Improvement of the physical stability of oil-in-water nanoemulsions elaborated with Sacha inchi oil employing ultra-high-pressure homogenization

Journal Pre-proof Improvement of the physical stability of oil-in-water nanoemulsions elaborated with Sacha inchi oil employing ultra-high-pressure homogenization Maria C. Rave, Juan D. Echeverri, Constain H. Salamanca PII:

S0260-8774(19)30445-5

DOI:

https://doi.org/10.1016/j.jfoodeng.2019.109801

Reference:

JFOE 109801

To appear in:

Journal of Food Engineering

Received Date: 17 August 2019 Revised Date:

24 October 2019

Accepted Date: 2 November 2019

Please cite this article as: Rave, M.C., Echeverri, J.D., Salamanca, C.H., Improvement of the physical stability of oil-in-water nanoemulsions elaborated with Sacha inchi oil employing ultrahigh-pressure homogenization, Journal of Food Engineering (2019), doi: https://doi.org/10.1016/ j.jfoodeng.2019.109801. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Improvement of the Physical Stability of oil-in-water Nanoemulsions Elaborated with

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Sacha Inchi Oil employing Ultra-High-Pressure Homogenization

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Maria C. Ravea, Juan D. Echeverria and Constain H. Salamancaa,b*

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a

Programa de Maestría en Formulación de Productos Químicos y Derivados, Facultad de Ciencias Naturales, Universidad Icesi, Calle 18 No. 122 -135, Cali 76003, Colombia. Departamento de Ciencias Farmacéuticas, Facultad de Ciencias Naturales, Universidad Icesi. Cali-Colombia, Calle 18 No. 122 -135, Cali 76003, Colombia. *Corresponding author: [email protected].,du.co (CHS).

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ABSTRACT

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This study focused on assessing the physical stabilization of several oil-in-water

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nanoemulsions obtained by Ultra-High-Pressure Homogenization (UHPH). For this, several

15

formulations were developed using Sacha inchi oil (∼9.3% w/w), ultra-pure water,

16

preservatives (0.44% w/w) and several emulsifier mixtures at 2% w/w, which were

17

combined in different proportions to provide surfactant blends with HLB values (HLBB) of

18

6, 8, 10, and 12. The conventional emulsions were then subjected to UHPH (40,000 psi)

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and underwent thermal stability assays for 4 weeks, where changes in creaming index,

20

droplet size,

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showed the required HLB (HLBr) for SI oil was approximately 8 and when UHPH was

22

utilized, high physical stability of emulsified systems was achieved. It was also found that

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the increase in HLBB for the emulsions submitted to UHPH leads to a less viscosity,

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smaller droplet sizes and more homogeneous system.

polydispersity, viscosity and zeta potential, were evaluated. The results

25 26

Keywords: Ultra high-pressure homogenization (UHPH), sacha inchi oil, required HLB,

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emulsion physical stability, nanoemulsion.

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1. Introduction

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Emulsions are among the most common colloidal heterodisperse materials and are used

31

across different sectors of the economy. As such, they represent many products used in

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everyday life, including personal care creams, lipstick, and UV light protectors (i.e.,

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sunscreen) (Lu and Gao, 2010; Otto et al., 2009; Salager et al., 2004; Tadros, 2009). The

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variety of emulsified products is even greater in the foodstuff industry, with common

35

products including milk, butter, mayonnaise, ice cream, and sauces (BeMiller, 2008;

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Dalgleish, 2010; Dickinson, 1989; Ding et al., 2019; Marrs, 1986; Milani and Maleki,

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2012; Muschiolik, 2007; Williams, 2004). In the pharmaceutical industry, emulsions are

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one of the most common ways to formulate medicines with hydrophobic active ingredients

39

(Barkat Ali Khan, 2012; Kale and Deore, 2016; McClements et al., 2007). Despite their

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wide and prominent use, emulsions can exhibit poor physical stability, including droplet

41

aggregation (flocculation and coalescence), creaming, sedimentation, and separation of

42

phases (breaking of the emulsion) (French et al., 2015; Han et al., 2001; Klinkesorn et al.,

43

2004). Thus, investigating the physical stability of these systems is an important and

44

ongoing area of research in the design and formulation of new emulsified systems.

45

Currently there are several processes to develop emulsions with different

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homogenization degrees, depending on the type of equipment used. Some of these

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mechanical devices are the colloid mills and the ultra-turrax® type dispersion tools, which

48

have been widely used to achieve suitable homogenization degrees, when an adequate

49

process optimization is done previously (Trujillo-Cayado et al., 2017, 2016). There is also

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high-energy emulsification equipment such as sonicators and microfluidizers, which have

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shown an excellent capability to homogenize and reduce the sizes of the dispersed phase

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(Bai and McClements, 2016; Cano-Sarmiento et al., 2015; Maa and Hsu, 1999; Mahdi

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Jafari et al., 2006).

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Other methodology is the ultra-high-pressure homogenization (UHPH), which has

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proven to be a promising alternative to achieve high physical stability in emulsified systems

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(Cha et al., 2019; Floury et al., 2000; Patrignani and Lanciotti, 2016; Pereda et al., 2010;

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Poliseli-Scopel et al., 2012). In this technique, which uses high energy and pressure, the

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coarse emulsion passes through a narrow nozzle, causing high turbulence, high shear, and

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cavitation, and leads to multiple breaks in the dispersed droplets until it reaches a high

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homogeneity with small particle sizes (10–500 nm) (Floury et al., 2000). Thereby, UHPH

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induces major changes in the physical appearance (from cloudy to fine emulsions) and

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physicochemical properties (viscosity, interfacial tension) of the processed material

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(Briviba et al., 2016; Lee et al., 2009; Zamora et al., 2010). As such, this technique

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represents a very interesting strategy for the manufacture of emulsions on the nanometric

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scale with low size polydispersity and high stability.

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Sacha inchi (SI: Plukenetia volubilis L.), better known as the mountain peanut, is a

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perennial plant native to the Amazon, and its oil is projected to be an important raw

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material for various industries thanks to its chemical composition. The oil is rich in omega-

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3, 6, and 9 fatty acids (FAs) and has a high amount of polyunsaturated FAs—such as alpha

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linoleic acid (18:3) and linoleic acid (18:2)—which constitute 80%–85% of the FA content.

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Likewise, this oil contains other unsaturated FAs like oleic acid (18:1), and even saturated

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FAs like palmitic acid (16:0) and stearic acid (18:0) (Gutiérrez et al., 2011; Hanssen and

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Schmitz-Hübsch, 2011; Liu et al., 2014; Maurer et al., 2012; Wang et al., 2018). These

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compounds contribute to the exceptional nutritional of this oil, in addition to the presence

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of antioxidants, which are also very popular for use in pharmaceuticals, cosmetics, and

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personal care products.

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To date, only a handful of studies have focused on a detailed physicochemical

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characterization of emulsions and nanoemulsions elaborated with SI oil, and even fewer

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employing UHPH (Tunkam and Satirapipathkul, 2016). For this reason, the main objectives

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of the study are focused on two specific aspects, (i) the determination of the required

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hydrophilic-lipophilic balance (HLBr), which is an important physiochemical parameter

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commonly used on the development of oil-in-water type emulsions (Orafidiya and

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Oladimeji, 2002; Robbers and Bhatia, 1961), and (ii) determining if UHPH can stabilize

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emulsions better and easier than the required HLB parameter.

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2. Experimental section

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2.1 Materials

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SI oil was obtained from a small agricultural cooperative located in the Municipality of

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Santander de Quilichao, Department of Cauca-Colombia. The industrial extraction process

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involved mechanical seed pressing (cold pressing) followed by appropriate storage to

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prevent oil oxidation before commercialization. Other ingredients used in the emulsified

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formulations were Steareth 2 (BrijTM S2, HLB = 4.9, melting point = 42 °C–46 °C),

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Steareth 20 (BrijTM S20, HLB = 15.3, melting point = 56 °C–60 °C), Glyceryl Stearate

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(CithrolTM GMS, HLB = 3.8, melting point = 57 °C–60°C), Polyoxyl 40 Stearate (MyrjTM

96

S40, HLB = 17.5, melting point = 44 °C–47 °C), Sorbitan oleate (SpanTM 80, HLB = 4.3,

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melting point = 10 °C–12 °C), and Polysorbate 80 (TweenTM 80, HLB = 15, melting point

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= –21 °C) purchased at CRODA (Snaith, United Kingdom). Methylparaben and 5

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propylparaben were acquired from Sigma-Aldrich (St. Louis, MO, USA). Water Type II

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(ultra-pure water) was obtained from a Millipore Elix Essential purification system (Merck

101

KGaA, Darmstadt, Germany).

102

2.2 Physicochemical quality control and lipid composition profile of Sacha inchi oil

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Physicochemical characterization and analyses of the fatty acid methyl ester profiles of

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the SI oil were conducted using methods recommended by the American Oil Chemists´

105

Society (AOCS) (American Oil Chemists’ Society, 2017) and United States Pharmacopeia

106

USP (USP, 2018). Determination of the refractive index, the saponification value, the

107

peroxide value, the iodine value, and the acid index were carried out, respectively,

108

according to the following guidelines: AOCS Cc 7-25, AOCS Cd 3-25, AOCS Cd 8-53,

109

(AOCS Cd 1c-85), and USP 40 <401>. Determination of the FA methyl ester profiles was

110

performed according to AOCS Ce 1-62. To determine the refractive index, a Refractometer

111

(VEE GEE Scientific Abbe Model C10, Vernon Hills, IL, USA.) was used. On the other

112

hand, the determination of the fatty acid methyl ester profiles was carried out according to

113

guideline AOCS Ce 1-62. Concerning refractive index, a refractometer Vee Gee C10 was

114

used. The analysis of fatty acid employed a gas chromatograph (Hewlett Packard HP 5890

115

– Series II, Palo Alto, CA, USA) equipped with a flame ionization detector and a BPX70-

116

ms capillary column (30 m x 0.25 mm x 0.25 µm) composed of 70%

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cyanopropylpolysilphenylene-siloxane. The initial temperature was 150 °C/min, which

118

increased by 5 °C/min up to 240 °C. The injector temperature was 240 °C and the detector

119

temperature was 280 °C, with a split ratio of 1:30. The carrier gas used was He at 1

120

mL/min, at a pressure of 11 psi.

121

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2.3 Elaboration of conventional emulsion and nanoemulsion

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Several heterodisperse formulations were prepared using SI oil, ultra-pure water,

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preservatives (methylparaben and propylparaben) (Fransway et al., 2019; Soni et al., 2005),

125

and binary mixtures of different type of surfactants at 2% w/w. These were combined in

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several proportions to provide hydrophilic-lipophilic balance values of mixture (HLBB) of

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6, 8, 10 and 12, as shown in Table 1.

128

Each emulsified system was prepared in triplicate in several stages. First, the SI oil and

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ultra-pure water were heated to 60 °C and 62 °C, respectively. Once the target temperatures

130

were reached, the preservatives were added to the ultra-pure water (aqueous phase), while

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the different binary surfactant blends (Steareth 2 and Steareth 20; Glyceryl Stearate and

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Polyoxyl 40 Stearate; or Sorbitan oleate and Polysorbate 80 at ratios described in Table 1

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were added to the SI oil (oily phase). Afterward, the oily phase was poured into the aqueous

134

phase and homogenized using an Ultra-Turrax homogenizer at 5,000 rpm for 10 min.

135

Subsequently, the system was cooled to room temperature to obtain the conventional

136

emulsions. The HLB of the blend of surfactants (HLBB), consisting of fraction x of A and

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(1-x) of B is assumed to be the algebraic mean of the two HLB numbers. This parameter

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was calculated according to the as follows:

139

‫ ܤܮܪ‬஻ = ‫ܤܮܪݔ‬௔ + ሺ1 − ‫ݔ‬ሻ‫ܤܮܪ‬௕

140

(1)

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where HLBB is the value of the binary surfactant blend and HLBa and HLBb are the HLB

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values of the respective surfactants according to their technical sheets.

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Nanoemulsions 7

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Once the conventional emulsions were obtained, 600 g of each was subjected to

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homogenization by ultra-high-pressure using a Nano DeBEE Laboratory Homogenizer

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(BEE international, South Easton, MA, USA). The operating conditions employed were:

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Zirconia nozzle with an orifice diameter of 0.20 mm, six zirconia reactors with orifice

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diameter of 1.75 mm, a pressure of 40,000 psi (2757.9 MPa) and a reverse flow

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configuration, with a total of four recirculation cycles. These conditions were previously

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established via a series of tests carried out before formulation of the nanoemulsions.

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2.4 Thermal stability assays of SI emulsions

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Each conventional emulsion and nanoemulsion was placed in a Falcon™ 15 mL conical

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centrifuge tube, which was subsequently incubated in one of two temperature conditions:

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40 ± 2 °C or 4.0 ± 0.5 °C. The stability test was carried out varying the temperature for four

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weeks. First, the samples were subjected to 40 °C during the first week, subsequently at 4

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°C during the second week, then at 40 °C during the third week and finally at 4 °C during

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the fourth week. The stability parameters evaluated were creaming index (CI), drop size,

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viscosity, zeta potential, electrical conductivity, and pH.

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2.4.1 Creaming index

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Fifteen-mL conical centrifuge tubes (Falcon™, diameter = 1.5 cm) were filled with

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freshly made emulsions and centrifuged at 3,000 rpm (150 RFC) for 4 h in a Wincon 80-2

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centrifuge (Changsha, China). The CI was calculated as

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CI =

HS × 100 HE

(2)

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where HS is the sediment height and HE is the sample height before centrifugation.

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2.4.2 Droplet size

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For the conventional emulsions, droplet size distribution was obtained using a

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Mastersizer 3000 (Malvern Instruments, Worcestershire, UK) equipped with a helium/neon

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laser at a wavelength of 632.8 nm, where ∼0.6 g of the emulsion was previously diluted

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with 10 mL of ultra-pure water at 25 °C ± 2 °C and stirred at 400 rpm before carrying out

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the measurement. The appropriate amount of sample was obtained when the obscurance

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level reached 2%–8%. Droplet size data were expressed as D[4,3] (Barth and Sun, 1993;

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Picandet, 2017; Scott, 2010). For the nanoemulsions, particle size and polydispersity index

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(PDI) were determined using a Zetasizer nano ZSP (Malvern Instrument, Worcestershire,

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UK) with a red helium/neon laser (633 nm), where 10 µL of each sample was dissolved in

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10 mL of distilled water. The particle size was measured using dynamic light scattering

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with an angle scattering of 173°, using a quartz flow cell (ZEN0023) at 25 °C. The

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instrument reports the particle size as the mean particle diameter (z-average) and PDI

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ranging from 0 (monodisperse) to 1 (very broad distribution). All measurements were

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performed in triplicate.

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2.4.3 Viscosity

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Viscosity was measured using a viscometer (micro-visc, RheoSense Inc., San Ramon,

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CA, USA), applying different shear stress (See support material file). All measurements

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were performed in triplicate.

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2.4.4 Zeta potential, electrical conductivity and pH measurements

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Zeta potential measurements were carried out using a zetasizer nano ZSP (Malvern

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Instruments, Worcestershire, UK) at 25 °C ± 2 °C, with equilibration times of 120 s in a

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DTS 1070 capillary cell. For these experiments, the attenuator position and intensity were

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set automatically. The samples were prepared using ∼130 mg of the emulsified material,

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which was diluted in 20 mL of ultra-pure water and manually stirred. A 50 µL aliquot was

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taken and diluted with 1 mL of ultra-pure water before each zeta potential measurement.

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Conversely, the electrical conductivity and the pH were determined using a CR-30

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conductivity meter and a Starter-2100 pH meter, respectively. All measurements were

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performed in triplicate.

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2.4.5 Graphs and statistical analysis

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The determination of the average values, the standard deviations and the graphs were made

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using the GraphPad prism 8 software.

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3. Results and discussion

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3.1. Physicochemical quality control and lipid composition profile of SI oil

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The chemical composition of oils can change based on various factors, such as genetic

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variety of the plant, farming techniques, environmental conditions (such as sun and rain),

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the type and amount of nutrients provided during growing, as well as techniques used for

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extraction, packaging, and storage. Before starting any study that involves the use of oil-

10

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like raw materials, it is common and best practice to perform detailed physicochemical

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quality control, thus ensuring that further study is useful and reliable. Moreover, this type

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of characterization can confirm that the obtained parameters agree with those reported in

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the technical sheet and identify if there are any adulterations in the oil. The results of the

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physicochemical characterization and fatty acid profile of the SI oil are summarized in

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Table 2, and were very similar to those previously reported (Niu, Li, Chen, & Xu, 2014;

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Saengsorn & Jimtaisong, 2017; Vicente, De Carvalho, & Garcia-Rojas, 2015) and listed in

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the oil data sheet.

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The results of refractive index, saponification value, peroxide value, iodine value, acidity

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index, amount of saturated fat (6.87%), amount of monounsaturated fat (9.73%), and

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amount of polyunsaturated fat (83.40%), as well as the composition of omega-3 (48.39%),

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omega-6 (35.01%), and omega-9 (9.64%) were very similar to the previously reported

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values for this oil (Vicente et al., 2015), suggesting that Sacha inchi oil used in this study

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has high purity.

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3.2 Thermal stability of SI emulsions

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An important aspect to highlight in these formulations is the proportion of SI oil (9.3%),

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the low proportion of emulsifiers (2%), and the absence of other stabilizing ingredients.

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These factors ensured that the obtained results depended exclusively on the formulation

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HLBB and the UHPH process, and not other factors. The results of the thermal stability

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assays are presented and discussed below, according to each parameter evaluated.

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3.2.1 Creaming index

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Along with thermal stability, CI is one of the most-employed physicochemical

239

parameters in the initial stages of developing emulsified products (preformulation). Assays

240

assess aggregation of the internal phase and separation of phases, which are extremely

241

useful parmeters for characterizing formulations and process conditions. Specifically, CI

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assays can quickly demonstrate whether formulations are optimal, particularly with regard

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to the amount of surfactant blend used. When a mixture of surfactants results in very low

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CI values, it is possible that the formulation is close to the desired stability zone, and the

245

HLBB value therefore matches the required HLB of the oil. CI results for the emulsified

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systems at different times and process conditions are shown in Fig. 1.

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For the conventional emulsions, the lowest CI values were achieved with HLBB = 8,

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except in system 1, where it was 10. In contrast, results from the emulsified systems passed

249

through UHPH suggest that CI is not affected by HLBB.

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With respect to the conventional emulsions of system 1, it was found that the lowest CI

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values at time zero were 4.3 ± 0.1 (HLBB = 8) and 12.0 ± 0.1 (HLBB = 10). However, over

252

the weeks of the thermal stability test, the only emulsion that remained stable (without

253

breaking) was that with a HLBB of 10, where the CI was 22.0 ± 0.1. For this reason, the

254

characterization of the emulsions of system 1 were carried out only for those with HLBB =

255

10. In contrast, systems 2 and 3 showed similar behavior, where emulsions with HLBB

256

values of 8 displayed CI values of 0 at time zero and 37.5 ± 1.5 and 14.7 ± 1.0 at the fourth

257

week, respectively. These results suggest that the HLBB value necessary to attain adequate

258

stabilization of emulsions elaborated with SI oil is around 8. This is very similar to a

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previously reported value (Saengsorn and Jimtaisong, 2017), where it was found that the

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required HLB value for SI oil was 8.5. However, it should be mentioned that the previous

261

study employed different conditions in this study, including a lower oil proportion phase 12

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(5% w/w), a higher concentration of emulsifiers (5% w/w), and incorporation of a

263

viscosifying agent as a co-stabilizer.

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The emulsions that underwent UHPH showed different and interesting results.

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Regardless of the duration in the thermal stability tests or the HLBB values of the materials

266

used in the formulation, these emulsions always achieved high physical stability. For

267

system 1, the emulsion with a value of HLBB of 6 presented a CI of 13.0 ± 1.1 at time zero,

268

while the emulsions with HLBB values of 8, 10, and 12 had CI values of zero. For system 2

269

at time zero, all CIs were zero; however, over the weeks of the thermal stability test, this

270

parameter increased, and was higher for emulsions with HLBB values of 6 and 8 and lower

271

in emulsions with HLBB values of 10 and 12. On the contrary, system 3 always reflected CI

272

values of zero, independent of HLBB values and the conditions employed in thermal

273

stability tests. These results are important because they suggest that exposing emulsions to

274

UHPH can prevent phase separation, one of the most common causes of emulsion

275

destabilization (Ivanov and Kralchevsky, 1997; Osipow et al., 1957).

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3.2.2 Drop Size and PDI The results of droplet sizes (D[4,3]) for conventional emulsion and z-average and PDI for

279

nanoemulsion at different times and process conditions are shown in Fig. 2.

280

The conventional emulsions displayed similar behaviors to those observed in the CI studies,

281

where the lowest droplet sizes were obtained with HLBB values of 8 and 10. These results

282

can be explained due to the formation of a compact interfacial film, which compresses the

283

droplet and reduces the internal phase size (Tabor, 1977; Yadav et al., 2008). For system 1

284

at time zero, the droplet sizes were 2.5 ± 0.2 µm and 3.3 ± 0.2 µm for the emulsions with

285

HLBB values of 8 and 10, respectively. However, over time, the emulsion with HLBB 13

286

values of 8 separated phases, while the emulsion with HLBB values of 10 changed the sizes

287

to 2.7 ± 0.2 µm. In contrast, systems 2 and 3 described a similar trend, where the drop size

288

increased. For emulsions with an HLBB value of 8 (at time zero) showed droplet sizes of

289

1.3 ± 0.1 µm and 2.6 ± 0.1 µm, respectively. These values increased over the 4 weeks of

290

the stability test to 11.9 ± 0.7 µm and 4.9 ± 0.1 µm. These results are consistent, taking into

291

consideration that thermal shock tests are designed to destabilize heterodisperse systems by

292

promoting migration of surfactants from interfacial areas. This leads to droplet aggregation

293

followed by coalescence, and finally, breaking of the emulsion.

294

On the contrary, the emulsions that were passed through UHPH showed a notable

295

decrease in droplet sizes (z-average) in all cases (between 112–441 nm) and lower PDI

296

values (0.05–0.29) with increasing HLBB values, which is why these systems were referred

297

to as nanoemulsions (Fig. 2). However, over time, a slight increase in droplet size was

298

observed, which was greater in emulsions with HLBB values of 6 and 8 than in those with

299

HLBB values of 10 and 12. These behavior is very similar to those observed in the CI

300

assays, suggesting that UHPH produces a new configuration in the heterodisperse system.

301

Accordingly, the effects of shear, cavitation, and impact generated during the process led to

302

a notable decrease in the internal phase of the emulsion, along with high uniformity.

303

Similarly, this technique also causes better incorporation of hydrophilic surfactants at the

304

oil-water interface, where their larger polar heads (compared to their hydrophobic regions)

305

lead to more compact and efficient interfacial films (Ivanov and Kralchevsky, 1997;

306

Ruckenstein, 1999).

307 308

3.2.3 Viscosity

14

309

The results of viscometric assays for the emulsified systems at different times and with

310

different process conditions are shown in Fig. 3.

311

In general, the results agree with those previously obtained in the CI and particle size

312

assays, where for conventional emulsions, the maximum viscosity was attained with a

313

HLBB value of 8. This suggests that a compact and organized interfacial film of emulsifiers

314

is generated at this HLBB value. In this case, the polyethoxylated groups of the hydrophilic

315

surfactants (Steareth 20, polyoxyl-40-stearate and polysorbate 80) form hydrogen bonds

316

that increase cohesiveness in the system and thus, increased viscosity. The differences

317

observed in viscosities of the different emulsions can be explained by the physicochemical

318

characteristics of the surfactants used (Ivanov et al., 1999; Klinkesorn et al., 2004; Pal,

319

1996).

320

For emulsions in systems 1 and 2 with HLBB values of 8 at time zero, the viscosity

321

values were 54.00 ± 0.60 cP and 5.50 ± 0.04 cP, respectively, and higher than for system 3

322

(1.50 ± 0.03 cP). This may be due to the physical state of the surfactants employed at room

323

temperature (25±1 °C). In the case of the surfactants used in systems 1 and 2 (Steareth

324

2/Steareth 20 and Glyceryl Stearate/Polyoxyl 40 Stearate), the emulsifiers are wax-like

325

solids, while the surfactants of system 3 (sorbitan oleate/polysorbate 80) are liquids. Hence,

326

during the elaboration of the conventional emulsions, and specifically in the cooling stages,

327

the surfactants solidified, turning the systems more viscous.

328

In the thermal stability assays of the conventional emulsions, different behaviors were

329

observed depending on the mixture of surfactants used. For system 1, the emulsion with a

330

HLBB of 8 displayed the highest viscosity, as described above. However, this emulsion

331

separated phases over time, while the emulsion with a HLBB of 10 remained stable, as

332

previously described in the CI assays. In these emulsions, different viscosity values were 15

333

observed over time, going from 29.00 ± 0.60 cP at time zero to 7.60 ± 0.14 cP at week two,

334

and finally to 10.80 ± 0.60 cP by the final week. In contrast, the emulsion of system 2 with

335

an HLBB of 8 displayed a fluctuating behavior, decreasing from 5.50 ± 0.04 cP at time zero

336

to 2.00 ± 0.16 cP at week two, and then increasing to 3.70 ± 0.16 cP by the fourth week.

337

The emulsion of the system 3 with an HLBB of 8 also exhibited different behavior, with a

338

slight increase in viscosity from 1.50 ± 0.03 cP to 2.30 ± 0.06 cP at week two, and then

339

remaining essentially constant at 2.40 ± 0.03 cP until the final week. These behaviors are

340

consistent considering the nature of the thermal shock assay, where the system is repeatedly

341

heated and cooled over four weeks. Various situations may occur under these conditions,

342

including fugacity of air bubbles incorporated during the dispersion, melting and

343

solidification of the wax-type surfactants, migration of hydrophilic surfactants from the

344

interface zone to the dispersed bulk phase, and precipitation of the hydrophobic surfactant.

345

All these situations could affect in different ways and degrees the rheological behaviors of

346

the conventional emulsions and could therefore account for behaviors without specific

347

trends.

348

On the other hand, the viscosity results for the nanoemulsions obtained by UHPH

349

showed more defined trends, with nanoemulsions with an HLBB of 6 reaching maximum

350

viscosities. Similarly, nanoemulsions with HLBB values of 6 and 8 (less hydrophilic) were

351

more viscous than those with HLBB values of 10 and 12 (more hydrophilic). This result is

352

interesting and is related to results observed for particle size and PDI, where the emulsified

353

systems with HLBB values of 6 and 8 showed larger particle sizes than the emulsions with

354

HLBB values of 10 and 12. This result confirms that UHPH actually leads to the formation

355

of a new configuration of the heterodisperse system, where hydrophilic surfactants are able

16

356

to generate more compact interfacial films and thus, the dispersing phase can flow more

357

easily (i.e., with less viscosity).

358 359 360

3.2.4 Zeta potential The results of zeta potential assays for the emulsified systems at different times and with

361

different process conditions are shown in Fig. 4.

362

The zeta potential results for the conventional emulsions and nanoemulsions in this study

363

are interesting because all the emulsifiers utilized were neutral and the expected values

364

should be close to zero. On the contrary, all the zeta potential values obtained were

365

negative. These results can be explained due to the spontaneous formation of a tiny

366

monolayer of hydroxyl ions at the oil-surfactant-water interface, which results from

367

autoprotolysis of water (Gao et al., 2014; Marinova et al., 2002, 1996). It is important to

368

highlight that the zeta potential results did not correlate with the type of emulsion

369

(conventional versus nanoemulsion), the HLBB values, or the thermal stability test

370

conditions.

371 372 373

3.2.5 Electrical conductivity The results of electrical conductivity assays for the emulsified systems at different times

374

and with different process conditions are shown in Fig. 5.

375

These results can be attributed to the presence of hydronium and hydroxyl ions from the

376

autoprotolysis of water, as well as the ions formed from the ionization of the carbonic acid

377

in the external phase. Different electrical conductivity behaviors were observed depending

378

on the emulsification system. For system 1, the conventional emulsions with HLBB values

379

of 8 and 10 at time zero presented the lowest electrical conductivity, with values of 4.4 ± 17

380

0.2 µS/cm and 3.8 ± 0.3 µS/cm, respectively. Among these emulsions, those with a HLBB

381

value of 10 showed that the electrical conductivity increased to 8.1 ± 0.5 µS/cm at the

382

fourth week. Conversely, the emulsions of systems 2 and 3 with a HLBB values of 8

383

displayed a decrease in electrical conductivity between the initial time and the final week,

384

going from 38.0 ± 0.2 µS/cm to 20.1 ± 0.9 µS/cm and from 57.1 ± 0.3 µS/cm to 38.0 ± 0.8

385

µS/cm, respectively.

386 387

Regarding to the nanoemulsions, the electrical conductivity showed different behaviors

388

from the conventional emulsions and among themselves. The nanosystems displayed

389

diverse behaviors depending on the system components and the HLBB values. For system 1

390

at time zero, the nanoemulsions with HLBB values of 6 and 8 displayed lower conductivity

391

(9.7 ± 0.6 µS/cm and 4.4 ± 0.2 µS/cm, respectively) than those with HLBB values of 10 and

392

12 (10.7 ± 0.3 µS/cm and 17.7 ± 0.5 µS/cm, respectively). On the contrary, for systems 2

393

and 3 at time zero, electrical conductivity decreased with an increase in HLBB values, and

394

there were no changes with respect to time in the thermal study. Therefore, the electrical

395

conductivity results for the conventional emulsions and nanoemulsions were very

396

consistent, taking into account the previous viscosity results where the emulsified systems

397

with high viscosity displayed lower electrical conductivity and vice versa. This result can

398

be explained by to the ion mobility in the dispersed phase, which move less with respect to

399

the rise in viscosity, according to the well-known Fick diffusion equation. (Balluffi et al.,

400

2006; Fick, 1855; Griskey, 2002)

401 402

3.2.6 pH

18

403

The results of pH study for the emulsified systems at different times and with different

404

process conditions are shown in Fig. 6.

405

The results of pH study at zero-time for each emulsified and nano-emulsified system were

406

very similar to each other, with values between 4.99 and 5.11. Such acidic values can be

407

attributed to two factors, (i) the chemical nature of some ingredients utilized in the

408

formulation and (ii) the acidification of the dispersing phase by the formation of carbonic

409

acid. In the first case, the preservatives used were alkyl esters of p-hydroxybenzoic acid,

410

which have a phenol substituent that can be ionized and therefore, decrease the pH of the

411

dispersing phase.

412

In contrast, the dispersion process of the oily phase in the aqueous phase can lead to the

413

incorporation of air bubbles, which in turn can contain CO2 (g) that is transformed to

414

carbonic acid in contact with water (Mook, 2000). Such effect could be evidenced by the

415

change in the pH of two ultra-pure water samples, where one was subjected to ultra-turrax,

416

while the other was not. The results were convincing because it was found that the sample

417

of water subjected to ultra-turrax had a more acidic pH than that sample not subjected.

418

Similarly, the pH of all emulsified and nano-emulsified systems decreased over the weeks,

419

which can be explained by the possible degradation of the alkyl parabens used in the

420

formulations. Such preservatives have shown susceptibility to acid hydrolysis, where the

421

alkyl ester group breaks forming benzoic acid, which can acidify the dispersing aqueous

422

phase of emulsions (Blaug and Grant, 1974; Halla et al., 2018). Although a similar

423

degradation effect could also be considered with the surfactants employed or with the

424

esterified fats present in sacha inchi oil; their high hydrophobicity degree would strongly

425

limit such degradation processes. Therefore, the observed changes in the pH of emulsions

19

426

and nanoemulsions could be attributed exclusively to the preservatives used and the

427

formation of carbonic acid.

428 429

4. Conclusions

430 431

The physicochemical results and quality control assays for the Sacha inchi oil were very

432

similar to those previously reported, indicating that the raw material was of high purity and

433

not adulterated. Likewise, the fatty acid composition of the SI oil, and especially for the

434

omega-3 (48.39%), omega-6 (35.01%) and omega-9 (9.64%) FAs, was very similar to that

435

reported on the provided data sheet. On the other hand, the HLBB value that led to a

436

maximum stabilization of the conventional emulsions was around 8. In relation to the

437

different mixtures of surfactants tested, the blend of Sorbitan 80 and Polysorbate 80

438

displayed the greatest ability to stabilize the oil-in-water emulsions prepared with SI oil.

439

The emulsifications subjected to UHPH displayed a decrease in size at nanometric scale

440

(between 100–500 nm), where emulsions with HLBB values of 6 and 8 presented larger

441

sizes and PDI than the emulsions with HLBB values of 10 and 12. It was also observed that

442

all the emulsions had negative zeta potential values, despite using only non-ionic

443

components. In addition, the emulsions showed different viscosities depending on the type

444

of surfactant employed and the HLBB values. On the other hand, the changes in pH

445

observed in emulsions and nano emulsions can be attributed to two possible effects

446

corresponding to the hydrolytic degradation of parabens and the formation of carbonic acid

447

in the dispersing aqueous phase.

448

Based on the results, it is possible to establish a starting point for the design of several

449

advanced formulations that could be applied in various economic sectors. Considering its 20

450

exceptional features, this oil will undoubtedly become an interesting raw material for the

451

development of new products in the coming decades. In the specific case of Colombia, this

452

oil may also be an excellent alternative to illicit crops, as it has high economical potential

453

and could therefore contribute to easing of the great socio-economic problems of Colombia.

454 455

Acknowledgments

456

This work was supported by the Icesi University (Internal grant No CA041368). The

457

authors thank to Nutresacha S.A. company from Colombia to provide the Sacha inchi Oil

458

used in this study.

459 460

Conflict of interest

461

The authors declare no conflict interest.

462

21

463

References

464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512

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24

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25

630 631

Table 1 Formulations of oil-in-water emulsion elaborated with SI oil. System HLB

632 633 634

Sacha inchi oil (%)

Preservatives

Surfactants blend at 2%

% Steareth 2 Steareth 20 water mp + pp (%) (%) (%) 6 1.79 0.21 8 1.40 0.60 1 9.30 0.30 + 0.14 q.s. 10 1.02 0.98 12 0.63 1.37 Glyceryl Polyoxyl 40 Stearate Stearate (%) (%) 6 1.67 0.33 8 1.38 0.62 2 9.30 0.30 + 0.14 q.s. 10 1.08 0.92 12 0.79 1.21 Sorbitan Polysorbate oleate (%) 80 (%) 6 1.68 0.32 8 1.31 0.69 3 9.30 0.3 + 0.14 q.s. 10 0.93 1.07 12 0.56 1.44 All % represent weight/weight, q.s.= quantity sufficient, mp =methylparaben, pp = propylparaben B

26

635 636

Table 2 Results of physicochemical characterization and fatty acid methyl ester profiles of SI oil Physicochemical parameter Value Refractive index 1.4810 Saponification value (mg KOH/g) 251.72 Peroxide value (meq O/kg) 14.77 Iodine value (g I2/100g) 195.05 Acid index (% oleic acid) 1.31 Profile of lipid composition (%w/w) Common name/shorthand IUPAC Name Value Myristc acid / C14:0 Tetradecanoic acid 0.02 Palmitic acid / C16:0 Hexadecanoic acid 3.89 Palmitoleic acid / C16:1(n-7) (Z)-hexadec-9-enoic acid 0.06 Heptadecanoic acid / C17:0 Heptadecanoic acid 0.07 Heptadecanoic acid / C17:1(n-7) (Z)-heptadec-9-enoic acid 0.03 Stearic acid / C18:0 Octadecanoic acid 2.80 Oleic acid / C18:1(n-9) (Z)-octadec-9-enoic acid 9.34 Linoleic acid (LA) / C18:2(n-6) (9Z,12Z)-octadeca-9,12-dienoic acid 35.01 Linolenic acid (ALA)/ C18:3(n-3) (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid 48.39 Arachidic acid / C20:0 Icosanoic acid 0.07 Gadoleic acid / C20:1(n-9) (Z)-icos-9-enoic acid 0.30 Behenic acid / C22:0 Docosanoic acid 0.02

637 638

27

639

Figure captions index

640 641

Fig. 1. Creaming index (CI) of oil-in-water emulsions and elaborated with SI oil at different

642

HLBB values and times of thermal stability. BE indicates that there is no value due to the

643

breaking the emulsion. (some pictures about the emulsified and nanoemulsified systems at

644

zero and final time are shown on the support material file).

645 646

Fig. 2. Droplet size of oil-in-water emulsions, z-average and and polidepersity index (PDI)

647

of nanoemulsions elaborated with SI oil at different HLBB values and times of thermal

648

stability. BE indicates that there is no value due to the breaking the emulsion.

649 650

Fig. 3. Viscosities of oil-in-water emulsions and nanoemulsions elaborated with SI oil at

651

different HLBB values and times of thermal stability. BE indicates that there is no value due

652

to the breaking the emulsion.

653 654

Fig. 4. Zeta potential of oil-in-water emulsions and nanoemulsions elaborated with SI oil at

655

different HLBB values and times of thermal stability. BE indicates that there is no value due

656

to the breaking the emulsion

657 658

Fig. 5. Electrical conductivity of oil-in-water emulsions and nanoemulsions elaborated with

659

SI oil at different HLBB values and times of thermal stability. BE indicates that there is no

660

value due to the breaking the emulsion

661 662

Fig. 6. pH of oil-in-water emulsions and nanoemulsions elaborated with SI oil at different

663

HLBB values and times of thermal stability. BE indicates that there is no value due to the

664

breaking the emulsion

665

28

666

667 668 669

Fig. 1

29

670 671 672

Fig. 2

673 674

30

675 676 677

Fig. 3

678

31

679 680

681 Fig. 4

682

32

Zeta potential (mV)

Zeta potential (mV)

683 684

685

Fig. 5

686

33

Conductivity ( S/cm)

Conductivity ( S/cm)

Conductivity ( S/cm)

Conductivity ( S/cm)

Conductivity ( S/cm)

Conductivity ( S/cm)

Conductivity ( S/cm)

pH

pH

pH

pH

pH

687 688 689

Fig. 6

690

34

Highlights

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The required HLB of sacha inchi oil is approximately 8

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The UHPH increases considerably the physical stability of oil-in-water emulsion

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The required HLB value for sacha inchi oil is not necessary when using UHPH

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The increase in HLBB in nanoemulsions leads to less viscosity, smaller and more homogeneous systems