Humectability and physical properties of hydroxypropyl methylcellulose coatings with liposome-cellulose nanofibers: Food application

Journal Pre-proof Humectability and physical properties of hydroxypropyl methylcellulose coatings with liposome-cellulose nanofibers: Food application Johana Lopez-Polo, Andrea Silva-Weiss, Marcela Zamorano, Fernando A. Osorio

PII:

S0144-8617(19)31370-0

DOI:

https://doi.org/10.1016/j.carbpol.2019.115702

Reference:

CARP 115702

To appear in:

Carbohydrate Polymers

Received Date:

2 September 2019

Revised Date:

20 November 2019

Accepted Date:

2 December 2019

Please cite this article as: Lopez-Polo J, Silva-Weiss A, Zamorano M, Osorio FA, Humectability and physical properties of hydroxypropyl methylcellulose coatings with liposome-cellulose nanofibers: Food application, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115702

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Humectability and Physical Properties of Hydroxypropyl Methylcellulose Coatings with Liposome-Cellulose Nanofibers. Food Application Johana Lopez-Poloa, Andrea Silva-Weissa, Marcela Zamoranoa, Fernando A. Osorio*a

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Department of Food Science and Technology, Universidad de Santiago de Chile, USACH.

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Avenida Ecuador 3769, Santiago, Chile. [email protected]; [email protected];

Corresponding author: Fernando A. Osorio: Department of Food Science and Technology,

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*

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[email protected].

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Universidad de Santiago de Chile, USACH. Avenida Ecuador 3769, Santiago, Chile.

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[email protected].

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The authors declare no competing financial interest.

Edible coating suspensions (ECS); Cellulose nanofibers (CN); Glycerol (G); Phosphatidylcholine (PC); Liposome with rutin (LR); Liposomal suspensions (LS); Potassium phosphate buffer (PPB); Polydispersity index (PDI); Zeta potential (ξ); Antioxidant suspension (AS); Rutin suspension (R); Coating suspensions with rutin (ECS-R); Coating suspensions of liposomes with rutin (ECS-LR); Contact angles (CA); Liposomes with rutin (LR); Potassium phosphate buffer (PPB).

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Graphical abstract

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Highlights

Edible coating suspensions (ECS) with hydroxypropyl methylcellulose are

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formulated

Liposomes with rutin are incorporated into HPMC based edible coating

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Liposomes caused significant changes in the physical properties of ECS

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Cellulose nanofibers caused significant changes in the physical properties of ECS

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Liposomes addition caused significant changes in the humectability of ECS

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The presence of liposomes increased the apparent viscosity of ECS

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The ECS partially hydrates the slice surfaces of almond and chocolate.

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

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Abstract The objective of this study was to investigate the physical, rheological and humectability properties of edible coating forming suspensions (ECS) based on hydroxypropyl methylcellulose (HPMC) containing: liposomes that encapsulate rutin, glycerol and cellulose

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nanofibers on sliced surfaces of almonds and chocolate. On average, liposomes measured between 110.6 ± 10.0 nm and were characterized as stable and homogeneous suspensions.

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Adding these liposomes to the edible coatings produced significant changes (p 0.05) in the

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density and surface tension, which favor the final appearance of the coating. The presence of liposomes increased the apparent viscosity of the ECS, showing a purely viscous and fluid

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behavior with a good fit (R2= 0.9996) with the Power Law model. The presence of liposomes

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and cellulose nanofibers decreased the value of the cohesive energy of the ECS. The studied ECS partially hydrate the surfaces of almond and chocolate as they showed contact angles

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under 90°.

Keywords: Edible coating; biodegradable polymer; liposomes; cellulose nanofibers; humectability.

1. Introduction

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Edible coating is a technology that extends the shelf life of food by preventing or delaying its deterioration, providing a partial barrier against moisture, oxygen and carbon dioxide, improving mechanical handling properties, and even as carriers of bioactive compounds (Dehghani, S., Hosseini, S. V., & Regenstein, 2018; Yousuf, Qadri, & Srivastava, 2018). Hydroxypropyl methylcellulose (HPMC), a biodegradable polymer derived from cellulose,

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when used as structural matrix of the edible coatings, represents an attractive alternative to form coatings that are transparent, odorless, insipid and resistant to oil (Rubilar, Zúñiga, Osorio, & Pedreschi, 2015). Due its hydrophilicity, HPMC offers little resistance to the transport of water vapor (Imran et al., 2012). This limitation can be overcome by developing a coating with the addition of nanometric fillers such as cellulose nanofibers (CN). For example, transport moisture reduction has been reported with the addition of CN in edible

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films based on mango puree (Henriette. Azeredo, Mattoso, Wood, Delilah Williams, Avena-

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Bustillos, & McHugh, 2009) and a film based on chitosan (Henriette. Azeredo et al., 2010).

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For the consumer, one of the key aspects for acceptance of covered food is its appearance. To achieve acceptable appearance of the food, the edible coating should be distributed evenly

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over the surface of the food, without defects in its structure such as irregularities in thickness, peeling, cracking or curling, as these can reduce or eliminate the protective properties of the

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coating (Kheshgi, 2011; Peressini, Bravin, Lapasin, Rizzotti, & Sensidoni, 2003). The coating process of a food depends on: the rheological behavior (viscosity, Young's

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modulus) of the edible coating suspension physical properties (density, surface tension, humectability, etc.), as well as the properties of the food to be coated (roughness, moisture content, percentage of fat, etc.), and the properties of the polymer used, the affinity between

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the surface of the food and the formulation of the coating, and method of application (Andrade, Skurtys, & Osorio, 2013). Thus, in order to obtain high quality edible coatings that are competitive with traditional plastics, each of the factors mentioned above must be optimized. The incorporation of bioactive compounds in the edible coatings provides an innovative approach to lengthen the shelf life of food. Edible coatings have been used as carriers of

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essential oils (Vital et al., 2018), bacteriocins, isothiocyanates, sorbic acid, antimicrobial enzymes, metal nanoparticles (H. Azeredo, 2013; Rhim, Park, & Ha, 2013) carotenoproteins (Hajji, Younes, Affes, Boufi, & Nasri, 2018) and antioxidant compounds (Bermúdez-Oria, Rodríguez-Gutiérrez, Rubio-Senent, Fernández-Prior, & Fernández-Bolaños, 2019; SilvaWeiss, Bifani, Ihl, Sobral, & Gómez-Guillén, 2013, 2014).

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Natural antioxidants such as flavonoids, are known for their important in vivo and in vitro free radical-scavenging properties. Rutin (quercetin-3-glucoside rhamnose) is a low-weight

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in fruits and vegetables (Yang, Guo, & Yuan, 2008).

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polyphenol compound, widely studied for its antioxidant properties and its wide distribution

However, its use as a bioactive compound in coatings is limited due to the reduced stability

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during the processing conditions and/or during storage (pH, temperature, light, oxygen), low solubility in aqueous media (0.13 mg/1 mL) and low bioavailability (Emami, Azadmard-

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Damirchi, Peighambardoust, Valizadeh, & Hesari, 2016; Vu et al., 2018).

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For this reason liposomes, as a technology of encapsulation, have been proven useful to overcome these drawbacks. Self-assembled spherical vesicles liposomes obtained with phospholipids, with hydrophilic heads and hydrophobic fatty acid tails, are biodegradable and non-toxic (Emami et al., 2016). Liposomes can be characterized through the study of

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their physical properties, such as the hydrodynamic diameter, the Zeta potential and the index of polydispersity, which also makes it possible to know its stability in the short and long term.

Different studies have been published on the combined use of edible coatings and technology of the liposomes. For example, nisin has been encapsulated in nanoliposomes of soy lecithin that were subsequently added to a solution of HPMC (6% w/v) to improve the bioavailability 5

of nisin and make biodegradable films with the active agent (Imran et al., 2012); quercetin and rutin have been encapsulated in liposomes and subsequently incorporated in coatings of carboxymethyl cellulose (2% w/v) in order to evaluate the impact of incorporating flavonol at different stages of the liposomal formulation on the efficiency of the encapsulation and the physical properties of liposomes (Silva-Weiss et al., 2018). A peptide fraction of shrimp has been encapsulated in nanoliposomes of phosphatidylcholine from soybeans in order to

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prepare a film of functional sodium caseinate (8% w/v) with good sensory properties

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(Montero et al., 2019); an edible film of chitosan 1% w/v containing phage encapsulated in liposomes of soy lecithin and cholesterol in order to improve the stability of the phages and

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their subsequent application in beef has also been developed (Cui, Yuan, & Lin, 2017).

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None of these works mentioned above has focused on the study of the effect of the use of liposomes on the physical, rheological and humectability properties of the ECS on surfaces

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of food. Therefore, the objective of this work was to study the physical, rheological and humectability properties of the ECS based on HPMC containing liposomes that encapsulate

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rutin, glycerol and cellulose nanofibers on surfaces of almonds and chocolates. 2. Material and Methods 2.1 Materials

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Rutin (Quercetin-3-rutinoside hydrate, ≥94% purity) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Soybean lecithin was provided by Dimerco (Chile). Glycerol (G) (> 99%), monopotassium and dipotassium phosphate were obtained from Winkler (Chile). Ethanol (99.9%, Merck), acetone (99.7%, Winkler) and ethyl acetate (99.8%, Winkler) were used as solvents. Hydroxypropyl methylcellulose (HPMC) (Methocel E19 Food Grade, Dow Wolff Cellulosics). Chocolate 62% cocoa (Costa, Carozzi S.A., Chile) and laminated almonds 6

(Prunus dulcis) (Frutexsa LTDA, Chile). Cellulose nanofibers (CN) were produced according to our previous work (Andrade et al., 2015) from agro-industrial residues (pineapple peel juice) and Gluconacetobacter swingsii sp. To produce bacterial cellulose, a culture medium pineapple peel juice (2.14 g/100 mL glucose, 2.4 g/100 mL fructose, 2.10 g/100 mL sucrose, 0.31 g/100 mL total nitrogen) was used. As shown in the SEM images of our previous work (Andrade et al., 2015) the typical size of the cellulose nanofibers was 50–

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60 nm wide. From tests performed at our laboratory, a length of 2821 nm for CN was

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determined by dynamic light scattering at a detection angle of 173° and the surface charge of the CN was - 45.6 mV was determined by dynamic light scattering at a detection angle of

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173°. FT-IR spectra of cellulose nanofiber are shown in Fig. S1. As we can see typical bands

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assigned to cellulose were observed in the region of 1645 - 900 cm−1. The peaks located at 1627 cm−1 correspond to the vibration of water molecules absorbed in cellulose. The

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absorption bands at 1424, 1371, 1318, 1057 cm−1 belong to stretching and bending vibrations of -CH2 and -CH, -OH and C-O bonds in cellulose. It would be expected that for

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hemicellulose, its FT-IR spectrum would be quite similar to that of cellulose because of its structural resemblance. However, in the hemicellulose there is a prominent band over the 1044 cm−1, which is uniquely assigned to C–O, C–C stretching or C–OH bending in xylan such as was reported by Galletti and coworkers (Raspolli et al., 2015). In our case, the FT-

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IR spectra of cellulose do not show this characteristic band. Therefore, in our work, we used cellulose nanofiber. 2.2 Preparation of Antioxidant Suspensions (AS) Purification of food grade crude soybean lecithin was obtained following the procedure described by Mosquera et al., (2014). Crude soybean lecithin (10 g) was dissolved into 50

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mL of ethyl acetate at 20°C. Then, distilled water (2 mL) was added slowly under manual agitation, resulting in the formation of two phases. The lower phase was separated and dispersed in 30 mL of acetone, forming clusters which were crushed using a glass stick. Then, acetone was separated by decanting it and a new aliquot of acetone (30 mL) was added, repeating the shredding process. The precipitate was vacuum filtered and dried in a desiccator at 20°C for 48 hr. Partially purified phosphatidylcholine (PC) was finally obtained. with rutin (LR) were prepared at laboratory scale by using a

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Liposomes

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heating/homogenization method with lamellarity and size reduction by an ultrasound rod (Alemán et al., 2016). PC (10 mg/mL) and rutin (5 mg/mL) were dissolved in ethanol (50%

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v/v): potassium phosphate buffer (PPB) (10 mM, pH 7.4) and kept in a water bath at 80 °C

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for 1 hr. All liposomal suspensions (LS) were prepared with addition of glycerol (0.76 % w/v) and kept in a water bath at 80 °C for 1 hr. After that, PPB was added until a rutin

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concentration of 1 mg/mL was reached. Suspensions were subjected to ten sonication cycles (1 minute of ultrasound and 1 minute in ground ice to allow sample cooling). Sonication

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cycles were applied using an ultrasonic cell disruptor (HIELSCHER UP100H, Germany, max 100 W) with MS7 Micro tip 7 sonotrode (7 mm diameter, 120 mm length 130 W/cm2 acoustic power density), working at 90 % amplitude and 22.5 Hz frequency. A free rutin suspension (R), that is, without encapsulating in liposomes, was prepared using

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the same concentration of rutin (1mg/mL) in ethanol (10%v/v) and PPB (90%v/v) to compare the effect that the rutin was trapped or not in liposomes on the properties of edible coatings suspensions (ECS). 2.3 Characterization of LS

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The characteristics of the liposomes with rutin were confirmed by determining their hydrodynamic diameter, zeta potential (ξ) and polydispersity index (PDI). Mean particle size, PDI and ξ analyses were performed by dynamic light scattering (DLS) technique using a Malvern instrument (Zetasizer Nano ZS, Malvern Instruments Ltd, England). Vertical cuvettes of 2000 μL capacity at 25 °C were used. Samples were diluted 100-fold in PPB

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before analysis. 2.4 Microstructure of LS

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The microstructure of the suspensions was studied using a Transmission Electron

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Microscope (TEM) (Tecnai 12, Philips, Amsterdam, Netherlands) with 80 kV using the negative-stain method. LS were diluted 10 times with PPB. Diluted samples were placed on

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Formvar-carbon coated copper grid (300 mesh, 3 mm diameter HF 36) and the liquid excess

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was drawn off using filter papers. A drop of uranyl acetate solution (2 % w/v) was placed on the samples and left 1 min at room temperature; the excess of liquid was drawn off. The micrographs were made after drying the grid on stove (LDO-150F model, LabTech, Korea)

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for 60 min at 25 °C.

2.5 Formulation of Edible Coating Suspensions (ECS) The ECS were prepared from HPMC (3 %w/v in relation to the ECS) Glycerol (G, 15 - 35

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% w/w with respect to the HPMC), cellulose nanofibers (CN, 3 - 5 % w/w with respect to the HPMC) and antioxidant suspension (AS, 30 - 60 % v/v in relation to the ECS) rutin suspension (R) or liposomes with rutin (LR). The HPMC was hydrated with distilled water at 90ºC using a magnetic stirrer at 800 rpm until its complete dissolution. Subsequently, G was added, with the CN, homogenized for 1 hr with magnetic agitation at 800 rpm. Then, the corresponding AS was added (R or LR), according to the concentrations that are reported in 9

Table 1, magnetically shaken for 1 hr. Finally, the suspensions for coating with rutin (ECSR) and coating suspensions of liposomes with rutin (ECS-LR) were obtained. 2.6 Physical Properties of the ECS: The physical properties of the ECS studied were density, surface tension, rheology and humectability.

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The density was measured pycnometrically. The surface tension was determined by the

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hanging drop method according to Skurtys et al., (2011). We used a high-speed camera (Pulnix Inc., San Jose, CA, USA) and a needle that provided a drop of the ECS controlled by

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programmable pump syringe. We used the Hot Shot software 1.3 (NAC Image Technology INC) and took between 10 and 30 photos for each sample that were analyzed using the

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MATLAB R2013A (8.1.0,604, The MathWorks Inc.). The surface tension is determined

𝑑 𝑠𝑒𝑛𝜃

Where

2

= − 𝑏

𝑔 ∆𝜌 𝛾𝐿𝑉

∗𝑧−

𝑠𝑒𝑛𝜃 𝑥

Eq. 1

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𝑑𝑥

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from the Laplace equation:

𝑥, 𝑧: Cartesian coordinates at any point of the drop. 𝑏: Radius of curvature in the vertex.

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𝑔: Acceleration of Gravity,

∆𝜌: Difference in density between the suspension and air at 20°C. 𝜃: Angle between the axis of the drop and normal in the interface of the drop. 𝛾𝐿𝑉 : Surface tension between the ECS and the air 2.7 Rheological Properties 10

A rheometer (Discovery Hybrid Rheometer HR2, TA Instruments, England) using a cone and plate configuration (cone angle 1.008°; diameter 60 mm) was used to carry out the experiments. The lower plate was equipped with a Peltier temperature control system. ECS sample was placed on the lower plate, and the upper plate was then lowered to the desired gap. To ensure that the samples reached and maintained the working temperature, a routine temperature stabilization for 5 min was applied. For steady measurements, shear rate was

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increased from 0.01 to 100 s−1 (upward ramp) and then decreased to 0.01 s−1 (downward

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ramp) at 25°C. Oscillatory tests were run in the linear region. Temperature sweeps from -8°C to 70°C to -8°C at a heating/cooling rate of 2°C/min, with 2% deformation and 1 Hz were

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applied. Also, frequency sweep from 0.01 to 3 Hz (0.01 to 20 rad s-1) were applied at 25°C

2.8 Measurements of Contact Angle

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with 2% deformation. Experiments were done in triplicate.

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For each ECS studied, contact angles (CA) were measured at ambient temperature (20 °C) on two types of foods, chocolate bars and laminated almonds, using an optical system which

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comprised a zoom video lens (Edmund Optics, NJ, USA). Small drops (2 μl) were manually deposited on the food surface using a precision microliter pipette (Gilson Pipetman U2). The apparent CA (the angle between the tangent plane to the surface of the liquid and the tangent

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plane to the food) was determined using ImageJ software (National Institutes of Health, USA). Spreading coefficient (𝑆𝑒𝑞 ), adhesion energy (𝑊𝑎 ) and cohesive energy (𝑊𝑐 ) were calculated from Eqs. 2; 3 and 4 respectively. 𝑆𝑒𝑞 = 𝑊𝑎 − 𝑊𝑐 𝑊𝑎 = 𝛾𝐿𝑉 (1 + 𝑐𝑜𝑠 𝜃)

Eq. 2 Eq. 3

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𝑊𝑐 = 2 𝛾𝐿𝑉

Eq. 4

Where: 𝛾𝐿𝑉 : Surface tension between the ECS and the air. 𝜃: Angle between the axis of the drop and normal in the interface of the drop.

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2.9 Statistical Analysis To determine the effect of the AS, G and CN concentration, and the presence of liposomes

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on the physical properties (ρ, μ, 𝛾𝐿𝑉 , 𝑆𝑒𝑞 𝑊𝑎 , 𝑊𝑐 ) of the ECS, a 24 factorial design was used,

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where it was determined that the effect of the AS concentration was not significant (p 0.05). Therefore, we decided to work with a 23 factorial design with G, CN and the presence of

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liposomes as factors. An analysis of variance (ANOVA) with a confidence level of 95%

the significant effects.

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3. Results and Discussion

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using the STATGRAPHICS Centurion XVI program (Version 16.2.04) was carried out on

3.1 Physical Properties

Liposomal suspensions that encapsulate rutin were obtained and incorporated into edible coating suspensions (ECS). Below, the physical properties obtained both from the liposomal

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suspensions and the suspensions for coating are described. 3.1.1 Liposome with Rutin (LR) Using the heating/homogenization method, unilamellar liposomal vesicles were obtained (Fig 1) by the DLS technique with a hydrodynamic diameter of 110.6 ± 3.0 nm. Utilizing a direct measurement of the liposome diameters from the micrographs obtained by TEM, a true

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diameter of 84 ± 26 nm was obtained, calculated as the average diameter of 22 liposomes randomly selected from two TEM images, with different magnification as shown in Fig. 1. Similar sizes were reported by Bonechi et al., (2018) for anionic, cationic and bipolar liposomes, loaded with quercetin or rutin using different lipids in a 1:1 M lipid to antioxidant relation. Babazadeh, Ghanbarzadeh, & Hamishehkar, (2017) reported sizes between 80-100 nm, which were prepared using the method of hydration of thin layer with different molar

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ratios of 1:PC (rutin:1, 1:2 and 1:3).

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The PDI and ξ of the LR was characterized as a measure of the stability of suspensions. The

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PDI of the LR was 0,333 ± 0.04 and ξ -52.9 ± 7.5 mV, which indicates that the LR are considered stable due to relatively high repulsive forces and the uniformity in the size of the

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liposomes, minimizing the aggregation or the sedimentation of its particles (Bhattacharjee,

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2016).

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a)

74 nm 73 nm 72 nm

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131 nm

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85 nm

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3.1.2 Edible Coating Suspensions (ECS)

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Table 1 presents the physical properties: density, surface tension and viscosity of suspensions of edible coating with rutin (ECS-R) and of the suspensions of edible coating of

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liposomes with rutin (ECS-LR). According to the multivariate analysis performed to assess the effect of factors, the normal probability plot (Figure not shown) indicated that the density,

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surface tension and viscosity of the ECS were significantly affected only by the presence or absence of liposomes and the concentration of CN in the formulation (p  0.05). 3.1.2.1 Density

In the case of density, as greater concentration of CN (5%w/w) were incorporated, ECS-R

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b)

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Figure 1. Transmission Electron Microscopy (TEM) of the liposomal suspensions with different resolutions. a) 26500x Magnification; (b) 60000x Magnification

suspensions showed higher density. On the contrary, in ECS-LR, a greater concentration of CN caused a significant decrease in density (p ≤ 0.05). A greater or lesser concentration of G did not significantly affect the density values of the ECS. In general, the ECS-LR have a higher density in comparison with the ECS-R (p ≤ 0.05), indicating that the presence of the liposomes increased the value of the density of most of the 14

ECS. The density of the ECS is an important parameter that influences the final thickness of the coating. Thus, for example, in the case of a coating application by the immersion method in accordance with the Landau-Levich: 𝑒 = 0.945𝑙𝑐 𝐶𝑎2/3 = 0.945𝜇

𝑈 √𝜌𝑔

𝛾 7/6 (Krechetnikov

& Homsy, 2005). An increase in the density of the ECS implies a decrease in the thickness 𝛾

of final coating formed over the food. Where 𝑒 is the thickness, 𝑙𝑐 = √

𝜌𝑔

𝜇𝑈 𝛾

is the number capillary, 𝛾 is the surface tension, 𝜇 is the dynamic viscosity,

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length, 𝐶𝑎 =

is the capillary

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𝜌 density and 𝑈 is speed.

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An increase in the density tends to decrease the thickness of the coating, which is favorable for the implementation of the ECS. In addition, a decrease of the thickness caused by the

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increase in density is directly related to the functionality of the coating, as important parameters such as permeability to water and gases, weight loss of food during storage,

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variation of color and opacity of the coating are affected (Ali, Maqbool, Ramachandran, &

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Alderson, 2010; Lin & Zhao, 2007; Silva-Weiss et al., 2013; Sobral, 2000).

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𝛾𝐿𝑉 (mN/m) ECS-R ECS-LR 56.84 ± 0.97ax 57.20 ± 0.05ax 54.70 ± 0.29ay 54.02 ± 2.21ay 56.43 ± 0.98ax 58.29 ± 0.76ax 53.22 ± 1.24ay 55.11 ± 1.82ay 56.02 ± 1.33ax 54.37 ± 1.14bx 54.02 ± 1.09ay 52.02 ± 1.05by 57.16 ± 0.20ax 54.00 ± 0.26bx 55.11 ± 1.24ay 52.33 ± 0.84by

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ρ (kg/m3) a 20°C ECS-R ECS-LR 990.3 ± 0.7by 1007.7 ± 3.8ax 993.7 ± 1.5bx 997.5 ± 0.3ay 989.3 ± 1.2by 1042.0 ± 1.4ax 993.1 ± 1.4bx 1006.1 ± 2.2ay 980.3 ± 0.7by 997.6 ± 4.7ax 993.4 ± 6.2bx 996.8 ± 1.4ay 956.4 ± 4.1by 1002.1 ± 1.8ax 980.9 ± 2.2bx 988.0 ± 2.1ay

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Sample AS G CN (%v/v) (%w/w) (%w/w) (N°) 1 30 15 3 2 30 15 5 3 30 35 3 4 30 35 5 5 60 15 3 6 60 15 5 7 60 35 3 8 60 35 5

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Table 1. Physical properties of the ECS-LR and ECS-R.

ηap (mPa s) ECS-R ECS-LR 58.07 ± 3.32bx 64.56 ± 1.42ax 53.96 ± 0.38bx 57.37 ± 1.46ay 56.49 ± 1.86by 65.64 ± 0.58ax 58.54 ± 2.63bx 60.20 ± 0.28ay 64.17 ± 0.88ax 71.20 ± 2.15by 47.31 ± 0.87by 67.10 ± 1.07ax 58.54 ± 2.59bx 68.89 ± 1.64ay 57.22 ± 1.80by 70.18 ± 1.20ax

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a and b indicate significant differences between ECS-R and ECS-LR with a same concentration of AS, G and CN. X and Y indicate significant differences between 3 and 5 % w/w CN with the same concentration of AS and G.

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3.1.2.2 Surface Tension (𝛾𝐿𝑉 ) A greater concentration of CN (5 %w/w) tends to significantly decrease (p ≤ 0.05) the values of surface tension of the ECS-R and ECS-LR (Table 1). This can be attributed to the fact that the CN accumulate at the coating-air interface, reducing the effective surface energy, which leads to a decrease in surface tension of the coating (Andrade et al., 2014). For

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example, a reduction of the surface tension has been reported by effect of incorporating CN into gelatin ECS (Andrade et al., 2014).

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The surface tension of the ECS is independent of the concentration of G. This result was

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expected since glycerol is not considered a surfactant (Rodríguez, Osés, Ziani, & Maté, 2006) and therefore the G does not cause a variation in the values of surface tension of the ECS.

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Table 1 also shows that only for the ECS with 60% v/v of AS, the presence of liposomes

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caused a significant decrease (p ≤ 0.05) in the values of surface tension, reaching the lowest values at 52.02 mN/m for the formulation with a low level of G (15%w/w) and a high level

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of CN (5%w/w) and 52.33 mN/m for the formulation with a high level of G (35%w/w) and high level of CN (5%w/w). This can be attributed to the fact that these formulations have a greater amount of phosphatidylcholine (partially purified from soy lecithin), so there are still traces of soya lecithin, which is known as a surfactant (Rodríguez et al., 2006). Considering

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that the surface tension decreases with the concentration of the surfactant because it interferes with the formation of hydrogen bridges and other forces involved in the adhesion between molecules (Raiger Iustman & López, 2009), a greater presence of soy lecithin would cause a decrease in the value of the surface tension of the ECS. It has also been reported that the addition of fatty acids, especially those of long-chain, influences the values of the surface tension of the ECS. Different authors have studied the 17

effect of adding different fatty acids to chitosan based edible matrices and concluded that the surface tension decreases as the length of chain of fatty acids increases (Fabra, Jiménez, Atarés, Talens, & Chiralt, 2009; Wong, Gastineau, Gregorski, Tillin, & Pavlath, 1992). Considering that in this study we used phosphatidylcholine of partially purified soy, for which the most abundant and present fatty acids are linoleic acid (C18: 2n6c), oleic acid (C18: 1N9c), palmitic acid (C16: 0), linoleic acid (C18: 3n3) y stearic acid (C18: 0) (Taladrid

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et al., 2017) which are long chain fatty acids and would be present in the formulations of

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ECS, therefore a decrease of surface tension with the greatest amount of soya lecithin would be expected. This is a desirable response for applying the coatings, since it has been reported

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that lower surface tension can prevent the coating from detaching from the surface of the

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food (Han, 2014; Raiger Iustman & López, 2009), resulting in a decrease in the cohesive forces between the molecules of coating suspensions. Rodríguez et al., (2006) developed

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potato starch films, glycerol as a plasticizer, Tween 20 and Span 80, soy lecithin as surfactants in the formulation, and found that lecithin was the surfactant that reached the

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lowest value of surface tension (49.6 mN/m) (p0.05). 3.2 Rheological Properties

Below we analyze the effect of incorporating liposomes, frequency and temperature on the

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rheological properties of the edible coatings. 3.2.1 Apparent Viscosity The apparent viscosity (defined as the ratio between the shear stress and the shear rate) of the ECS increased significantly (p ≤ 0.05) with the presence of liposomes (Table 1). The average apparent viscosity of the ECS-LR (65.63 ±1.53 mPa*s) was greater than the apparent viscosity of the ECS-R (56.78±2.02 mPa*s). This can be attributed to the presence of a large 18

number of spherical vesicles (liposomes) in the suspension, which, when subjected to a shear rate presented greater interaction or friction between them, leading to an increase in the apparent viscosity. In addition, it has been reported that liposomes become more rigid when they encapsulate polyphenols, which would also result in an increase in the values of apparent viscosity of the ECS-LR (Mourtas, Haikou, Theodoropoulou, Tsakiroglou, & Antimisiaris,

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2008). All of the ECS studied behaved as pseudoplastic type fluids (𝑛 < 1) (data not shown) for a

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deformation gradient between 0.1 and 100 𝑠 −1 and showed non thixotropic effects when

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comparing the ascending and descending flow curves. For all formulations in study, the flow

  K n 

Eq. 5

is the shear rate (𝑠 −1 ); and 𝐾 it is the consistency

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Where 𝜎 is the shear stress (𝑃𝑎);

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curves were described by the Power Law model (Eq. 5):

coefficient (𝑃𝑎𝑠 𝑛 ) and 𝑛 is the flow behavior index (dimensionless).

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3.2.2. Effect of Frequency

Fig. 2 shows the frequency dependence (0.01 to 3 Hz) of the storage (𝐺 ′ ) and loss (𝐺′′) modules of the formulations with 60% v/v of AS, 3% w/w CN and 15 and 35% w/w G. 𝐺′′

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was higher than 𝐺 ′ in the entire frequency range applied and 𝐺′′ showed a sharp increase and therefore greater dependence on frequency, taking values around 0,006 Pa with 0.01 Hz and 0.91 Pa with 2.5 Hz. Meanwhile, 𝐺 ′ was maintained between 0.01 and 0.03 Pa throughout the frequency range studied. The other formulations of ECS presented a similar behavior.

19

□ G'' (Pa)

1

a)

b)

c)

d)

0.1

■ G'

0.01

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0.1

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0.01

0.001 0.01

0.1

1

10 0.01

0.1

1

10

Frequency (Hz)

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Frequency (Hz)

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■ G'

□ G'' (Pa)

1

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0.001

Figure 2. Variation of storage modules (G', full) and loss module (G'', empty) with regard to the frequency of edible coatings with 60%v/v AS. a) ECS-R 15% w/w G, b) ECS-R 35% w/w G, c) ECS-LR 15% w/w G, d) ECS-LR 35% w/w G. = 2% Strain; T=25°C.

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Due to the fact that 𝐺′′ was greater than 𝐺 ′ for all ECS viscous behavior is dominant over the elastic behavior, and independent of the frequency that is being applied (Rao, 2003). This could be explained based on what is informed by Osorio, Molina, Matiacevich, Enrione, & Skurtys, (2011) who reported a gelling temperature (TG) over 30°C for edible HPMC films

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(0.5; 1.0 and 3.0 % w/w). Meanwhile, for this work, as these analyzes were carried out at 25°C, one would not expect ECS to present a gel-type structure, but a viscous behavior. For industrial applications, this indicates that at lower processing times, for example, at the level of a mixer, the ECS will behave as liquid. This implies that to deform the material, the supplied energy would dissipate entirely (Powles, Rickayzen, & Heyes, 1999). A similar

20

behavior was reported by Lizarraga, Piante Vicin, González, Rubiolo, & Santiago, (2006), which in their study with whey protein concentrate 2.0-32.0% w/w of total solids and with different concentrations of λ- carrageenan 0.1-2.0% (w/w) found that all of the studied concentrations showed a similar behavior to that of a liquid at low frequencies with a loss module (𝐺′′) greater than the storage module (𝐺 ′ ). On the contrary, Ganesan, Munisamy, & Bhat, (2018), in their study with edible films of semi-refined carrageenan and glycerol 20%

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w/w as a plasticizer, found for their samples a gel-type behavior with a high elastic module

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(𝐺 ′ > 100 Pa) and a low viscous modulus (𝐺′′ > 60 Pa).

Both storage 𝐺 ′ and loss 𝐺′′ modules were modeled and they showed a good fit following the

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Power Law model. 𝐺 ′ adjusted well to the Eq. 6 taking values 𝑎 between 0.02-0.19 Pa*sb

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and 𝑏 between 0.42-0.76 for the ECS-R; and values 𝑎 between 0.01-0.05 Pa*sb and 𝑏 between 0.12 and 2.73 for the ECS-LR. 𝐺′′ was modeled according to the Eq. 7 and the adjusted

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parameters obtained for ECS-R are 𝑐 from 0.09 to 0.24 Pa*sd and 𝑑 between 0.71 and 0.89; and for the ECS-LR values 𝑐 between 0.05 and 0.07 Pa*sd and 𝑑 between 0.90 and 0.98 were

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obtained. All the adjusted curves mentioned above showed coefficient of determination of 𝑅2 = 0.9996.

Eq. 6

G″ = 𝑐𝑤 𝑑

Eq. 7

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G′ = 𝑎𝑤 𝑏

Where 𝑤 is the angular frequency, and 𝑎; 𝑏; 𝑐 and 𝑑 are the parameters of the Power Law respectively.

3.2.3. Effect of Temperature

21

Fig. 3a shows the effect of temperature on the variation of storage (G') and loss (G'') modules of suspensions ECS-R and ECS-LR with 60% v/v AS, 35% w/w G and 3% w/w CN for a heating rate of 2°C/min at 2% deformation and 1 Hz frequency. A viscoelastic behavior is observed in the range of -8 to 70°C, characteristic for all formulations in study. As the temperature increases, the suspensions show a slow decline in both modules in the range of 1 - 100 Pa. In this region of low temperature G'' is greater than G', which means that the

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suspensions presented a similar behavior to a fluid between 0 and 30°C because the system

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is hydrated, causing low polymer-polymer interaction (Zhang et al., 2015). As temperature increases, G' tends to be equal to G'' and tan α is equal to 1; gelation of the system occurs at

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36°C for the ECS-R and 42°C for the ECS-LR. This substantial increase in both modules is

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due to the kinetics of sol-gel transition and is associated with the gelation temperature (TG) of HPMC suspensions. At low temperatures, the molecules of the HPMC are hydrated and

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there is little interaction between the chains of this polymer. However, as the suspension heats, the molecules gradually lose the water of hydration and the apparent viscosity drops.

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Later, when the temperature of the ECS reaches the temperature of gel formation, viscosity increases and the gel structure develops. In the ECS, gelling mechanism is due to the hydrophobic interaction between molecules that contain substitution with methoxyl, because water molecules of the outer layers are expelled from the chains of cellulose ether with the

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increase of temperature. Therefore, greater polymer-polymer interaction occurs and an infinite reflected network forms due to a sharp increase in the apparent viscosity (N. Sarkar, 1979; Zhang et al., 2015). It has been reported that HPMC solutions (10 - 20% w/w) without the addition of plasticizers gels between 55 and 62°C. For edible coatings prepared with a mixture of HPMC (0.5 - 3%

22

w/w), K-carrageenan and emulsion of carnauba wax the TG has been reported between 29.9 and 35.5°C (Osorio et al., 2011; Nitis Sarkar, 1995; Zhang et al., 2015). The melting temperature for phospholipids such as phosphatidylcholine (16:0) has been reported at 41.8°C, and liposomes made of dipalmitoylphosphatidylcholine, at 41.3°C (Biltonen & Lichtenberg, 1993; Rowe, 1985). In addition, studies report that the incorporation of compounds such as glycerol can cause a decrease in the TG of the suspensions due to its

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greater affinity for water, therefore, they accelerate the dehydration of the polymer (Hanawa

>

TG of ECS-R, 36°C, this greater TG in the ECS-LR can be

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The TG of ECS-LR, 42°C

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et al., 1995; Mitchell et al., 1990).

attributed to the presence of the liposomes in these samples, for which a glass transition

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temperature around 40°C has been reported (Koynova & Caffrey, 1998; Pippa et al., 2018). When it reaches this temperature of transition in the liposomal structures, a phase change in

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the liposomal membrane occurs, which passes into the liquid crystalline phase, a disordered state, in which the phosphatidylcholine is released laterally, causing damage in in the bilayer

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in some cases (Ulrich, 2002). For DPPC liposomes that encapsulate rutin and quercetin prepared by the method of thin film hydration, a transition temperature of 40.5 and 39.1°C has been reported, respectively (Goniotaki, Hatziantoniou, Dimas, Wagner, & Demetzos, 2004). Whereas, in liposomes of egg phosphatidylcholine prepared using the method of

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thin film hydration reported a transition temperature of 40°C (Cacela & Hincha, 2006). On the other hand, in the cooling curve (Fig. 3b) there is a reversible behavior.

23

1000000

a) 100000

Heating G' G'' (Pa)

10000

1000

of

100

ro

10

1 0

10

20

30

40

50

60

70

80

-p

-10

Temperature (°C) 100000

lP

Cooling

1000

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G' G'' (Pa)

10000

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b)

100

10

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1

-10

0

10

20

30

40

50

60

70

80

Temperatura (°C)

Figure 3. Effect of temperature of the ECS-LR (□) and ECS-R () on the modules △G' (full) and G'' (empty) with a concentration of 60% v/v AS, 35% w/w G and 3% w/w CN. With a rate of warming of 2°C/min; 2% of deformation and 1 Hz of frequency.

24

3.3 Humectability For optimization and proper functioning of the ECS, parameters such as the coefficient of dispersion or humectability (Seq) should be studied, which represents the ability of a given liquid to spread on a solid surface; the energy of adhesion (Wa) that promotes diffusion of the liquid and cohesive energy (WC) which promotes the contraction of the coating (Ribeiro, Vicente, Teixeira, & Miranda, 2007). The control of these coefficients enables obtaining

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coatings with a good affinity with the food to be coated.

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To evaluate these coefficients in the ECS, two foods with a high fat content were used: almonds (52.5%) and chocolate (44%). The dispersion coefficient (Seq), the adhesion energy

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(Wa) and the cohesive energy (WC) obtained for the different formulations are shown in

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Tables 3 and 4. Here it is observed that the ECS-R with a 60% v/v AS have a higher value of Wc in comparison with the ECS-LR, which indicates that the presence of liposomes

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significantly decreased (p ≤ 0.05) the value of the cohesive energy of the ECS with greater percentage of AS (60%v/v). The latter results are favorable because it causes the ECS not to

ur na

contract and to diffuse better on the surface to be covered (Ribeiro et al., 2007). In addition, the value of Wc is independent of the concentration of glycerol because glycerol is not regarded as a surfactant. However, an increase of the concentration of CN tends to reduce the values of Wc of most of the formulations. This is due to the fact that the CN accumulate

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in the air-to-formulation interface (Andrade et al., 2014), reducing the forces of cohesion that hold ECS together.. Humectability was studied by determining the values of the dispersion coefficient (Seq). In the case of the surface of chocolate, no significant effect (p ≤ 0.05) was found in the values of Seq between the ECS-R and ECS-LR. For the ECS with a 30%v/v AS it is observed that

25

the increase of the concentration of CN (5%w/w) causes a significant decrease (p ≤ 0.05) in the values of Seq. On the contrary, in the formulations with 60%v/v AS, increasing the concentration of the CN (5%w/w) significantly increases (p ≤ 0.05) the values of Seq. Therefore, a greater concentration of AS and CN improves the humectability of the coating on the surfaces studied. In the case of the surface of the laminated almond, no significant

of

effects were observed of the presence of liposomes, CN and G. The best value (closest to zero) of Seq. was determined for each food in study. In the case of

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almonds (Table 3) with ECS-R, the best value was for the formulation with a 60%v/v AS,

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35%w/w G and 5%w/w CN (25.4 mN/m) and with ECS-LR, the best value was the formulation with a 30% v/v AS, 15% w/w G and 5% w/w CN (76.8 mN/m). For the case of

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chocolate (Table 2) the best values of Seq were of -42.0 mN/m for the formulation with 60% v/v AS, 35% w/w G and 5% w/w CN and 34.0 mN/m, for the formulation with 60% v/v AS,

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15% w/w G and 5% w/w CN. Therefore, in order to improve the humectability of the ECS in the surfaces in study it is more convenient to work in parallel with 60% v/v AS and a

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greater concentration of CN (5%w/w).

With regards to the values of the contact angle for the ECS-R and the ECS-LR, it is noted that for both chocolate and almonds the values obtained are less than 90°; therefore, it can be

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said that the ECS partially hydrates the studied surfaces (Choi, Park, Ahn, Lee, & Lee, 2002). However, it can be seen in Fig. 4 that the contact angle to the surface of almonds is lower than for the surface of chocolate. This indicates a greater affinity of the coatings to the surface of almonds, which can be attributed to the almonds having a higher percentage of fat (55%) and a low percentage of moisture (8%).

26

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Table 2. Parameters associated with the humectability of ECS in chocolate

Seq (mN/m) ECS-R ECS-LR bx -55.0 ±3.0 -41.1ax ±5.2 -57.0ay ±2.2 -71.0by ±1.0 -42.2ax ±3.3 -49.0bx ±2.1 -50.0ay ±3.1 -75.0by ±0.3 -61.0ay ±4.1 -63.2ay ±7.0 -52.0bx ±3.0 -34.0ax ±1.3 -76.0ay ±0.4 -75. 3ay ±5.4 -42.0ax ±6.4 -39.0ax ±5.2

lP

re

-p

ro

Sample AS G CN Wc (mN/m) Wa (mN/m) θ (°) (N°) (%v/v) (%w/w) (%w/w) ECS-R ECS-LR ECS-R ECS-LR ECS-R ECS-LR ax ax bx ax ax 1 30 15 3 114.0 ± 2.0 114.4 ± 0.1 59.1 ± 3.3 73.2 ± 1.3 76.5 ±4.3 63.6bx ± 2.5 ay ay ay by 2 30 15 5 109.4 ± 1.0 108.0 ± 4.4 52.3 ± 2.6 37.1 ± 4.3 73.5ax ± 3.0 58.6bx ±3.2 3 30 35 3 112.0ax ± 2.0 117.0ax ± 2.0 106.2ax ± 6.3 67.3bx ± 2.0 69.5ax ±1.0 69.4ax ± 4.6 ay ay ay by 4 30 35 5 106.4 ± 3.0 110.0 ± 4.0 57.0 ± 3.2 30.0 ± 5.0 79.8ax ±3.0 72.9bx ± 6.2 5 60 15 3 112.0ax ± 3.0 109.0bx ± 0.1 47.4bx ± 3.2 50.1ay ± 4.2 63.7ay ±2.2 64.3ay ± 1.8 ax by bx ax 6 60 15 5 112.0 ± 2.2 104.0 ± 2.1 52.0 ± 2.0 78.0 ± 9.4 77.4ax ± 2.9 67.1bx ±5.2 7 60 35 3 114.3ax ± 0.4 108.0bx ± 1.0 38.2ªy ± 0.1 33.0by ± 2.2 70.7ax ±4.4 58.8bx ± 1.7 ay by bx ax 8 60 35 5 110.2 ± 2.0 105.0 ± 2.0 62.4 ± 1.4 71.1 ± 6.0 74.5ax ± 2.1 59.2bx ±2.2 a and b indicate significant differences between ECS-R and ECS-LR with a same concentration of AS, G and CN. x and y indicate significant differences between 3 and 5 % w/w CN with the same concentration of AS and G.

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Table 3. Parameters associated with the humectability of ECS in almonds

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Sample AS G CN Wc (mN/m) Wa (mN/m) θ (°) (N°) (%v/v) (%w/w) (%w/w) ECS-R ECS-LR ECS-R ECS-LR ECS-R ECS-LR ax ax ax by ax 1 30 15 3 114.0 ± 2.0 114.4 ± 0.1 80.00 ±6.0 55.82 ± 8.0 55.7 ±3.1 25.6bx ± 0.5 ay ay by ax 2 30 15 5 109.4 ± 1.0 108.0 ± 4.4 72.90 ±2.0 102.24 ± 4.0 29.3ay ±1.6 24.7bx ± 1.4 ax ax ax by 3 30 35 3 112.0 ± 2.0 117.0 ± 2.0 82.94 ±7.3 77.30 ± 0.1 35.5ax ±1.1 29.6by ±0.3 ay ay by ax by 4 30 35 5 106.4 ± 3.0 110.0 ± 4.0 44.20 ±2.3 48.12 ± 3.4 40.6 ±2.1 20.3bx ±3.1 5 60 15 3 112.0ax ± 3.0 109.0bx ± 0.1 62.30by ±2.0 66.10ax ± 1.2 30.5ax ±2.2 20.2by ±0.9 ax by by ax ax 6 60 15 5 112.0 ± 2.2 104.0 ± 2.1 52.74 ±8.4 64.83 ± 8.0 29.7 ±1.2 23.4by ±0.7 ax bx by ax ay 7 60 35 3 114.3 ± 0.4 108.0 ± 1.0 61.60 ±6.0 91.70 ± 3.0 30.4 ±1.9 21.1by ±1.0 ay by by ax ax 8 60 35 5 110.2 ± 2.0 105.0 ± 2.0 77.21 ±3.0 80.71 ± 12.3 40.5 ±2.3 24.8bx ±2.0 a and b indicate significant differences between ECS-R and ECS-LR with a same concentration of AS, G and CN. x and y indicate significant differences between 3 and 5 % w/w CN with the same concentration of AS and G.

Seq (mN/m) ECS-R ECS-LR ax -34.0 ± 1.1 -59.0by ±18.0 -101.2by ± 1.0 -15.8ax ±1.4 -30.0ax ± 7.0 -39.2bx ±0.7 -90.2by ± 0.2 -56.3ay ± 6.0 -47.0ax ± 2.0 -90.0by ±1.1 -51.2by ± 1.0 -47.2ax ±17.0 -53.0by ± 7.0 -16.3ax ±4.0 -27.4ax ± 0.2 -30.0ay ±8.1

27

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Figure 4: Contact angle between the surfaces of food and the ECS. Conclusions

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The incorporation of unilamellar liposomes showed significant changes (p 0.05) in density,

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surface tension, cohesive energy, and humectability of suspensions of HPMC based edible coating applied to surfaces of fatty foods (almonds and chocolates). Incorporation of

lP

liposomes caused a significant increase (p 0.05) in the apparent viscosity of the ECS; showing pseudoplastic behavior. Throughout the frequency range studied, the suspensions

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showed viscous behavior at 25°C. Storage and loss modules showed a good agreement (p 0.05) with the Power Law model, presenting a viscoelastic behavior with regard to temperature variation in the range of -8 to 70°C, where the ECS-R gellified at 36°C and the ECS-LR, at 42°C. For future work it is recommended to study the effect of incorporating

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liposomes into the barrier and texture properties of HPMC edible coatings.

28

Author Contributions

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Johana Lopez-Polo: performed the experiments; analyzed the data; helped writing the paper; Andrea Silva: helped analyzing the data; Marcela Zamorano: helped performing experiments; Fernando Osorio: Conceived and designed the experiments; contributed reagents; wrote the paper.

Acknowledgments: The authors would like to thank the support given by FONDECYT-

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Chile Project 1161079, and Project DICYT 081971OL. J.L.P. acknowledges both the Academic Vice-rectory of the Universidad de Santiago de Chile USACH for her Ph.D.

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