplets by replacing the protein at interfaces, permitting discrimination of flocculated droplets from coalesced droplets. When droplets flocculated, the emulsion size distribution shifted to smaller sizes (smaller mode value). In contrast, the addition of SDS had no influence on the size distribution of coalesced droplets. Figure 7 shows that the size of the particles in emulsions increased with time without SDS, especially for emulsions from CA suspensions containing TSC. For example, the mode value of E2 st increased from 12 to 33 μm after 21 days of storage and from 12 to 90 μm for E4 st . In the presence of SDS, the size distribution of the droplets did not evolve over time, the mode being 12 μm, similar to the size determined after the preparation of the emulsions (data not shown). These constant values indicated that E2 st , E3 st and E4 st were destabilized by flocculation but were stable against coalescence. The E1 st emulsion, which maintained a constant mode value throughout storage, was stable against both flocculation and coalescence phenomena.

The corresponding micrographs of each emulsion at each time-point were in good agreement with laser light scattering data (Fig. 7). Each emulsion maintained the same droplet size during storage. However, some micrographs showed contrast differences, with bright milkfat droplets at the foreground of the image and dark red droplets at the back. This Around 24 ± 1% of the total protein present in the emulsions was adsorbed at the interface, whatever the type of CA suspension used to make the emulsion, which corresponded to a casein surface concentration of around 17.4 ± 0.7 mg m -2 .

The interfacial dilatational moduli (E * ,E' and E'') were determined at the melted milkfat/CA suspensions interface. All suspensions presented similar values: 14.6 ± 0.4, 14.4 ± 0.4 and 2.9 ± 0.2 for complex (E * ), elastic (E') and viscous (E'') moduli, respectively. The contribution of E' to E * was higher than the E'' contribution, reflecting solid-like behavior of the adsorbed casein aggregate layers.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 Discussion

The results are discussed in two stages, with first a focus on the characterization of the CA suspensions in terms of mineralization and colloidal properties. The second stage consisted of investigation of the emulsifying and emulsion-stabilizing capacity of the CA used as emulsifying agents in two types of model dairy emulsions.

Characterization of the different CA suspensions

Addition of TSC resulted in progressive casein micelle demineralization

Analysis of the distribution of minerals confirmed that TSC had an influence on the mineralization of the casein micelle. By chelating the diffusible calcium, citrate ions induced the progressive removal of the colloidal calcium (Gaucheron, 2004). This was in accordance with results reported by many authors who recorded citrate chelation of calcium either by determining calcium activity (de Kort et al., 2011;[START_REF] Johnston | Effects of some calcium-chelating agents on the physical properties of acid-set milk gels[END_REF][START_REF] Udabage | Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk[END_REF], or diffusible calcium and/or colloidal calcium concentrations (Le [START_REF] Ray | Heat stability of reconstituted casein micelle dispersions: changes induced by salt addition[END_REF][START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript Mizuno | Effects of emulsifying salts on the turbidity and calciumphosphate-protein interactions in casein micelles[END_REF][START_REF] Mohammad | Influence of some polyvalent organic acids and salts on the colloidal stability of milk[END_REF][START_REF] Odagiri | Complexing of calcium by hexametaphosphate, oxalate, citrate, and ethylenediamine-tetraacetate in milk. II. Dialysis of milk containing complexing agents[END_REF][START_REF] Ozcan-Yilsay | Effect of trisodium citrate on rheological and physical properties and microstructure of yogurt[END_REF][START_REF] Vujicic | Interaction of polyphosphates and citrate with skim milk proteins[END_REF]) in milk or micellar suspensions.

The simultaneous and correlated decrease in the colloidal inorganic phosphate concentration (Fig. 2) was attributed to the solubilization of the colloidal calcium phosphate (Le [START_REF] Ray | Heat stability of reconstituted casein micelle dispersions: changes induced by salt addition[END_REF][START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript Mizuno | Effects of emulsifying salts on the turbidity and calciumphosphate-protein interactions in casein micelles[END_REF][START_REF] Mohammad | Influence of some polyvalent organic acids and salts on the colloidal stability of milk[END_REF]. Increasing the concentration of TSC therefore led to progressive calcium phosphate demineralization of the CA suspensions.

Furthermore, the correlation observed between the colloidal concentrations of calcium and sodium (Fig. 2) suggested that the negative charges induced by the calcium demineralization (presence of free phosphoseryl residues) were screened by monovalent sodium ions, potentially explaining the constant zeta potential observed for each CA suspension (Table 2).

Mineral content was also modified by the casein powder resuspension and dialysis steps.

Determination of colloidal and diffusible calcium in S1 (prior to dialysis -data not shown) and

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
demineralization (24%, reported in Table 1) was attributed to the resuspension of the purified casein micelle powder in water and to the dialysis step.

The dialysis step also permitted removal of the added citrate and established a similar diffusible phase in the four suspensions (Table 1). As the result, the ionic strengths of all the suspensions were taken to be similar in the four suspensions.

TSC demineralization resulted in disaggregation of the casein micelle

Structural modifications of the CA were observed parallel to the micellar demineralization.

The quantity of sedimentable proteins was reduced and that of non-sedimentable proteins consistently increased ( (2011).

AsFlFFF characterization was performed in order to evaluate the sizes of the dissociated CAs. This revealed that three populations of particles of different sizes and proportions were simultaneously present in the CA suspensions (Fig. 3). Population A consisted of large CA with MW and R h comparable to those of the casein micelle (MW between 5 x 10 7 and 1 x 10 10 g.mol -1 and r rms of 50 -350 nm), as previously reported (Glantz, Håkansson, Lindmark Månsson, Paulsson, & Nilsson, 2010;[START_REF] Pitkowski | Structure and dynamical mechanical properties of suspensions of sodium caseinate[END_REF]. The addition of TSC induced dissociation of these aggregates and increased the proportion of population B. This population consisted of aggregates similar to sodium caseinate particles with MW of 4 to 9 x 10 6 g.mol -1 and R h between in 10 -20 nm, as reported by [START_REF] Lucey | Characterization of commercial and experimental sodium caseinates by multiangle laser light scattering and size-exclusion chromatography[END_REF]. Using 50 times more TSC per gram of protein than in our study, [START_REF] Panouillé | Heat induced aggregation and gelation of casein submicelles[END_REF] reported slightly smaller CAs (MW = 2 x 10 5 g.mol -1 and R h = 12 nm). Finally, population C corresponded to the smallest particles in our suspensions. The percentage of these small particles was also increased by the increased addition of TSC. This suggested that population C corresponded to casein monomers dissociated from the larger CAs.

According to Guyomarc'h et al. (2010) and Glantz et al (2010), they could also be attributed to residual whey protein monomers.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

As demonstrated by [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | Scattering and turbidity study of the dissociation of the casein by calcium chelation[END_REF], [START_REF] Lin | Effect of calcium ion on the structure of native bovine casein micelles[END_REF] and [START_REF] Marchin | Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle x-ray scattering/ultrasmall-angle x-ray scattering[END_REF] with polyphosphate and EDTA calcium chelation, the dissociation of casein micelles by calcium chelating agents is a "cooperative process" in which the structure of the casein micelle remains intact (large aggregates) or becomes fully dissociated (small aggregates of the same size are produced). In other words, the dissociation of the casein micelle does not provide aggregates of intermediate sizes. The three populations of particles (casein micelle-like aggregates, sodium caseinate-like aggregates, and protein monomers) and their dependence on the amount of TSC added confirmed that the "cooperative process" can be applied to the TSC dissociation of casein micelles.

The hydration measurements of S1 d , S2 d , S3 d and S4 d pellets (Table 2) differed from the findings of Le [START_REF] Ray | Heat stability of reconstituted casein micelle dispersions: changes induced by salt addition[END_REF] who reported that the water content of the sedimented CAs increased with the addition of TSC. This was also supported by the voluminosity data determined by De Kort et al. (2011). Compared to our study, these authors did not monitor the diffusible phases of their suspensions. The dialysis step and thus the diffusible environment of the sedimentable casein aggregates therefore seemed to have an impact on their hydration.

As expected, TSC demineralized and dissociated the casein micelle to different extents in order to produce four suspensions containing various CAs. The effects of a calcium chelating agent on the casein micelle seemed to be in good agreement with the use of an ionexchange resin to sequestrate the calcium ( [START_REF] Xu | Effect of calcium sequestration by ion-exchange treatment on the dissociation of casein micelles in model milk protein concentrates[END_REF][START_REF] Ye | Functional properties of milk protein concentrates: emulsifying properties, adsorption and stability of emulsions[END_REF]. [START_REF] Xu | Effect of calcium sequestration by ion-exchange treatment on the dissociation of casein micelles in model milk protein concentrates[END_REF] reported a similar dissociation of the casein micelle into smaller CAs and a decrease in the total calcium content of their casein micelle suspension. These authors also reported that, beyond a level of 20% of calcium demineralization (which is lower than the demineralization rate of our four suspensions), the dissociated caseins present in the ultracentrifuged supernatant (non-sedimentable proteins) were of similar composition to that of the native

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
casein micelle. This suggests that the micelle-like CAs and the mixture of sodium-caseinate CAs and the "free" casein monomers have the same composition.

This first step of our study was necessary to characterize and control our CA suspensions accurately in order to elucidate their emulsifying and emulsion-stabilizing capacity. To summarise, suspension S1 d mostly contained highly mineralized and large casein micelle-like CAs. Intermediate suspensions (S2 d and S3 d ) contained a mixture of both large and small sodium caseinate-like CAs, with a small quantity of "free" casein monomers. Finally, S4 d mainly consisted of poorly mineralized small CAs, "free" casein monomers and residual traces of large CAs (Fig. 8A).

Investigation of CA capacity as emulsifying agents

Decreasing the size of the CA increased its emulsifying capacity

The emulsifying capacity of a protein (or a protein aggregate) can be characterized by measuring the emulsion droplet size at a particular protein concentration: the smaller the droplet, the better the protein aggregate as an emulsifier (Euston & Hirst, 1999). Differences in emulsifying capacity can generally be attributed to the surface activity and/or to the size of the emulsifying agent: the higher the surface activity and/or the smaller the size, the greater the emulsifying capacity.

Emulsion size distribution profiles and micrographs (Figs. 4,5) clearly indicated differences in emulsifying capacity which depended on the CA suspension used. The presence of small CAs facilitated the blending of the milkfat, making it possible to form emulsions with a smaller droplet size, and protected the emulsions against the appearance of bridging flocculation between the milkfat droplets (Fig. 8B).

The surface tension, , is characteristic of the surface activity of the CA, i.e. how effective CAs are at reducing unfavorable interactions between the milkfat and the suspension [START_REF] Mcclements | Food emulsions principles, practices, and techniques[END_REF]. For the concentrations used here, the surface tension measurements at the milk fat/CA suspension interfaces revealed that large micelle-like and small sodium caseinate-like CAs had the same ability to reduce the unfavorable interactions between the

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
two phases (5 -6 mN m -1 ). All the samples had the same surface tension at equilibrium (obtained after 5 min) and hence the same surface coverage.

Our results, showing that there was no difference in equilibrium between our samples, differed from those of [START_REF] Courthaudon | Surface active and emulsifying properties of casein micelles compared to those of sodium caseinate[END_REF], who found that sodium caseinate was more surface active than casein micelles. However, this strongly depends on the concentrations studied: in the study reported here we used a fairly high concentration (20 g/kg) and the interfacial layer was obtained at equilibrium by the combined adsorption of the free casein monomers, the sodium caseinate-like CAs and the micelle-like CAs, which ruptured once adsorbed. Indeed, measurements at a concentration of 1.2 g/kg also provided the same surface tensions and rheological properties (whatever the state of aggregation -data not shown), meaning that at a concentration of 20 g/kg there was a large reservoir of proteins in the bulk, compared to the quantity that could be adsorbed.

It is not possible from these measurements to simply ascribe the differences in emulsifying properties to different surface activities of the types of aggregates. Nevertheless, as we also report here, [START_REF] Courthaudon | Surface active and emulsifying properties of casein micelles compared to those of sodium caseinate[END_REF], [START_REF] Ye | Functional properties of milk protein concentrates: emulsifying properties, adsorption and stability of emulsions[END_REF], [START_REF] Mulvihill | Surface active and emulsifying properties of caseins/caseinates as influenced by state of aggregation[END_REF] and Euston & Hirst (1999) established a correlation between the state of aggregation of the caseins and their emulsifying capacity.

For further analysis, it is important to note that we were not able to monitor the dynamics of adsorption at short timescales t (typically for t < 2 s). However, our results showed that the surface tension had already decreased significantly during this short non-monitored period.

There might therefore have been differences in the dynamics of adsorption between the samples at very short timescales (those having the highest concentrations of monomers reducing the surface tension more rapidly).

In fact, the emulsion production process was rapid, and the associated timescale was also in the order of 1 s. Understanding the differences between emulsifying properties may therefore require monitoring of the surface coverage at such short timescales (less than 1 s). Many small, mobile casein units, such as casein monomers and sodium caseinate-like CAs are

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
thus available for rapid adsorption and to emulsify greater amounts of milkfat/suspension interface at high concentrations of TSC. In contrast, when not enough casein units were present in the suspension to adsorb on the generated interface rapidly (e.g. E1 ec ), the milkfat droplets coalesced until all their surfaces were covered, thus making the emulsions coarser.

Large micelle-like CAs also had the ability to share between two independent droplets and induce bridging flocculation (Fig. 8B).

Emulsions were stable against coalescence but creamed and flocculated

Destabilization of emulsions can result from three phenomena i.e. creaming, flocculation and coalescence. The E st emulsions were designed to have identical droplet sizes, despite the differences in CA suspension emulsifying capacity used to prepare them. This approach removed the influence of the droplet size on the creaming, flocculation and coalescence phenomena.

Visual observations (Fig. 6) and emulsion size measurements (Fig. 7) as a function of time showed that the emulsions remained stable against coalescence throughout storage.

However, emulsions were destabilized by creaming and flocculation (Fig. 7).

4.2.3

The adsorbed CAs contributed to coalescence stability whatever their state of aggregation.

The stability of the emulsions against coalescence is generally correlated with the characteristics of the CA layers adsorbed at the droplet surface. Interfacial casein concentrations and surface tension values provided information on the extent of casein adsorption at the interface, and dilational rheology determined how strongly proteins were adsorbed and interacted at the interface [START_REF] Mcclements | Food emulsions principles, practices, and techniques[END_REF].

As with the surface tension data, the surface casein concentrations were also similar (17.4 mg m -2 ) and independent of the type of CA used to form each emulsion. Our values were between those found by Euston & Hirst (1999) on milk casein concentrate (21 mg m -2 ) and [START_REF] Courthaudon | Surface active and emulsifying properties of casein micelles compared to those of sodium caseinate[END_REF]). Our measurements thus fall within the highest reported values for interfacial concentration, and can be interpreted as a thick layer of adsorbed proteins, in agreement with the fact that we were using high protein concentrations, such that the interfacial properties did not depend on the concentration and that we had a large excess of proteins in bulk.

Dilatational rheology measurements demonstrated that adsorbed layers of CAs had similar solid-like behaviors (E' >> E'') whatever the state of aggregation of the casein used to form the emulsion. Large CAs spread out at the interface, and intermolecular interactions within the adsorbed layers were similar. The wide contribution of E' to E* (E >> E'') was in agreement with the literature on sodium caseinate at diverse oil/water interfaces [START_REF] Amine | Investigation of emulsifying properties and emulsion stability of plant and milk proteins using interfacial tension and interfacial elasticity[END_REF][START_REF] Benjamins | Viscoelastic properties of triacylglycerol/water interfaces covered by proteins[END_REF]. These two results suggested that the state of aggregation of the casein was not decisive for the stability of the emulsions against coalescence.

Creaming and flocculation enhanced each other

Creaming is due to the difference in density between the milkfat and the aqueous suspension phases of the emulsions. This phenomenon was enhanced by the large size of the individual milkfat droplets (12 μm). In our case, creaming (Fig. 6) and flocculation (Fig. 7) only occurred during the first week of storage, suggesting that these two concomitant phenomena influenced each other. On the one hand, creaming was intensified by the formation of milkfat droplet combination due to flocculation. On the other hand, flocculation was favored by the creaming that moved the droplets forward and encouraged their contact, which is a necessary step for the final destabilization of flocculation to occur (Dauphas, Amestoy, , Anton, & Riaublanc, 2008). However, the nature of the CA and the environment also had a role in the appearance of flocculation.

Llamas

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

Unabsorbed CAs induced depletion-flocculation of the emulsion droplets

Depletion-flocculation is an instability mechanism that occurs in emulsions and is induced by the presence of unabsorbed particles. It takes place when two neighboring droplets are close enough to exclude any unabsorbed particles from the gap that separates them.

Consequently, an osmotic pressure gradient is induced that causes net attraction between the emulsion droplets [START_REF] Asakura | Interaction between particles suspended in solutions of macromolecules[END_REF]Dickinson & Golding, 1997[START_REF] Ray | Heat stability of reconstituted casein micelle dispersions: changes induced by salt addition[END_REF]Dickinson et al., 1997;[START_REF] Radford | Depletion flocculation of caseinate-stabilized emulsions: what is the optimum size of the non-adsorbed protein nano-particles?[END_REF]. This phenomenon was observed in our emulsions because of the presence of unabsorbed CAs.

The evolution of the milkfat droplet sizes in the emulsion as a function of time (Fig. 7) revealed that increases in percentage of small CAs in the emulsions augmented flocculation of the milkfat droplets. This was in agreement with the results obtained on native casein micelles, calcium-depleted casein micelles, calcium caseinate and sodium caseinate (Dickinson & Golding, 1998;Euston & Hirst, 1999;[START_REF] Srinivasan | Creaming stability of oil-in-water emulsions formed with sodium and calcium caseinates[END_REF][START_REF] Ye | Functional properties of milk protein concentrates: emulsifying properties, adsorption and stability of emulsions[END_REF]. These reported studies demonstrated that the depletion-flocculation process was strongly dependent on the state of aggregation of the casein. Furthermore, small CAs were of the optimum size (20 nm) to cause the greatest depletion-flocculation of emulsion droplets [START_REF] Radford | Depletion flocculation of caseinate-stabilized emulsions: what is the optimum size of the non-adsorbed protein nano-particles?[END_REF].

CA environment (mineral equilibrium and storage temperature) influenced the sticking of the emulsion droplets

Finally, storage temperatures higher than 37°C can induce gelation by flocculation of sodium caseinate and casein emulsions if a sufficient amount of added calcium is present in the emulsion aqueous phase (Dauphas et al., 2008;Dickinson & Casanova, 1999;Dickinson & Eliot, 2003;Eliot & Dickinson, 2003). Added calcium reduces the steric repulsion between the emulsion droplets by binding to the adsorbed caseins, and high temperature encourages hydrophobic interactions between caseins and promotes sticking behavior (Dauphas et al., 2008;Dickinson & Casanova, 1999). However, no calcium was present in the diffusible phases of the emulsions as it was not present in the CA suspensions (Table 1). Moreover, This suggested that sodium ions reduced steric repulsion between the emulsion droplets in our system. This hypothesis was supported by the increased colloidal sodium content in the CA suspensions (Table 1) and the constant zeta potential values (23.1 ± 1.4 mV) measured on E st emulsions throughout storage. Because of their highly aggregated state, large and strongly mineralized CAs were also less inclined to link with their counterparts adsorbed on separated milkfat droplets or suspended in the bulk emulsion phases.

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 Conclusion

Varying the concentration of added TSC in pure casein micelle suspensions produced four CA suspensions that were progressively demineralized and dissociated. The diffusible phases of these suspensions were monitored with a dialysis step. The use of these CAs as emulsifying agents in our model dairy emulsions revealed differences in emulsifying and emulsion-stabilizing properties. The smaller CAs had better emulsifying capacity as their presence favored the formation of emulsions with smaller droplet sizes. The surface activity of the four CA suspensions was similar and the differences in emulsifying capacity were attributed only to variation of the state of aggregation of the CAs. With regard to the stabilizing capacity of the CAs, all the emulsions were unstable under our storage conditions (21 days, 50°C). Creaming was promoted by the prese nce of large droplets in the emulsions and favored the occurrence of flocculated droplets. Flocculation was also enhanced by the presence of small, demineralized CAs. However, all the emulsions remained stable against coalescence during storage. This was probably due to the presence of similar quantities of adsorbed CAs at the surface of the emulsion droplets that formed protective layers with similar viscoelastic properties. Combining the results obtained on the CAs in suspension with the emulsion properties revealed that the state of aggregation of the CAs had a major impact on their emulsifying capacity and emulsion-stabilizing properties. Modulating the mineral content of the casein micelle is therefore an interesting method for optimization of emulsion functionality. Further studies on CA composition and nanostructure, both in suspension and adsorbed at the milkfat/water interface, would improve understanding of the differences between the emulsifying and emulsion-stabilizing properties. In this case, the destabilization of the emulsions in the early stages should be studied for a better understanding of the involved phenomena. As an extension of this work, investigation of the rheology of the creamed layers of the emulsions is planned as well as the assessment of other functionalities of newly formed CAs.

  produced to facilitate the creaming during storage and foster the appearance of flocculation and coalescence. All chemicals used for this study, hydrochloric acid (HCl) and tri sodium citrate (TSC) (Carlo Erba reagent, Val de Reuil, France), sodium azide (NaN 3 ) (Riedek-de Haën, Seelze, Germany), sodium hydroxide (NaOH), sodium dodecyl sulfate (SDS), D(+)-saccharose (saccharose) (VWR international, Leuven, Belgium), calcium chloride dihydrate (CaCl 2 .2H 2 O) (Sigma-Aldrich, St. Louis, USA), sodium di-hydrogen phosphate 2-hydrate (NaH 2 PO 4 .2H 2 O) (Panreac, Barcelona, Spain), Fast Green FCF (FG) (Sigma-Aldrich, St. Louis, USA) and Nile Red (NR) (5H-Benzo -phenoxazine-5-one, 9-diethylamino, Sigma-Aldrich, St. Louis, USA)

  sizes and proportions in the CA suspensions (AsFlFFF) The molecular weights (MW) and hydrodynamic radii (R h ) of the CAs were determined in suspensions S1 d , S4 d (extreme points), and S2 d (intermediate point) using asymmetrical flow field-flow fractionation (AsFlFFF) coupled to multi angle laser light scattering (MALLS) as described in Guyomarc'h, Violleau, Surel & Famelart (2010) with slight modifications. A solution saturated in calcium and phosphate (similar to the solution used for the dialysis step)

(

  A, B and C) were observed by UV:Population A (19 -27 min) corresponded to particles with MW from 7.5 x 10 7 to 2.5 x 10 9 g mol -1 in S1 d , S2 d and R h from 30 to 100 nm. Population A in S4 d had MW ranging from 2.2 x 10 7 to 3.7 x 10 9 g mol -1 , and R h between 130 and 250 nm. Differences between population A in S4 d and in the other samples must be interpreted with caution because the Rayleigh ratio for this population in S4 d was weak and the MW and R h values deduced from this signal might be less accurate. Moreover, according to the UV and Rayleigh ratio signals, the largest particles of S4 d suspensions were eluted simultaneously with the largest particles of other suspensions, i.e. S1 and S2 d (peaks are superimposed), and therefore these particles had similar MW and R h . had Rh between 21 and 26 nm (evaluated on S1 d and S2 d only). Corresponding MW were between 1.4 x 10 7 and 3.15 x 10 7 for S1 d and S2 d and between 3.4 x 10 6 and 1 x 10 7 for S4 d . Finally, population C (14-15 min) had R h between 18 and 22 nm (evaluated on S1 d and S2 d only) and MW between 7.0 x 10 6 and 1.5 x 10 7 g.mol -1 for S1 d and S2 d and between 1.4 x 10 6 and 3.4 x 10 6 for S4 d . As for population A, differences between MW in S4 d and in the other samples must be interpreted with caution. Again, UV signals indicated that for all suspensions, the B and C populations of particles eluted simultaneously in S1 d , S2 d , and S4 d . According to the quality of the Rayleigh ratios signals of the suspensions, different data treatments were applied which could explain the differences in the MW values observed.

Figure 4 .

 4 Figure 4. Given that the size distribution profiles were monomodal, the mode values (i.e. the

  attributed to the appearance of 3D milkfat droplet flocs in the emulsions that coexisted on different focal planes of the micrographs. According to the microscopy observations, E3 st and E4 st emulsions were the most highly flocculated under our storage conditions.The zeta potential of individual emulsion droplets and flocculated droplets did not evolve significantly during the 21 days of storage (23.1 ± 1.4 mV).

  flocculation increased when the colloidal calcium content of the CAs decreased.

  d < S2 d < S3 d < S4 d . This progressive casein micelle demineralization, expressed as a calcium demineralization rate (Table 1), was 24, 35, 56 and 81% for suspensions S1 d , S2 d , S3 d and S4 d , respectively.

Table 2 )

 2 , which showed progressive dissociation of the CAs. Similar trends were reported by[START_REF] Udabage | Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk[END_REF], Le[START_REF] Ray | Heat stability of reconstituted casein micelle dispersions: changes induced by salt addition[END_REF] and De Kort et al.

Acknowledgements:

The authors thank the French Dairy Interbranch Organization (CNIEL) for their financial support, Rachel Boutrou for assistance with writing the manuscript and Anne-Laure Chapeau for proof reading the article.

M A N U S C R I P T A C C E P T E D

ACCEPTED MANUSCRIPT Dalgleish, D. G., & Corredig, M. (2012). The structure of the casein micelle of milk and its changes during processing. Annual Review of Food Science and Technology, 3 (1), 449-467. Dauphas, S., Amestoy, M., Llamas, G., Anton, M., & Riaublanc, A. (2008). Modification of the interactions between -casein stabilized oil droplets with calcium addition and temperature changing. Food Hydrocolloids, 22(2), 231-238.

de Kort, E., Minor, M., Snoeren, T., van Hooijdonk, T., & van der Linden, E. (2011). Effect of calcium chelators on physical changes in casein micelles in concentrated micellar casein solutions. International Dairy Journal, 21(12), 907-913. Dickinson, E. (1999). Caseins in emulsions: interfacial properties and interactions.

International Dairy Journal, 9(3-6), 305-312. Dickinson, E., & Casanova, H. (1999). A thermoreversible emulsion gel based on sodium caseinate. Food Hydrocolloids, 13(4), 285-289.


D Ben Hassan

Walid Rekik

A Otero-De-

La Roza

F Ben Mefteh

Thierry Roisnel

Thierry Bataille

Houcine Naïli
email: houcine_naili@yahoo.com

Dhouha Ben Hassan

Alberto Otero-De-La Roza

Ben Fedia

Mefteh

Non-covalently generated 3D supramolecular crystal structure in a new family of hybrid nitrates templated by piperazine: thermal behavior and in vitro antimicrobial potential

Keywords: hybrid nitrate, H-bonded system, non-covalent interactions, biologic activity. ϰ

come

Non-covalently generated 3D supramolecular crystal structure in a new family of hybrid nitrates templated by piperazine: Thermal behavior and in vitro antimicrobial potential

Introduction

Recently, the crystal arrangement and the design of solid-state materials have become areas of great interest [1][2][3][4][5]. During the last few years, several types of non-covalent interactions of varying strengths, such as coordination bonding [4,5], versatile hydrogen interactions [6][7][8][9], ʌ-ʌ stacking [10,11] and electrostatic interactions [12,13] have been recognized and used in constructing extended supramolecular architectures. Until now, the most important driving forces within crystal structures are coordination bonding and hydrogen bonding interactions. Materials having supramolecular architectures, in which transition metal complexes are linked through anions via hydrogen bonds, have attracted attention over the last few years due to their fascinating structures and their exciting properties such as their ability to catalyze specific reactions [START_REF] Lehn | Supramolecular Chemistry. Concepts and Perspectives[END_REF]. In this type of compounds, the anionic groups have shown to play a very important role in the synthesis of a wide range of compounds. So that, creating structures by the use of anions has become a well known synthetic strategy. The literature is rich with a large number of hybrids involving different anionic groups that play a major role in the supramolecular networks especially as hydrogen bonds donors. We report for example a few number of hybrids containing sulfates [START_REF] Rekik | [END_REF][16][17][18][19], selenates [20][21][22], perchlorates [23][24][25] etc… Nitrate anion NO 3 -has also been used in a large number of structures due to its ability to form non-covalent interactions such as hydrogen bonds by donating oxygen atoms via their lone electron pairs [26][27][28][29]. A literature search shows that, nitrate anion is present in a very large number of organometallic compounds as ligand [ML(NO 3 ) x ]•nH 2 O [30][31][32], free anion

[ML](NO 3 ) x •nH 2 O [33][34][35][36][37], [M(L-H)](NO 3 ) x •nH 2 O [38], or both [ML(NO 3 ) x ](NO 3 ) y •nH 2 O [39,40] with n = 0 for anhydrous compounds. The complexes characterized by the presence of nitrate as counterion showed diverse types of crystal structures with interesting biological, catalytic and magnetic properties [36,38]. Thus, having supramolecular structures with free entities like nitrates and free organic molecules is of great interest. We report in the present paper, a new family of compounds whose structures involve free nitrates, metallic complexes and protonated piperazine molecule with hydrogen bond networks forming the 3D supramolecular structures.

ϱ

Experimental

Chemical preparation

All the title compounds were prepared by the slow evaporation method. In the first step, the piperazine hexahydrate (1 mmol / 0.086 g) was dissolved in a minimum amount of distilled water to afford a colorless solution. Under magnetic stirring, the nitric acid HNO

Spectroscopic analysis

The infrared measurements were obtained using a Perkin Elmer FT-IR spectrometer. The samples were diluted with spectroscopic grade KBr and pressed into a pellet and the scans were run over the range of 500-4000 cm -1 .

X-ray single crystal diffraction and structure determination

The title materials crystallize in the triclinic system, space group PƯ. X-ray diffraction data were collected at low temperature (T=150K) on single crystal using an APEXII Bruker-AXS diffractometer. The reflection data were acquired using graphite-monochromated KĮ(Mo) radiation (Ȝ = 0.71073 Å). The peak integration, frame scaling and corrections for Lorentz-polarization were carried out with the program Bruker SAINT [START_REF] Bruker-Nonius | SAINT version 7.23A[END_REF]. A multi-scan absorption correction was applied to the intensity values using SADABS program [START_REF] Sheldrick | [END_REF].

ϲ

The metallic atoms were located using the direct methods with the SIR97 program [START_REF] Altomare | [END_REF]. The positions of all the rest non-hydrogen atoms were determined from successive Fourier calculations using SHELXL-2014 program [44]. These programs are available in the WinGX interface [45]. All the hydrogen atom positions of water molecules were located in a difference map and refined with distance restraints OʊH and HʊH of 0.85 and 1.38 Å, respectively. For the piperazine moiety, all the hydrogen atoms bonded to the carbon were positioned geometrically where those bonded to the nitrogen atoms were located after successive Fourier calculations.

Thermogravimetric (TG) and temperature-dependent X-ray diffraction (TDXD) analysis

The TG measurements for the three compounds were performed using a Perkin-Elmer Pyris 6 TGA instrument. The samples of the three compounds were heated from 30 to 500 °C with a heating rate of 5 °C.min -1 under air atmosphere.

The temperature-dependent X-ray powder diffraction (TDXD) was carried out with a ș-ș Bruker AXS D8 Advance powder diffractometer which is equipped with a high-temperature Anton Paar HTK1200 oven camera and a LynxEye detector. The powder patterns were collected using the Cu (KĮ 1 ) radiation (Ȝ = 1.5406 Å) selected with a graphite monochromator from ambient to 500 °C with a heating rate of 21.6

°C.h -1 .

The NCI method

The NCI calculations, as a computational technique recently applied to solid state compounds, use qualitative density functional theory and allow for the location and visualization of the non-covalent ϳ interactions, like hydrogen bonds and weak ionic and Van Der Waals interactions, which occur in regions characterized by small electron density (ȡ) and reduced density gradient (S) as its first derivative.

The electron densities for the title compounds at their experimental crystal structures were calculated using density-functional theory (DFT) with the Quantum ESPRESSO program [46]. The calculation parameters were 80 Ry for the plane-wave expansion cutoff, 800 Ry for the density cutoff, and a 4x4x4 kpoint grid. The Perdew-Burke-Ernzerhof functional [47] was used. For the Co crystal only, we used cold smearing [48] with a smearing parameter of 0.01 Ry. The calculated self-consistent electron densities were used with the NCI plot technique [49,50] as implemented in the Critic2 program [51,52].

Biological study

The in vitro biological activity of the title compounds was studied to evaluate their potential as antibacterial and antifungal agents using the well diffusion method against eight bacteria and three fungi.

The used Gram-positive bacteria are Bacillus subtilis JN934392 (Bs), Bacillus ceureus JN934390 (Bc),

Staphylococcus aureus ATCC6538 (Sa), Listeria monocytogenes ( Lm) and Micrococcus luteus (Ml), and the Gram-negative ones are Salamonella enteric serotype Enteritidis ATCC43972 (Se), Escherichia Coli ATCC25922 (Ec) and Klebsiella pneumonia (Kp). The antifungal test was performed against Fusarium sp JX391934 (Fsp), Fusarium oxysporum AB586994 (Foxy) and Pythium sp AY598675 (Psp) fungi. In a typical procedure, wells were made on the agar medium after inoculation with microorganism's cultures in petri plates containing, respectively, Mueller-Hinton Agar and Potato-Dextrose Agar for antibacterial and antifungal tests. Each well was filled with 60 ȝL of the test aqueous solutions at different concentrations (50, 100 and 150 mg/mL) of each compound. After diffusion for two hours at 4 °C, the plates were incubated at 37 °C for 24 h and at 30 °C for 72 h respectively for antibacterial and antifungal tests. In order to prove the activity of the synthesized complexes, aqueous solutions concentrated at 150 mg/mL of the piperazine, manganese nitrate, copper nitrate and cobalt nitrate were tested against the used bacteria and fungi with the same procedure and at the same conditions. The antimicrobial activity was estimated by measuring, in millimeters, the diameters of the inhibition zones formed around the wells.

ϴ

Results and discussion

Spectroscopic analysis

The FTIR spectra analysis shows similar curves for the three compounds. In the present paper, the infrared spectrum of (1) only is presented (Fig. 1). The most important absorption bands of the all compounds as well as their assignments are given in Table 1. The broad band around 3400 cm -1 in the spectrum can be attributed to stretching vibrations of water molecule. The nitrate vibration bands are observed around 800 cm -1 (Ȟ 2 ) and 700 cm -1 (Ȟ 4 ) [53,54] as well as a very intense band located near 1300 cm -1 (Ȟ 3 ) indicating the presence of free NO 3 -groups [START_REF] Nakamoto | Infrared Raman Spectra Inorg. Coord. Compd[END_REF]. Concerning the organic molecule, the two bands situated closed to 3000 and 1580 cm -1 can be assigned to the stretching and bending vibration modes of NH 2 +

, respectively [START_REF] Saha | [END_REF]. The asymmetric and symmetric stretching vibration modes of methyl groups are represented by two near bands located around 2800 and 2760 cm -1 which can be assigned to the asymmetric and symmetric vibration modes respectively [57,58]. A number of bands appearing in the spectral region of low wave number (500-600 cm -1 ) are attributed to the different vibration modes of the organic ring.

Crystal structures

All the crystal data and structure refinement details are listed in Table 2. Selected bond distances and angles as well as hydrogen bond geometry are given in tables 3 and 4 respectively. All of the studied hybrid nitrates crystallize isotypically in the triclinic system, centrosymmetric space group PƯ. For this reason, only figures relative to the manganese based compound are shown. Figure 2 represents the asymmetric unit extended by symmetry while the figures of the asymmetric units relative to the two other materials are given in SI1 and SI2. Figure 3 and Figure 4 show projections of the crystal structure of (1) along a and b-axes, respectively. Figure 2 Å. These value ranges are in accordance with many studied structures involving free nitrate groups [33][34][35][36][37]. The environment around the metallic cations can be described as a slightly distorted octahedron: polyhedra distorsion values have been calculated to be equal to 3 10 -4 , 7.2 10 -3 and 2 10 -4 for Mn, Cu and Co compounds, respectively. These irregular octahedra, situated in the center of the cells (Fig. 3) are separated by intermetallic distances M II …M II equal to 7.1795 (6), 7.2389 (6) and 7.1379 (4) Å for compounds (1), ( 2) and ( 3), respectively according to the crystallographic a-axis.

The deviation from the ideal octahedral geometry is revealed by the range of angles observed around the metallic center (from 86.97 (5) to 90.17 (6) ° for (1), from 87.15 (5) cation displays the so-called [4+2] coordination type which is associated with the well-known Jahn-Teller effect [16,20,[61][62][63] and then the geometry around this metal can be described as square bipyramidal and on the edges parallel to the crystallographic b-axis. The free nitrate anions are involved in a hydrogen bond system formed by OWʊH⋅⋅⋅O and NʊH⋅⋅⋅O to generate the three dimensional structures. The oxygen atoms, which are acting as acceptors, are H-bonded to the oxygen atoms of the six water molecules surrounding the metallic cations and nitrogen atoms of the piperazine moieties. Indeed, the OW-H⋅⋅⋅O created between the aqua molecules and the nitrate groups are considered as weak H-bonds [64,65] except OW2-O7 for (1) (table 4). Within each structure and together with NʊH⋅⋅⋅O hydrogen bonds, these interactions are responsible of the 3D architecture which proves the important role of free nitrate anions in the supramolecular network. In addition, the nitrate ions are found to be stacked to form anionic layers parallel to the (0 1 2) plane. These layers alternate, along the crystallographic b-axis, with the cationic layers formed by the positively charged metallic octahedra and piperazine moieties (Fig. 3).

[2 0 Ư] is a second direction of the anionic-cationic alternation established between the anionic and cationic layers parallel to the (1 0 Ư ) plane (Fig. 4). As seen in this figure, the cationic-cationic alternation between the protonated piperazine and the metallic octahedra is observed along the crystallographic b-axis.

Thermal analysis

Thermal analysis of (C 4 H 12 N 2 ) 3 [Mn(H 2 O) 6 ](NO 3 ) 8 (1) The TG curve presented in Figure 5 (a) shows a small rise caused by a weight increase of 0.9 % which indicates that the material incorporated half a water molecule (theoretical weight increase: 0.97 %) due to ϭϭ the hygroscopic nature of the compound. The representation of the powder X-ray diffraction patterns TDXD (Fig. 6) shows that the resulting phase is crystalline.

The decomposition phenomenon begins with dehydration by the departure of 5 water molecules giving rise to an amorphous monohydrate phase (Fig. 6). The observed weight loss, 9.69 %, agrees closely with the expected value of 9.75 %. This step occurs within a large temperature range (until 200°C). At the higher temperatures, the precursor is totally decomposed by losing, simultaneously, the remaining water molecules, the organic part and the nitrate anions. The two processes are inseparable under these conditions. The expected weight loss is 91.46 % while the experimentally observed value is 91.05 %.

Along the degradation, the intermediate phase is amorphous to X-rays. In fact, the TDXD plot shows no X-ray diffraction peaks between 200 and 400 °C. In the end of the decomposition phenomenon of the manganese based complex, the final product is the manganese oxide Mn 2 O 3 . Based on the TDXD representation, this phase is crystallized and stable beyond 400 °C.

Thermal analysis of (C 4 H 12 N 2 ) 3 [Cu(H 2 O) 6 ](NO 3 ) 8 (2)

The copper based complex decomposes in two steps (Fig. 5 (b)). The first step corresponds to the dehydration. In fact, from 80 up to around 180 °C, five water molecules are eliminated from the crystal structure giving rise to a monohydrated phase. Through this step, there is formation of crystallized phases as shown in the TDXD representation (Fig. 7).

The dehydration occurs with a weight loss of 9.53 % very close to the calculated value of 9.65 %. The next step is attributed to the total decomposition of the resulting phase. The total expected weight loss of 89.35 % agrees well with the observed value of 90.38 %. The decomposition finished by the formation of the copper oxide Cu 2 O 3 crystallized between 200 and 450 °C as it is clearly seen in the three-dimensional X-ray diffraction pattern. This oxide is not stable and turns into a more stable crystalline phase which is the copper oxide CuO. This transformation is proved by the apparition of new diffraction peak at around ϭϮ

Thermal analysis of (C 4 H 12 N 2 ) 3 [Co(H 2 O) 6 ](NO 3 ) 8 (3)

Referring to the TG curve presented in Figure 5 (c), the initial behavior of compound ( 3) is slightly different compared to those observed for (1) and ( 2). Indeed, the precursor starts with a total dehydration phenomenon by evaporation of all the six water molecules present within the unit cell around the metallic cation (observed mass loss 11.91 %, calculated mass loss 11.7 %). This phenomenon takes place until around 180 °C and gives rise to the anhydrous phase (C 4 H 12 N 2 ) 3 Co(NO 3 ) 8 which totally decomposes, in a next step, to end with the cobalt oxide Co 2 O 3 . The observed mass loss during this decomposition is 90.87

% while the theoretical one is 91.05 %.

It is interesting to note that the thermal decomposition phenomenon of the nitrate anion and formation of the metal oxide may be initiated through breaking of the N-O bond in M-O-NO 2 followed by departure of gaseous NO 2 .

NCI analysis

NCI calculations were carried out on the title compounds because they have nearly identical compositions and connectivity architecture only differing by the nature of the metallic cation. The purpose is the direct observation of ionic attractions and repulsions, hydrogen bonding and van der Waals attractions in these compounds to determine the main forces that govern their formation. Figure 8 shows the NCI domains around the metal complex in the Mn crystal. The plots for the other compounds are very similar, and are not shown for simplicity. The most important feature in these plots is the extended three-dimensional hydrogen bond network formed between the aqua ligands and the adjacent nitrate molecules. These hydrogen bonds are revealed in the NCI plot as round dotted domains. To further examine the noncovalent interactions as a whole, plots of the reduced density gradient (RDG) against the electron density multiplied by the second eigenvalue of the density Hessian are used. In previous works, it has been found that the separation between the attractive and repulsive interactions depend on the sign of the second eigenvalue, Ȝ 2 , of the electron-density Hessian where the positive (Ȝ 2 > 0) and negative (Ȝ 2 < 0) signs ϭϯ correspond to the repulsive and attractive regions, respectively [49]. Figure 9 represents the NCI analysis of the low and moderate density attractions relative to the three compounds based on Mn (a), Cu (b) and Co (c) respectively. The peaks at low RDG in the negative x-axis (Ȝ 2 < 0) correspond to intermolecular attractive contacts, and can be grouped, as indicated in the plots, in three categories: NʊH⋅⋅⋅O and OWʊH⋅⋅⋅O hydrogen bonds, weak directional interactions between piperazine and water ligands and weak non-specific interactions (van der Waals). Clearly, the hydrogen bonds sustain the threedimensional structure of the crystal. Interestingly, the strength of the hydrogen bonds, which can be estimated by the electron density of the peaks, differs only slightly depending on the metal atoms as shown in Figure 9. Despite to isotype structures, this may be due to the variation of the atomic radius values of the metallic cations in their six fold coordination state [66].

Biological study

The antibacterial and antifungal tests were performed against five Gram-positive, three Gram-negative bacteria and three fungi at different concentrations of the three synthesized compounds. In preliminary screening, it has been found that the inhibition zones become larger when the concentrations of the compounds increase. This relation is generally observed in this type of tests [67,68]. Probably, this is due to the effect of the metal ions. For this reason, the results founded for the concentration of 150 mg/mL were used to discuss the biological activity of the three hybrid materials. The three present compounds were effective against the tested bacteria with variable ranges of diameters of inhibitory zones (Fig. 10).

The cobalt based complex showed the highest activity. In fact, it presented very large inhibitory zones with different diameters varying from 30 to 43 mm. Referring to the literature, the organometallic complexes based on Co are founded to be very effective against bacteria [68,69]. This can be due to the high toxicity of the divalent cobalt metal against these species. The Mn based compound presented moderate activity against three Gram-positive and only one Gram-negative bacteria. This can be due to the differences in cell wall structure of the bacteria which can produce differences in antibacterial susceptibility. In fact, Gram-positive bacteria possess a thick cell wall containing many active layers but ϭϰ in contrast, Gram negative bacteria have a relatively thin cell wall consisting of a few active layers [70].

Concerning the antifungal activity, it was observed that the Mn based compound had no activity against the tested fungi while the two others were effective. The Co (II) complex is found to be more fungi toxic than the Cu (II) based one with diameters of the inhibition zones ranging from 15 to 22 mm and from 20 to 30 mm respectively for compound (2) and (3). Based on these results, the antimicrobial activity of the investigated hybrid compounds follows the order: Co (II) > Cu (II) > Mn (II).

The separated fragments which are the piperazine and all metal nitrates were tested towards the same bacteria and fungi. All the results are regrouped in table 5. In fact, the tested standards exhibit very lower antibacterial activity as compared to the corresponding hybrid materials. The piperazine is found to be ineffective against the majority of the used bacteria. The manganese nitrate is the less active compound where the cobalt nitrate presents the highest antibacterial activity. Thus, the same order observed for the hybrid materials is followed by the metal nitrates. Concerning the antifungal activity, all the same tested standards are ineffective against the used fungi. The obtained results evidently show that the activity of the separated components becomes more pronounced when they are regrouped together in a whole complex. Thus, the structure and the assembly manner of these groups seem to be a major factor influencing the biological activity.

Conclusion

In summary, a new family of hybrid nitrate compounds has been prepared. Three new compounds based on Mn (II), Cu (II) and Co (II) metals and containing in their structures free nitrate ions and protonated piperazine cations have been successfully synthesized and characterized. The X-ray diffraction analysis showed that the materials are isotypically crystallized with a similar arrangement of different cationic and anionic entities in the space directions. A 3D hydrogen bonded system constructed by cationic (piperazine-H 2 ) 2+ and [M II (H 2 O) 6 ] 2+ entities as donors and nitrate anions as acceptors has been established. The thermogravimetric analysis of the three complexes showed that their degradation proceeds in two steps: a dehydration followed by a total decomposition leading to the formation of the metal oxides M 2 O 3 . The degradation steps and their associated temperatures were comparative from one complex to another due to the close geometry as well as the similar covalent and non-covalent bonds ϭϱ existing within each one. The non-covalent interaction (NCI) plots using the calculated electron densities from density-functional calculations at the experimental crystal geometries were used to characterize the interactions in the crystal. The three-dimensional network is supported by hydrogen bonds between the metal complex and the nitrate anions. These hydrogen bonds show very slight differences depending on the metal center. Biologically, the three synthesized hybrid compounds were active against the tested bacteria and fungi with a highest potential showed by the cobalt based compound.

Supplementary data

CCDC Numbers: 1447621, 1433261 and 1433259 contain the supplementary crystallographic data respectively for Mn, Cu and Co compounds. These data can be obtained free of charge from the Cambridge Crystallographic data centre, 12 Union Road, Cambridge CB2 1EZ, UK. E-mail: deposit@ccdc.cam.uc.uk.

Tables Table 1: Characteristic IR absorption bands (cm -1 ) of ( 1), ( 2) and ( 3) ------------Foxy ------------Psp ------------

Band

  geometry. The third Figure shows that the metallic octahedra occupy the center of the cell. The piperazine cations are placed in the center of the faces formed by a and c-axes (position of the centroid: (½, 0, ½))

Figure 1 :Figure 2 :Figure 3 :Figure 4 :

 1234 Figure 1: FTIR spectrum of (C 4 H 12 N 2 ) 3 [Mn(H 2 O) 6 ](NO 3 ) 8 (1) Figure 2: Asymmetric unit of (C 4 H 12 N 2 ) 3 [Mn(H 2 O) 6 ](NO 3 ) 8 (1) extended by symmetry to complete (C 4 H 12 N 2 ) 2+ and [Mn(H 2 O) 6 ] 2+ entities. The thermal ellipsoids are plotted at the 50% probability level. H bonds are represented by dashed lines. (Symmetry codes: (i): -x+1, -

Figure 5 :Fig. 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 1 Figure 2 Figure 3

 5678910123 Figure 5: The TG curve representations of the thermal decomposition of

Figure 4 Figure 5

 45 Figure 4

Figure 6

 6 Figure 6

Figure 7 Figure 8 Figure 9 Figure 10

 78910 Figure 7

  The chemical formula obtained after the structural refinement is (C 4 H 12 N 2 ) 3 [M II (H 2 O) 6 ](NO 3 ) 8 with M

II :

Mn (1), Cu

(2)

and Co

(3)

. Tris(piperazinediium) hexaaquamanganese(II) nitrate, tris(piperazinediium) hexaaquacopper(II) nitrate and tris(piperazinediium) hexaaquacobalt(II) nitrate were the full names relative to the Mn, Cu and Co compounds.

  II (H 2 O) 6 ] 2+ , four free nitrate anions NO 3 -and isolated piperazine molecules (C 4 H 12 N 2 ) 2+ . The metallic cations M 2+ (½, ½, ½) occupy a special position coinciding with an inversion center so that, among the six water molecules forming the metallic octahedron, three water molecules (OW1, OW2 and OW3) are crystallographically independent. The piperazine molecules, with a chair conformation, are doubly protonated. The ring centroid of one of them (completed by symmetry) lies on inversion center while the centroid of the other is placed in a general position. As it can be seen in figure, NʊH⋅⋅⋅O and OWʊH⋅⋅⋅O relate the different entities to create an extensive H-bond system being the origin of the 3D supramolecular structures. The two nitrogen centers in the piperazinediium cations

, representing the asymmetric unit extended by symmetry (Symmetry codes :(i): -x+1, -y+1, -z+1; (ii) -x+3, -y, -z+1), shows that the molecular structures of the complexes consist of the divalent metallic cation surrounded by six water molecules in an octahedral ϵ fashion forming cationic entities [M are protonated. The CʊC distances take the values of 1.507 (2) and 1.518 (2) Å while the CʊN distances are in the range 1.482 (2)-1.497 (2) Å. The angles CʊCʊN and CʊNʊC are close to the ideal tetrahedral angle 109 ° (see SI3). Referring to the literature, the values of the different distances and

angles are in good agreement with those reported in numerous previous works containing piperazinediium cations incorporated in hybrid materials

[START_REF] Rekik | [END_REF]22,59,60]

. The angles within the uncoordinated nitrate anions vary from 116.86

(17)

to 122.7 (3) ° (table

3

). The NʊO distances are between 1.219 (2) and 1.285

(2)

  Ǽ, respectively. Concerning the Cu based compound, the distorted octahedral arrangement ϭϬ around the metallic cation produce two equatorial bond distances of 1.9553 (12) and 2.0028(12) Å which are very close and an axial one measuring 2.3554(12) Å which is considerably longer. Therefore, Cu 2+

	to 93.81 (5) ° for (2) and 86.84 (7) to
	91.74 (7) ° for (3)), while the interaxial angles OWʊM II ʊOW i are equal to 180 ° (table 3). The
	MnʊOW and CoʊOW bond distances vary in the ranges 2.1382 (14)-2.2206 (13) and 2.0609 (18)-
	2.1257 (18)

Table 2 :

 2 Crystal data and structure refinement details of (1), (2) and(3)

	assignment Compound (1) Compound (2) Compound (3)
	Ȟ(O-H)	3355	3344	3367
	Ȟ(NH 2 )	3026	3029	3084
	Ȟ asy (CH 2 )	2833	2835	2884
	Ȟ sy (CH 2 )	2768	2773	2790
	į(NH 2 )	1587	1587	1594
	Ȟ 2 (NO 3 )	806	812	815
	Ȟ 3 (NO 3 )	1323	1324	1316
	Ȟ 4 (NO 3 )	700	714	707
	organic ring	1100-850	1100-850	1100-850

Table 3 :

 3 Selected bond distances (Å) and angles (°) within the metallic octahedra and the nitrate anions for compounds (1), (2) and(3)

		Compound (1)	Compound (2)	Compound (3)
	Metallic octahedron
	MʊOW1	2.2206 (13)	2.3554 (12)	2.1257 (18)
	MʊOW2	2.1382 (14)	1.9553 (12)	2.0609 (18)
	MʊOW3	2.1968 (13)	2.0028 (12)	2.0930 (17)
	OW1ʊMʊOW2	90.17 (6)	93.81 (5)	91.74 (7)
	OW1ʊMʊOW3	86.99 (6)	87.15 (5)	86.84 (7)
	OW2ʊMʊOW3	87.97 (5)	92.92 (7)	88.37 (7)
	OW1ʊMʊOW1 i	180.0	180.0	180.0
	OW2ʊMʊOW2 i	180.0	180.00 (7)	180.0
	OW3ʊMʊOW3 i	180.00 (6)	180.00 (7)	180.0
	Nitrate groups
	N1ʊO1	1.2556 (19)	1.2572 (18)	1.255 (3)
	N1ʊO2	1.2522 (19)	1.2568 (18)	1.252 (3)
	N1ʊO3	1.2518 (18)	1.2428 (18)	1.246 (3)
	N2ʊO4	1.2296 (19)	1.2335 (18)	1.218 (3)
	N2ʊO5	1.2709 (19)	1.2567 (19)	1.264 (3)
	N2ʊO6	1.2565 (19)	1.2621 (18)	1.250 (3)
	N3ʊO7	1.285 (2)	1.2777 (18)	1.277 (3)
	N3ʊO8	1.219 (2)	1.2428 (18)	1.229 (3)
	N3ʊO9	1.238 (2)	1.234 (2)	1.229 (3)
	N4ʊO10	1.2738 (18)	1.2715 (18)	1.268 (3)
	N4ʊO11	1.2585 (19)	1.2623 (18)	1.257 (3)
	N4ʊO12	1.2381 (17)	1.2316 (17)	1.235 (3)
	O1ʊN1ʊO2	118.68 (13)	118.43 (13)	118.26 (19)
	O1ʊN1ʊO3	119.53 (15)	120.09 (14)	119.9 (2)
	O2ʊN1ʊO3	121.78 (16)	121.48 (14)	121.8 (2)
	O4ʊN2ʊO5	121.12 (16)	121.78 (15)	121.2 (2)
	O4ʊN2ʊO6	121.47 (16)	120.57 (15)	121.4 (2)
	O5ʊN2ʊO6	117.40 (14)	117.65 (14)	117.4 (2)
	O7ʊN3ʊO8	119.90 (19)	119.56 (15)	119.7 (2)
	O7ʊN3ʊO9	116.86 (17)	118.62 (14)	117.6 (2)
	O8ʊN3ʊO9	123.2 (2)	121.80 (15)	122.7 (3)
	O10ʊN4ʊO11	118.40 (13)	118.07 (13)	118.38 (18)
	O10ʊN4ʊO12	120.47 (14)	120.98 (14)	120.7 (2)
	O11ʊN4ʊO12	121.13 (14)	120.95 (14)	120.94 (19)
	Symmetry code: (i): -x+1, -y+1, -z+1

Table 5 :

 5 Diameters of inhibitory zones, measured in mm for aqueous solutions of the piperazine, manganese nitrate, copper nitrate and cobalt nitrate at the concentration of 150 mg/ml

		Piperazine	Mn	Cu	Co
	Bs	---	9	17	28
	Bc	---	7	15	---
	Sa	---	6	19	21
	Lm	---	---	16	19
	Ml	---	---	20	25
	Se	---	4	14	---
	Ec	10	3	---	21
	Kp	8	7	20	31
	Fsp

Acknowledgements

AOR thanks the Spanish Malta/Consolider initiative (no. CSD2007-00045).

Formatting and funding sources

This research did not receive any specific grand from funding agencies in the public, commercial or notfor-profit sectors.

Conflicts of interest

The authors declare no competing financial interest.