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\begin{document}


\def\RunningAuthor{Aicha Fahry et al.}
\firstPage{7182}
\articleType{Original Article}
\receivedDate{02 Aug 2020}
\acceptedDate{25 Sep 2020}
\revisedDate{07 Sep 2020}
\journalVolume{2020, 11}
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\title{\textbf{Influence of the span 80/Gelatin B combination on the formulation and stabilization of Argan oil-in-water emulsions}}
\author{Aicha~Fahry\textsuperscript{*},  
            Yassir~El~Alaoui,  
            Younes~Rahali,  
            Nawal~Charkaoui, 
            Abdelkader~Laatiris~\\[5pt]{Faculty of Medicine and Pharmacy of Rabat, Laboratory of Pharmaceutics,  University Mohamed V, 10000-Rabat, Morocco}}

\begin{abstract}
The present study aims to evaluate the effect of the combination of a linear protein: Gelatin type B and an oil-soluble emulsifier Span 80 (sorbitan monooleate) in the stabilization of argan oil-in-water emulsions. For this purpose, the emulsifiying properties of Gelatin itself with Argan oil as lipid phase were investigated first, by preparing oil-in-water (O/W) emulsions containing 10  wt\%.  Argan oil and varying Gelatin concentrations (0.5-2 \% w/w), we have also formulated Argan O/W emulsions by span 80 alone at levels ranging from 1 to 6 wt\%. Subsequentely, we explored the influence of the simultaneous application of the Span 80 and the gelatine on the stability properties and on the droplets size of Argan O/W emulsions, using different mixtures of the two emulsifiers. We compared the stability properties (flocculation, creaming, and phase separation) of argan O/W emulsions prepared with type B gelatine as the only emulsifier with those of emulsions prepared with span 80 and mixture of gelatin/Span. For stable emulsions, our analysis was completed with measurement of droplets size and Zeta Potential. Finally, all of the experimental results and the storage time showed that the emulsions prepared by 10 wt\% argan oil and 2 (w/w) \% Gelatin + 3 wt\% Span 80 were the most stable with optimum conditions for minimal creaming, small droplets size (size {\textless}1\ensuremath{\mu}m) and high net droplet charge (absolute value of ZP {\textgreater} 23). The presence of span 80 in coexistence with gelatin, even in small quantitiees, has a profound influence on the stability of the argan O/W emulsions. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Argan oil,\newline Gelatin,\newline Oil in water emulsion,\newline Span 80,\newline Stability
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Aicha~Fahry~\\ Phone:\ (212)661493289 ~\\  Email:\ aichafahry@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{ Introduction}
 Argan oil is a natural product extracted from the fruit of Argania spinosa, an endemic tree in Morocco. Argan tree, of the family Sapotaceplays an essential ecological and socio-economic role in south western Morocco \unskip~\citep{922240:20671333}. This oil contains a high level of both oleic acid and linoleic acid, making it an excellent source of basic polyunsaturated fatty acids. Also, it is particularly rich in polyphenols and tocopherols that exhibit significant antioxidant activity. Argan oil is used in the food, pharmaceutical and cosmetics industries and supports the treatment of many diseases \unskip~\citep{922240:20671331}.  During the last decades, scientific work came to support its use \unskip~\citep{922240:20671333}. In this context, increased attention has been paid recently to the application of Argan oil in emulsions, which might further enhance its beneficial properties.

An emulsion is a dispersion of two immiscible liquids; generally oil and water. These systems are thermodynamically unstable and can be stabilized by the addition of emulsifiers which can be synthetics surfactants, fine solid particles or even biopolymers. 

The development and application of emulsifiers has drawn much attention to the increased stability of emulsions over the past two decades \unskip~\citep{922240:20671344}. Amphiphilic macromolecules or biopolymers such as the proteins can be used to decrease interfacial tension and to form steric elastic films. Among these proteins, gelatin has been applied to stabilize emulsions due to its excellent biocompatibility and biodegradability. It has been widely explored in the fields of food, pharmacy, bioimaging and tissue engineering \unskip~\citep{922240:20671344}.  Indeed, gelatine is a versatile biodegradable polymer derived from animal collegen; it has a structure of a linear chain with very little ramifications \unskip~\citep{922240:20671334}.  The amphoteric characteristic combined with the hydrophobic sites, on the gelatine chain give gelatin surface active and emulsion stabilizing properties.Gelatin is capable of adsorbing at the oil/water interface and foaming a continuous viscoelastic film on the surface of oil droplets, thus improving then emulsions stability \unskip~\citep{922240:20671334}. However, gelatin alone as sole emulsifier produce relatively large droplet size. This imparts the need of gelatine to either be used together with anionic surfactants or modified hydrophobically by attachment of non-polar side-groups \unskip~\citep{922240:20671345}.  Recently, acid or alkaline pretreated gelatins, particles of crosslinked gelatin \unskip~\citep{922240:20671344}, gelatin / chitosan or gelatin / glucomannan / tannic acid nanocomplexes have been developed to increase the stability of emulsions. 

Several studies have explored the emulsifying capacity of gelatine used alone or in combination with a surfactant, using different types of oils, synsthetic or mineral, as the dispersed phase. But, to our knowledge, the study of the effect of the combination of gelatine with an oil-soluble nonionic surfactant (Span 80) on the behaviour of emulsions based on Argan oil has not been fully exploited. This is the reason why, for our work, we have chosen to study gelatin, span 80 and their combinations to prepare Argan oil-in-water emulsions. 

For this objective, we first prepared Argan O/W emulsions stabilized only either by gelatin or by span 80 as the sole emulsifier. Then secondly we prepared argan O/W emulsions by combining the two emulsifiers, protein and surfactant, with different mixtures of Gelatin and Span80 by keeping constant the same amount of oil, then we compared the different emulsions in order to determine the effect of the presence or the absence of the surfactant on the behaviour of the argan emulsions stabilized with gelatin. The observation of physical stability against coalescence and creaming throughout the storage period at room temperature of these emulsions were complemented with droplet size and zeta potential measurement.
    
\section{Materials and Methods}
\textbf{Materials}

Gelatin was a commercial type-B-gelatin, obtained from Riedel de Ha{\"{e}}n AG-, in the form of the powder. The non ionic surfactants Span{\textsuperscript{\textregistered}} 80(Sorbitan monool{\'e}ate) was bought from Sigma Aldrich laboratoire. Moroccan Argan oil was purchased locally. Freshly distilled and filtered water was utilized for the preparation of all solutions. A dynamic scattering of Zetasizer light 3000HS (Malvern Instruments, France) was used for measuring the size of dispersed droplets and zeta potential.

\textbf{Preparation of Gelatin solutions}

Gelatin solutions were prepared at total final concentration of 0.5-1 and 2 (w/w) \%. The powder was dissolved in distilled water at 40\ensuremath{^\circ}C\ensuremath{\pm}1 under gentle stirring (300 rpm) for at least 45 minutes. Homogeneous colloidal solutions are obtained.

\textbf{Preparation of Argan oil-in-water emulsions}

Three series of O/W emulsions were prepared using Argan oil as the oily dispersed phase at a fraction of 10 wt \% and three types of emulsifiers: the gelatine solution used alone at concentrations of 0.5- 1 and 2 (w/w) \%; Span 80 alone at concentrations of 1-2-3 and 6 wt \%; and the combination (Gelatin +span 80) in different proportions.

For the non-ionic surfactant, the concentration used does not exceed 6wt\%. This value was set from a preliminary study on the formulation and stabilization of argan oil by a mixture of two chemical surfactants, span 80 and Tween 80 at a total concentration of 6 wt\% \unskip~\citep{922240:20671330}. In our study we tried to formulate O/W emulsions of argan oil with a single liposoluble surfactant: span 80 alone at 6wt\%, value fixed by the aforementioned study, then this value was reduced at 3-2 and 1 wt\%. At these same concentrations, the surfactant was combined with gelatine at concentrations ranging from 0.5 to 2 (w/w) \%.

The adopted preparation protocol is the same for all emulsions: Span 80 is always dissolved in the oily phase; the two phases are heated, when they are at 70 \ensuremath{^\circ} and are homogeneous, the aqueous phase is stirred at 400 rpm using a magnetic stirrer, then the fatty phase is gradually added dropwise in the aqueous phase with stirring for one minute. The whole is mixed and homogenized at 9500 rpm for 5 min with Polytron type homogenizer (Polytron PT MR 3100 Kinematica AG, Switzerland. Rotor-stator: 92/ Polytron PT-DA 3012/2S).

Each emulsion is identified by a code indicating its composition: GS (G: gelatin; S: Span and a number indicating the percentage by weight, (Table~\ref{tw-ae6ab339d569})).

\textbf{Characterization}

\textbf{Visual observation of emulsions}

The characterization of the appearance of the emulsions and of their physical stability were studied by visual examination on a black background after 24 hours, 1, 2 and 3 week of storage at room temperature. 

\textbf{Particle size and ZP measurement }

Zeta potential (ZP) and particle size were measured for three individual samples with a dynamic scattering of Zetasizer light 3000HS (Malvern Instruments, France instrument). The measurements were carried out at 25 \ensuremath{^\circ} C. 
    
\section{Results and Discussion}
\textbf{Effect of the nature and concentration of the emulsifier}

The results of visual observation of the prepared emulsions are summarized in Tables~\ref{tw-a6c4418991d6} and~\ref{tw-84304e38e079}.


\begin{table*}[!htbp]
\caption{\boldmath {Composition and sample code of emulsions formulated by 10 wt \% Argan oil, Gelatin (0.5-1-2 and 4 w/w (\%)), Span 80 (0-1-2-3 and 6 wt (\%)), homogenization at 9500 rpm for 5min} }
\label{tw-ae6ab339d569}
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\tbltoprule \rowcolor{kwdboxcolor}Concentration Span 80 \mbox{}\protect\newline Gelatin (w/w) \%  & \multicolumn{5}{p{\dimexpr(.801\linewidth-2\tabcolsep)}}{\cAlignHack Span 80}\\
\rowcolor{kwdboxcolor} & 0\% & 1\% & 2\% & 3\% & 6\%\\
\tblmidrule 
0.5  &
  G05S0 &
  G05S1  &
  G05S2  &
  G05S3  &
  G05S6\\
1 &
  G1S0 &
  G1S1  &
  G1S2  &
  G1S3  &
  G1S6\\
2 &
  G2S0  &
  G2S1  &
  G2S2  &
  G2S3 &
  G2S6\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Comparison of the stability properties of Argan O/W emulsions (10 wt \% oil) made with differents concentration (0.5-1 and 2 \%( w/w)) of Gelatin solutions without Span 80} }
\label{tw-a6c4418991d6}
\centering 
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\tbltoprule \rowcolor{kwdboxcolor}Gelatin \mbox{}\protect\newline (\%w/w) & Sample  \mbox{}\protect\newline code & Creaming (a) \mbox{}\protect\newline Stability & Phase (b) \mbox{}\protect\newline separation\\
\tblmidrule 
0.5 &
  G05S0 &
   X (24h) &
   Ѵ  \\
1 &
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   X (24h) &
   Ѵ  \\
2 &
  G2S0 &
   X (72h) &
   Ѵ  \\
\tblbottomrule 
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\begin{tablenotes}\footnotesize 
    
\item{(a)Key: Ѵ = no creaming evident; X= creaming detected }
    
\item{(b) Key: Ѵ  = no phase separation evident; X= phase separation detected}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Comparison of the stability properties of Argan O/W emulsions (10\% w/w oil) made with combination Gelatin + Span 80 at differents concentration} }
\label{tw-84304e38e079}
\centering 
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\tbltoprule \rowcolor{kwdboxcolor}Gelatin  \mbox{}\protect\newline Concentration \mbox{}\protect\newline  (\%w/w) & Span80 \mbox{}\protect\newline  \% & Sample  \mbox{}\protect\newline code & Creaming (a) \mbox{}\protect\newline Stability & Phase (b) \mbox{}\protect\newline separation\\
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\multicolumn{1}{p{\dimexpr(.2163\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{0.5}} &
  1 &
  G05S1 &
   X  &
   Ѵ  \\
 &
  2 &
  G05S2 &
   X  &
   Ѵ  \\
 &
  3 &
  G05S3 &
   X  &
   Ѵ  \\
 &
  6 &
  G05S6 &
   Ѵ   &
   X \\
\multicolumn{1}{p{\dimexpr(.2163\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{ 1}} &
  1 &
  G1S1 &
   X  &
   Ѵ  \\
 &
  2 &
  G1S2 &
   X  &
   Ѵ  \\
 &
  3 &
  G1S3 &
   X  &
   Ѵ  \\
 &
  6 &
  G1S6 &
   X  &
   Ѵ  \\
\multicolumn{1}{p{\dimexpr(.2163\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{ 2}} &
  1 &
  G2S1 &
   X  &
   Ѵ  \\
 &
  2 &
  G2S2 &
   X  &
   Ѵ  \\
 &
  3 &
  G2S3 &
   Ѵ +++ &
   Ѵ +++\\
 &
  6 &
  G2S6 &
   Ѵ +  &
   Ѵ + \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{(a)Key: Ѵ  = no creaming evident; X= creaming detected }
    
\item{(b)Key: Ѵ = no phase separation evident; X= phase separation detected}
    
\item{+++:   Stable for 3 months at room temperature; +  :   Stable for 1 month at roomtemperature.}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Comparison of the stability properties of Argan O/W emulsions (10 wt \% oil) made with Gelatin 4w/w \% alone and combination (Gelatin 4w/w \% + Span 80 at differents concentration)} }
\label{tw-bf379909ab22}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{LLLL}
\tbltoprule \rowcolor{kwdboxcolor}Sample \mbox{}\protect\newline Code & Creaming \mbox{}\protect\newline Stability (a) &  Phase \mbox{}\protect\newline Separation (b) & Average droplet size (\ensuremath{\mu}m) , after 1 week of storage\\
\tblmidrule 
G4S0 &
   X  &
   Ѵ  &
  ND (c)\\
G4S1 &
   Ѵ  &
   X  &
  ND\\
G4S2 &
   Ѵ  &
   X  &
  ND\\
G4S3 &
   Ѵ (4wk) &
   Ѵ (4wk) &
  2.26\\
G4S6 &
   Ѵ (2wk) &
   Ѵ (2wk) &
  2.62\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{(a) Key: Ѵ = no creaming evident; X= creaming detected }
    
\item{(b) Key: Ѵ = no phase separation evident; X= phase separation detected}
    
\item{(c) Key: ND = not determined because the emulsions destabilized within a week; wk =week}
\end{tablenotes}
\end{threeparttable}

\end{table*}
The stability of argan oil-in-water emulsions formulated by gelatine alone as an emulsifying solution depends on the concentration of the gelatin. At 0.5 and 1 w/w \%, the emulsions are formulated but their stability does not exceed 24 hours, on the other hand at 2 w/w \% gelatin, the emulsion has remained stable for more than 72 hours. This result is in agreement with \unskip~\citet{922240:20671346}  who reported that typically a protein concentration of 1 to 3 w/w \% is used to stabilize emulsions.

Table~\ref{tw-84304e38e079}  shows that the stability of all the emulsions formulated with the combination (gelatin + span) depends on the concentration of the two emulsifiers. The emulsions which were prepared by the combination (gelatin 0.5 or 1 w~/ w~(\%) + span at 1-2 or 6 wt\%), creamed 24 hours after their preparation while at 3wt\% span, creaming appears after 48 hours.

Gelatin at 2 w/w (\%), combined with 1 or 2 wt\% of span, allowed to obtain stable emulsions for 72 h, while combined with 3 or 6wt\% of span we obtained an even better stability which lasted more than one month for G2S6 (6wt\% span) and three months for G2S3 (3wt\% span), stored at room temperature.

It was noted that whatever the concentration of gelatin (0.5-1 and 2\%) combined with the span at (1-2-3 and 6\%) it is at 3wt\% of span that the emulsions are obtained more stable (G2S3, G1S3 and G05S3). It was also noticed that during the period of storage at room temperature, the emulsions did not develop any signs of degradation of the organoleptic qualities due to oxidation; this may be due to the nature of the oil. Indeed, Argan oil is stable oil because it contains a significant amount of natural antioxidants, mainly tocopherols \unskip~\citep{922240:20671330}.

To properly study the effect of the combination of the two emulsifiers (protein + surfactant) on the formulation and stabilization of argan oil-in-water emulsions, the two most stable emulsions of these series, G2S3 and G2S6 were selected and reformulated without gelatin, but only with the same surfactant (span 80), at the same concentration (3 and 6 wt\%), still with 10\% argan oil and following the same protocol cited above, emulsions named respectively G0S3, G0S6.

Visual inspection throughout the storage period at room temperature showed no creaming, flocculation or phase separation of these emulsions (G0S3-G0S6) which remained stable for more than two weeks.


\bgroup
\fixFloatSize{images/2a18306e-c1da-4078-a709-f75cbec06fbc-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/2a18306e-c1da-4078-a709-f75cbec06fbc-upicture1.png}{\includegraphics{images/2a18306e-c1da-4078-a709-f75cbec06fbc-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Evolution of the size of the droplets as a function of time}}
\label{f-b5db21c3702d}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/b79e3aaf-cecc-4062-b06e-cac83618b17b-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/b79e3aaf-cecc-4062-b06e-cac83618b17b-upicture2.png}{\includegraphics{images/b79e3aaf-cecc-4062-b06e-cac83618b17b-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Particle size distribution of the stable emulsion G2S3}}
\label{f-84fe028c04e9}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/9daefe84-e110-4c85-a8dd-fd77942ced0e-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/9daefe84-e110-4c85-a8dd-fd77942ced0e-upicture3.png}{\includegraphics{images/9daefe84-e110-4c85-a8dd-fd77942ced0e-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Zeta-Potential distribution of G2S3 emulsion}}
\label{f-3c17e6db55fa}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/96b15ccc-a3ca-494c-a01e-2c056ca67561-upicture4.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/96b15ccc-a3ca-494c-a01e-2c056ca67561-upicture4.png}{\includegraphics{images/96b15ccc-a3ca-494c-a01e-2c056ca67561-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Effect of weight fraction (10-30) of argan oil on the time stability of O~/ W~emulsions prepared with respectively gelatin 2 w/w\% alone as emulsifier (G2S0); gelatin 2w/w\% +Span 80 (1 wt\%) (G2S1); gelatin 2w/w\% + Span 80 (3 wt \%) (G2S3) and gelatin2w/w\% + Span 80 (6 wt \%) (G2S6). Homogenizer speed 9500 rpm, 5 min}}
\label{f-a970b7711f18}
\end{figure*}
\egroup
\textbf{Particle size measurement}

To assess the effectiveness of each emulsifier in stabilizing argan oil emulsions, used alone or in combination, a measurement of the droplet size of stable emulsions, formulated with or without gelatin (G2S3; G2S6; G0S3; G0S6) was realized. The size of the droplets of the emulsion stabilized with 2w/w\% gelatin alone (G2S0) was also measured. This measurement was carried out immediately after emulsification, then after 1, 2, 3 and 4 weeks of storage at room temperature with dynamic light scattering Zetasizer 3000HS (Malvern Instruments, France instrument). From Figure~\ref{f-b5db21c3702d}, Evolution of the size of the droplets as a function of time of 10 wt \% Argan O/W emulsions prepared with respectively gelatin 2 w/w\% alone as emulsifier (G2SO); gelatin 2w/w\% + Span 80 (3wt\%) (G2S3); gelatin 2w/w\% + Span 80 (6 wt \%) (G2S6) and Span 80 alone 3wt\% (G0S3) and 6 wt \% (G0S6). Homogenizer speed 9500 rpm, 5 min.

The average droplet size of the emulsion formulated by gelatine alone (G2S0) is 2,308 \ensuremath{\mu }m, measured immediately after emulsification at T0; the evolution of this size is not shown on the graph because the emulsion became destabilized within a week.

By comparing this value with those obtained with the other emulsions, it was observed that at T0, the G2S0 emulsion exhibited the largest average droplet size. All other emulsions were less than 2,303 \ensuremath{\mu }m in size. This finding is in agreement with the results of the work carried out on gelatine used as an emulsifier. Gelatin alone as an emulsifier has been reported to produce relatively large droplets \unskip~\citep{922240:20671345}. In another study, the same authors confirmed that O/W emulsions obtained with fish gelatine contained a small fraction of relatively large droplets ( {\textgreater} 10 \ensuremath{\mu }m). They explained it by the relatively low surface activity of gelatin. Used alone, it showed the lowest capacity to reduce interfacial tension and it was not adsorbed efficiently to the surface of droplets \unskip~\citep{922240:20671345}.

In contrast, as shown by the curves in the figure above, the average particle size of emulsions prepared by span 80 alone was smaller than those formulated by gelatine alone due to the excellent adsorption rate of the surfactant (G0S3 \textless\ G0S6 {\textless}G2S0). Due to their smaller size and lower molecular weight, surfactants diffuse, adsorb, and stabilize an interface faster than proteins. The small size of the surfactant allows for higher droplet curvature (reduced steric hindrance) and globules can generally be smaller than with biopolymers \unskip~\citep{922240:20671346}. When gelatine was used with span 80, the average particle size of the emulsions was significantly reduced. For the combination (gelatin2\% + Span3\%), the mean particle size was the smallest (0,303 \ensuremath{\mu}m) with a narrow particle size distribution (Figure~\ref{f-84fe028c04e9}) and which did not exceed 1 \ensuremath{\mu}m after one month of storage at room temperature.

While for the other emulsions (G0S3-G0S6 and G2S6), the particle size was {\textgreater} 1 \ensuremath{\mu}m for the most part but it did not exceed 2.3 \ensuremath{\mu}m during the same period and under the same storage conditions as G2S3. So this comparison of argan oil emulsions formulated with 2w/w\% gelatin in the presence or not of span 80, allowed us to note that the size of the droplets decreases when the span and the gelatine are combined, whether at 3 or 6wt\% with a marked reduction in droplet size in the simultaneous presence of gelatine 2\% and span at 3\% (303 nm).

This improvement in stability in the simultaneous presence of 2w/w\% gelatine and 3\% span can be explained by a synergistic interaction at the level of the argan oil-water interface of the two emulsifiers. Our results also agree with a recent study carried out a few months ago on the mechanism of interaction of gelatine with low molecular weight surfactants\unskip~\citep{922240:20671344}. Instead of argan oil, they used fish oil and explored the preparation and storage of gelatin / surfactant stabilized emulsions. They demonstrated that gelatine and the two types of surfactants adsorb synergistically (Span 80 and soy lecithin) on the oil / water interfaces of emulsions \unskip~\citep{922240:20671344,922240:20671340}. 

Indeed, the behaviour of O/W emulsions stabilized by combinations of proteins and low molecular weight surfactants is controlled by the nature of the interactions between proteins and surfactants at the oil-water interface \unskip~\citep{922240:20671339,922240:20671342}. In this sense, several authors have intensively studied in recent years the interaction or the effect of the addition of the surfactant on the behaviour of O/W emulsions stabilized by proteins and the mechanisms of interfacial interactions of these two types of emulsifiers, especially in the food industry\unskip~\citep{922240:20671332,922240:20671338}. These studies reported that for the mechanism of this synergistic interaction, proteins have been shown to stabilize emulsions by forming a viscoelastic layer adsorbed on oil droplets, which form a physical barrier to coalescence \unskip~\citep{922240:20671332,922240:20671348}  while emulsifiers form a fluid and tight layer at the interface with low interfacial tension. This results in an emulsion with a small droplet size distribution \unskip~\citep{922240:20671332}. Another study \unskip~\citep{922240:20671347,922240:20671339} confirmed this mechanism and reported that in cases of coexistence of proteins and surfactants at the interface, surfactant molecules can fill in the gaps of the protein layers adsorbed or interacted with protein molecules, leading to a more interfacial, stronger and more viscoelastic layer \unskip~\citep{922240:20671335}. 

\textbf{Zeta Potential}

Zeta potential characterizes the surface charge of the droplets and reflects the repulsive force between the emulsion droplets (Wang Bo et al., 2020). The magnitude of the potential gives an indication of the potential stability of the dispersion system. Therefore, if the particles have high potential values, either positive or negative, the colloidal system will be stable ~ (Elgart A et al., 2012). Flocculation therefore occurs only in the case where the repulsion is minimum that is to say at zero zeta potential. In our case, we measured the zeta potential of the most stable emulsion G2S3 which was carried out at 25 \ensuremath{^\circ} C with a dynamic scattering of Zetasizer light 3000HS (Malvern Instruments, France instrument). The results are shown in Figure~\ref{f-3c17e6db55fa}.

According to this graph, the zeta potential of emulsion G2S3 was negative and it was found to be -23, 7 mV. This value reveals that the G2S3 emulsion droplets therefore have a fairly high zeta potential, which indicates the stability of the system since no aggregation is expected. The negative zeta potential can be attributed to the ionization of the carboxylic moieties (COOH) giving rise to carboxylate groups (COO-) \unskip~\citep{922240:20671349}. Particles with negative zeta potential repel each other very strongly. This is why colloids are very stable and inhibit any agglomeration. And it is this negative charge which gives the gelatine its stabilizing power in synergy with the Span. So there is an electrostatic repulsion, which is one of the main mechanisms of stabilization with proteins, and which plays a crucial role \unskip~\citep{922240:20671337}. The charge of the protein also plays a role in its adsorption at the interface. The more charged the protein, the more soluble it is in water and the higher the diffusion and adsorption rates at the interface \unskip~\citep{922240:20671341}. This finding showed that the gelatin in combination with span could be used as emulsifiers to provide better electrostatic repulsion between emulsion droplets \unskip~\citep{922240:20671343}. 

\textbf{Effect of protein concentration} \textbf{\space }

In the emulsions already formulated with gelatine concentrations of 0.5-1 and 2w / w\%, only those at 2 w/w\% which made it possible to obtain the most stable emulsions, and to determine the effect of an increase in gelatine concentration on the physical properties of argan emulsions, a series of argan O/W emulsions was prepared, always keeping the same amount of oil (10 wt \%) with a 4 w~/w \% solution of gelatine alone (G4S0); then by combining it respectively with Span 80 at 1 wt \% (G4S1); at 2 wt \% (G4S2); at 3 wt\% (G4S3) and at 6 wt \% (G4S6). The results of the observation of the stability properties of these emulsions during their storage at room temperature are presented in Table~\ref{tw-bf379909ab22}.

Only emulsions prepared by a combination (gelatin 4w/w\% + 3wt\% span) or (gelatin 4w/w\% + 6wt\% span) which remained stable for two weeks for G4S6 and one month for G4S3 at room temperature. G4S0 creamed 24 hours after preparation while G4S1 and G4S2 exhibited phase separation.

The emulsions formulated with 4w/w\% gelatin (G4S3 and G4S6) have droplets of sizes greater than 2 \ensuremath{\mu }m (G4S6{\textgreater} G4S3{\textgreater}2\ensuremath{\mu}m), therefore greater than those obtained with gelatine at 2w/w\% (G2S3 \textless\ G2S6 {\textless}2 \ensuremath{\mu }m); therefore, according to these results, for argan oil emulsions, the droplet size increases with increasing protein concentration. This can be explained as it has been reported by \unskip~\cite{922240:20671337},  that proteins can form one or more layers at the interface depending on their concentration in the aqueous phase, which can increase the size of the droplets.

\textbf{Effect of the amount of oil}

Argan O~/ W~emulsions were prepared with the 2 w/w (\%) gelatine solution as the sole emulsifier and then in combination with the span 80 at 1-3 and 6 wt\% respectively; but this time by increasing the quantity of oil to 20 and 30wt\%. The results obtained are illustrated in Figure~\ref{f-a970b7711f18}.

The figure shows that the stability time of the emulsions decreases when the quantity of argan oil increases and this regardless of the emulsifiers used (gelatin in the absence or presence of the surfactant). At 30wt\% argan oil, all emulsions prepared showed phase separation 1 day after emulsification. The most stable emulsions are those prepared with 10\% argan oil. This result is in agreement with the work of \unskip~\cite{922240:20671330}, who reported that the most stable emulsions prepared were at an argan oil fraction \textless\ 0,1. An increase in the oil fraction ( {\textgreater} 0.15) had a strong negative effect on the stability of the emulsion.

\textbf{Effect of the preparation protocol}

We kept the two emulsifiers: gelatin 2w/w\% and span 80 with argan oil still at 10wt\% and we changed the preparation protocol, we changed the order of addition of the surfactant; instead of adding it simultaneously with the gelatin, it was added after formulation of the emulsions with the gelatin. For this, the emulsions were prepared in two stages. First, argan O~/ W~emulsions were formulated with the 2w / w\% gelatin solution alone and 10 wt\% argan oil. After mixing the two phases with magnetic stirring at 400 rpm for one minute and homogenization at 9500 rpm / 5 min, span 80 (1-6 wt \%) was added to the emulsions already stabilized by gelatin. Then the emulsions were rehomogenized at the same speed (9500 rpm / 5min). 

The freshly prepared emulsions all had a milky and homogeneous appearance but their stability did not exceed 24 hours, even for the emulsion which exhibited the greatest stability with the first protocol (formulated with 2\% gel + 3\% span). This shows that the addition of span 80 after formulation and stabilization of an O~/ W~argan emulsion by a protein such as gelatine has caused its destabilization; this can be explained by a competitive interaction between the surfactant and the protein at the interface. In fact, by this second emulsification protocol, the span, perhaps, displaced the gelatine from the argan oil-water interface, this has been reported by several studies, for example that of \unskip~\cite{922240:20671332}, which predicts that in food emulsions there is generally a mixture of proteins and emulsifiers competing for the interfacial zone. This can produce a finer emulsion, however, the emulsifiers break down the viscoelastic layer adsorbed on the proteins, resulting in an emulsion with reduced stability \unskip~\citep{922240:20671332}.  It was also reported in another study \unskip~\citep{922240:20671347}, that the observed effects can be attributed to the gradual displacement of a high molecular weight component (protein), responsible for the elasticity of the emulsion, the stabilizing layers (interfacial) by the non-ionic surfactant \unskip~\citep{922240:20671347}.  According to the results of this test, the preparation protocol can be considered as a main factor in the type of interfacial interaction (competitive or synergistic) between the two emulsifiers gelatine and span 80 in the formulation of argan O/W emulsions.
    
\section{Conclusions}
This study shows that Span 80 can be used in combination with gelatine under suitable conditions to produce argan oil emulsions with good stability and a lower concentration of synthetic compounds. According to this study, the main factors influencing the formulation and stabilization of argan oil emulsions were the type of emulsifier, its concentration, the oil fraction and the preparation protocol. Finally, this association of argan oil with a natural biopolymer and a liposoluble surfactant can be exploited, for possible cosmetic, food or pharmaceutical applications because of the great stability of this oil, which can, in certain cases, be used substitute for other oils, in oil-in-water or water-in-oil emulsion preparations with a lifetime of which can be longer without the need to add antioxidants synthetic.

\textbf{Funding Support}

The authors declare that they have no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest for this study.
    

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