Accepted Manuscript

Comparison of natural and synthetic surfactants at forming and stabilizing

nanoemulsions: Tea saponin, Quillaja saponin, and Tween 80

Zhenbao Zhu, Ying Wen, Jianhua Yi, Yungang Cao, Fuguo Liu, David Julian
McClements

PII: S0021-9797(18)31212-8
DOI: https://doi.org/10.1016/j.jcis.2018.10.024
Reference: YJCIS 24179

To appear in: Journal of Colloid and Interface Science

Received Date: 4 September 2018

Revised Date: 9 October 2018
Accepted Date: 10 October 2018

Please cite this article as: Z. Zhu, Y. Wen, J. Yi, Y. Cao, F. Liu, D. Julian McClements, Comparison of natural and
synthetic surfactants at forming and stabilizing nanoemulsions: Tea saponin, Quillaja saponin, and Tween 80,
Journal of Colloid and Interface Science (2018), doi: https://doi.org/10.1016/j.jcis.2018.10.024

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Comparison of natural and synthetic surfactants at forming

and stabilizing nanoemulsions: Tea saponin, Quillaja saponin,

and Tween 80
a, * a a a c
Zhenbao Zhu , Ying Wen , Jianhua Yi , Yungang Cao , Fuguo Liu ,

David Julian McClements b, *

a
School of Food and Biological Engineering, Shaanxi University of Science and

Technology, Xi’an, Shaanxi 710021, PR China

b
Department of Food Science, University of Massachusetts, Amherst, MA 01003,

United States
c
College of Food Science and Engineering, Northwest A&F University, Yangling

712100, PR China

* Corresponding authors

Email: zhuzhenbao@sust.edu.cn

mcclements@foodsci.umass.edu

Journal: Journal of Colloid and Interface Science

Submitted: September 2018

1
Abstract
Hypothesis: This study compared the interfacial and emulsification properties of

tea saponins, quillaja saponins, and Tween 80. We hypothesized that tea saponins are an

effective and sustainable source of plant-based emulsifiers that could replace synthetic

or animal-based emulsifiers in many commercial applications.

Experiments: Interfacial tension measurements were used to characterize the

behavior of the three surfactants at an oil-water interface. The emulsifying properties

of the surfactants were determined by preparing oil-in-water emulsions containing 10

wt% medium chain triglycerides (MCT) and varying surfactant levels (0.1-2 wt%)

using high-pressure homogenization (pH 7). The impact of surfactant type on

emulsion formation and stability was determined by measuring particle size,

zeta–potential, microstructure, and creaming stability.

Findings: The tea saponins were capable of producing nano-scale droplets (d32 <

200 nm) at low surfactant-to-oil ratios (SOR < 0.1). Emulsions containing tea saponins

remained stable to droplet aggregation when exposed to various temperatures (30-90℃),

salt levels (0-200 mM NaCl), and pH values (3-9). However, droplet flocculation

and/or coalescence occurred under highly acidic (pH 2) and high ionic strength

(300-500 mM NaCl) conditions. Tea saponin-coated oil droplets appeared to be

mainly stabilized by a combination of electrostatic and steric repulsion. The tea

saponins behaved similarly or better than the other two emulsifiers under most

conditions. These results suggest that tea saponins are effective plant-based

surfactants that may have applications in commercial products.

Keywords: Tea saponin; quillaja saponin; biosurfactant; nanoemulsion; Tween 80

2
1. Introduction
Saponins are secondary metabolites found in a majority of plant species [1,2].

Many saponins are surface-active because they are comprised of one or more

hydrophilic sugar moieties covalently attached to a hydrophobic steroid or triterpene

backbone [3]. Recently, there has been considerable interest in using saponins as

natural emulsifiers due to their ability to efficiently form and stabilize emulsions [4].

Until now, most of the studies in this area have focused on extracts isolated from the

Quillaja Saponaria Molina tree [5]. Indeed, these extracts are already commercially

available for application in the food and other industries. Numerous researchers

have reported that quillaja saponin (QS) is an efficacious emulsifier for forming and

stabilizing oil-in-water (O/W) emulsions and nanoemulsions. Yang et al. reported that

QS formed nanoemulsions (d<150 nm) with good physical stability over a range of

environmental conditions [6]. QS has also been used to formulate orange oil

nanoemulsions (d<100 nm) with low turbidities, which are suitable for application in

beverage products [7]. More recently, Tippel et al. reported that QS had a high surface

activity and could be used to solubilize hydrophobic carotenoids, such as lutein esters,

in aqueous solutions [8].

Although QS has proved to be an effective emulsifier, there is interest in

identifying alternative sources of saponins because of their potential benefits in terms

of taste, cost, or sustainability. The genus Camellia Lutchuensis belongs to the

family Theaceae Mirbel, which comprises of more than 325 species [9]. Camellia

seeds are traditionally used as a source of tea oil in China. However, in the past few

decades, researchers have focused on increasing the utilization of Camellia seed oil

press-cake as a by-product for economic and sustainability reasons. Previously,

researchers have mostly focused on extracting, isolating, and characterizing the

saponins isolated from Camellia seeds. Over seventy saponins have been identified

in Camellia seeds with most of them being oleanane-type triterpene monodesmosides

with three or four sugar residues attached at C-3 [10]. Camellia seed cake is generally

3
disposed of as waste after extracting the oil, even though it is a rich source of tea

saponins (TS). It would therefore be advantageous to turn these residues into

value-added functional ingredients that can be used in a variety of products in the

food, supplement, cosmetic, personal care, detergent, and pharmaceutical industries.

However, to the best of our knowledge, there have been few studies on the utilization

of TS as natural emulsifiers to form and stabilize emulsions.

Recently, there has been consumer pressure for commercial products that are

more natural, sustainable, and environmentally friendly, which has prompted industry

to replace synthetic or animal-based surfactants with natural plant-based alternatives,

such as phospholipids, biosurfactants, polysaccharides, amphiphilic proteins, and

bioparticles [11,12]. Compared to other types of natural emulsifiers, saponins can be

utilized at relatively low levels to form fine oil droplets that remain stable over a wide

range of environmental conditions [5,13]. A recent study showed that the interfacial

activity of saponins differs depending on their botanical origin and molecular

structure [10]. The aim of the present study was therefore to compare the performance

of QS, TS, and a widely used synthetic surfactant (Tween 80) on the formation and

stabilization of nanoemulsions using a high-pressure homogenization method.

Furthermore, we characterized the interfacial properties of the three emulsifiers and

studied the impact of common environmental stresses (pH, ionic strength, and

temperature) and long-term storage on the stability of the nanoemulsions. This

research provides valuable information on the potential of tea saponins to be used to

formulate nanoemulsions suitable for use in commercial products. Moreover, it may

facilitate the establishment of an important outlet for the application of these

underutilized byproducts.

2. Materials and methods

2.1 Materials

Tea saponin (TS) was obtained from Han Qing Biological Technology Co. Ltd.

4
(Hunan, China). This product is supplied as a yellowish powder that contains around

51.8% saponins, 36.1% total carbohydrate, 5.6% crude protein, 2.7% ash, and 3.7%

water according to the manufacturer. Quillaja saponin (Q-Naturale® 200) was

provided by Ingredion Incorporated (Bridgewater, NJ, U.S.A). It was reported to

contain 20-22% (w/w) Quillaja saponaria extract soluble solids and 78-80% (w/w)

water. A non-ionic surfactant, Tween 80 (T80), was purchased from Sigma-Aldrich

Co. (St. Louis, MO, U.S.A). Medium chain triglyceride (MCT) oil (Miglyol 812) was

purchased from Coletica (Northport, NY, U.S.A). Sodium chloride and sodium

phosphate monobasic were purchased from Aladdin Co. Ltd. (Shanghai, China). All

other reagents were of analytical grade. Distilled and deionized water was used in all

experiments.

2.2 Emulsion preparation

Oil-in-water emulsions were prepared according to the previous method used by

our laboratory [6]. Briefly, 10 wt% oil phase and 90 wt% aqueous phase was blended

together. The oil phase consisted of MCT while the aqueous phase consisted of

surfactant (0.1-2 wt% T80, QN, or TS) in buffer solution (10 mM sodium phosphate

buffer, pH 7.0). First, the oil and aqueous phases were added to a beaker, and then a

coarse emulsion was prepared by blending them together using a high-shear blender

(Fluko superfine homogenizer F6/10, Fluko Equipment Co. Ltd, Shanghai, China) for

2 min at ambient temperature. The coarse emulsion was then passed through a

high-pressure homogenizer (AH-BASICI, ATS Engineering Limited, Ontario, Canada)

for three cycles at a homogenization pressure of 50 MPa. The concentration of

surfactant used is reported on an active ingredient basis (rather than a total ingredient

basis).

2.3 Emulsion stability testing

The impact of a series of environmental stresses that may be encountered in

5
commercial applications on the stability of the nanoemulsions was investigated.

Thermal processing: Emulsions were transferred into glass test tubes that were

then incubated in water baths set at temperatures ranging from 30 to 90℃ and held

there for 30 min. The samples were then cooled to room temperature and stored for 24

h prior to analysis.

pH: Emulsions were placed in glass beakers, adjusted to different pH values

(2.0-9.0) using NaOH and/or HCl solutions, transferred to glass test tubes, and then

stored for 24 h at room temperature prior to analysis.

Ionic strength: The ionic strength of emulsions was adjusted to different levels by

adding salt (100-500 mM NaCl) and then the samples were stored for 24 h at room

temperature prior to analysis

Storage stability: Emulsions containing 0.02% (w/w) of sodium azide (NaN3) as a

preservative were stored at different temperatures of 5, 37, and 55℃ for 30 days and

samples periodically taken for analysis.

2.4 Determination of particle size

Information about the dimensions of the particles in the emulsions was obtained

using laser diffraction (Mastersizer 2000, Malvern Instruments, Worcestershire, UK).

Samples were diluted using 10 mM phosphate buffer (pH 7.0) to avoid multiple

scattering effects. The refractive indices used for the dispersed and continuous pahses

were 1.53 and 1.33, respectively. All measurements were carried out at 25 °C.

2.5 Determination of Zeta–potential

Particle surface charge characteristics (Zeta-potential) were determined using an

electrophoresis instrument (Zetasizer Nano-ZS90, Malvern Instruments,

Worcestershire, UK). Samples were diluted with 10 mM phosphate buffer solutions at

pH 7.0 prior to analysis to avoid multiple scattering effects. If a sample had

phase-separated due to creaming, then it was evenly dispersed by stirring before

6
collection of an aliquot for analysis [14].

2.6 Interfacial Tension Measurements

Interfacial tension versus surfactant concentration profiles were determined using

an interfacial tensiometer (DCAT21, Data Physics Co., Germany) equipped with a

9.95 mm long and 0.2 mm thick platinum sheet. MCT was used as the oil phase and

the aqueous phase was prepared by dissolving an appropriate amount of surfactant

(0.0005-2wt%) into phosphate buffer solution (10 mM, pH 7). The platinum sheet and

container were carefully cleaned according to the manufacturer’s instructions prior to

use. First, the aqueous phase was poured into the sample cup. Then, the platinum

sheet was immersed into the aqueous phase and pulled up to the interface. The oil

phase was then slowly added to the water phase until the platinum sheet was

completely submerged in oil. The interfacial tension of each sample was then

determined from measurements of the force required to pull-out the platinum sheet.

2.7 Microstructure Analysis

The microstructure of the emulsions was characterized at room temperature using

fluorescence confocal laser scanning microscopy (LSM800, Carl Zeiss, Germany). A

lipid stain was prepared by dissolving 0.02% Nile red in a mixture of 1,2-propanediol

and water (volume ratio of 50: 1), mixing well, and then storing at 4℃ in the dark.

Prior to analysis, 1 mL of the emulsion samples was dyed with 40 μL of this Nile Red

solution to highlight the location of the lipid phase. A wavelength of 488 nm was used

to detect the fluorescence signal from the Nile red. The sample was transferred onto a

glass microscope slide and covered with a glass coverslip. All images were captured

with a 10× eyepiece and a 63× objective lens (oil immersion).

2.8 Statistical Analysis

All measurements were performed on at least two freshly prepared samples with

7
three measurements per sample. The results are reported as means ± standard

deviations. Statistical analysis was performed by ANOVA using SPSS statistic

Software (IBM, USA). A P-value of <0.05 was considered significant.

3. Results and discussion

3.1 Interfacial characteristics

Based on their structure, tea saponins would be expected to be highly

surface-active because they have both a hydrophobic aglycone part and a hydrophilic

carbohydrate part (Figures 1a and 1b). The interfacial characteristics of surfactants

plays a major role in determining their ability to fabricate and stabilize emulsions [15].

We therefore measured the interfacial tension vs. surfactant concentration profiles of

the three surfactants to better understand their interfacial behavior at MCT oil-water

interfaces (Fig. 2). Oil droplet disruption inside a homogenizer is known to increase as

the magnitude of the interfacial tension decreases [16]. In the absence of surfactant,

the interfacial tension (γ0) was 24.42 ± 0.03 mN/m. The interfacial tension

decreased steeply as the surfactant level was first increased, which is indicative of the

surfactant molecules adsorbing to the oil-water interface and screening the

thermodynamically unfavorable molecular interactions between the oil and water

phases. There interfacial tension decreased less steeply as the surfactant level was

increased further, until a relatively constant value was attained, indicating that the

interfaces had become saturated [17]. In general, the three surfactants had fairly

similar interfacial tension (γ) vs. surfactant profiles, suggesting they had similar

surface activities. The interfacial tension at saturation was around 4.8 mN/m for TS,

2.8 mN/m for QS, and 6.4 mN/m for Tween 80, which may reflect differences in the

packing of the molecules at the interface, as well as their ability to screen unfavorable

interfacial interactions.

More insight into differences in the interfacial characteristics of the surfactants

was obtained by calculating their surface activity (k) using the following expression

8
[15]:

Here C1/2 is the surfactant concentration where half the adsorption sites at the interface

are occupied by surfactant molecules, ads

is the free energy change associated with

transfer of surfactant molecules from the bulk phase to the interfacial layer, R is the

gas constant, and T is the absolute temperature. C1/2 can roughly be taken to be the

surfactant level where the surface pressure (π = γ0-γ) is half its maximum value (π∞).

In this study, we calculated the following parameters from the surface pressure vs.

surfactant profiles of the different systems (Figure S1, Supporting Information):

C1/2=0.0033 wt% (26.6 μM)，k=3759 M-1, and ads

=-8.23RT for TS; C1/2=0.0029wt%

(18.2 μM)，k=5495 M-1, ads

=-8.61RT for QS; and, C1/2=0.0037wt% (28.3 μM)，

k=3533M-1, and ads

=-8.16RT for T80. The molecular weights of TS, QS, and T80

used in these calculations were 1241, 1650, and 1309 g/mol, respectively, obtained

from literature values [6,18]. These results indicate that TS has a strong affinity for

an oil-water interface and exhibits fairly similar interfacial characteristics as the other

surfactants used.

3.2 Influence of surfactant types and concentration on emulsion formation

The impact of surfactant type and level on the particle size of emulsions produced

under fixed homogenization conditions was then investigated. As expected, there was

a decrease in mean particle diameter (d32) with increasing surfactant level for all three

surfactants (Fig. 3a). The d32-surfactant profiles exhibited the two-regime trend

commonly observed during homogenization of emulsions: a steep decrease at low

surfactant levels, followed by a relatively constant value at high surfactant levels [15].

This dependence can be divided into two regimes: a “surfactant-limited” regime and a

“surfactant-rich” regime [19,20]. At low surfactant levels, there is not enough

surfactant present to protect all the oil droplets generated inside the homogenizer.

Consequently, small oil droplets collide with each other and coalesce, increasing the

9
droplet size. The dimensions of the droplets in the surfactant-limited regime is

therefore primarily determined by the type and level of surfactant present. At

relatively high surfactant concentrations, there is enough surfactant present to protect

all the small oil droplets generated by the homogenizer, so the droplet size is limited

by the strength of the disruptive forces produced by the homogenizer rather than by

the surfactant properties [6].

There were some differences in the particle size versus surfactant level profiles

for the three surfactants (Fig. 3a). The smallest droplets were produced using high

levels of TS (d32≈186 nm). Moreover, the lowest surfactant level required to create

small droplets was also for TS. These experiments indicate that all three surfactants

were effective at producing small droplets during homogenization, but that TS was

slightly more efficient. The differences in the effectiveness of the three surfactants

may be due to various factors that impact droplet fragmentation and coalescence

within homogenizers, including: (ⅰ) the rate at which surfactants attach to oil droplet

surfaces; (ⅱ) the ability of surfactants to depress the interfacial tension; (ⅲ) the

impact of surfactants on the rheology of the interface; and, (ⅳ) the effectiveness of the

surfactants at generating strong repulsive interactions between the droplets [5,21,22].

The surface load of the surfactants at saturation (Γ) was estimated using the

following equation [23]:

here, Cs is the surfactant concentration in the emulsion (in kg m-3), φ is the dispersed

phase volume fraction, Γ is the surface load (in kg m-2), and d32 is surface-weighted

mean droplet diameter (m). This approach assumes that all the surfactant is bound to

the oil droplet surfaces. The above expression was used to calculate the surface load

of TS based on the known disperse phase volume fraction (φ ≈ 0.1), measured

minimum droplet diameter (d32 ≈ 186 nm), and a fixed surfactant concentration of 1%

(Cs≈10kg m-3). The results suggest that the surface load of TS was approximately 3.1

mg m-2, which is close to the value of 2.9 mg m-2 reported previously for other

10
saponins [24,25]. Consequently, one would expect the tea saponins to form smaller

droplets than many other natural emulsifiers (such as phospholipids or

polysaccharides) when used at the same level [5].

3.3 Influence of pH

TS- and QS-emulsions were stable to droplet aggregation and phase separation

from pH 3 to 9, but unstable at pH 2 (Figs. 4b and 4c). The TS-emulsions exhibited

both flocculation and coalescence at this low pH value, whereas the QS-emulsions

only exhibited flocculation. The T80-emulsions were stable from pH 2 to 9, with no

change in microstructure, no visible phase separation (Fig. 4c), and no evidence of

droplet growth (Fig. 4b).

To provide some insights into the mechanism of acid-instability in the emulsions

fabricated using saponins, the pH-dependence of the droplet Zeta-potential was

determined (Fig.4a). The surface potential of both the TS- and QS-coated oil droplets

changed appreciably with pH, with the Zeta-potential changing from strongly negative

at high pH to weakly negative at low pH (Fig.4a). However, the magnitude of the

negative charge on the QS-coated droplets under acidic conditions was appreciably

higher than that on the TS-coated droplets, which may account for their better

coalescence stability. These results suggest that the saponin-coated droplets were

mainly stabilized by electrostatic repulsion. Thus, the poor stability of the emulsions

to droplet aggregation at low pH can be attributed to a weakening of the electrostatic

repulsion between the droplets [26]. These results are in good agreement with those of

previous studies on the electrical characteristics of saponin-coated droplets [6,13,27].

The negative surface potential on these droplets is a result of carboxylic acid groups

on the adsorbed saponin molecules [4,6,28]. The reason that the emulsions containing

T80-coated droplets were stable across the whole pH range, even though they had a

relatively low negative charge, is because they are mainly stabilized by steric

repulsion due to their large polymeric head groups [5]. The relatively poor

11
acid-stability of the saponins has also been demonstrated for a number of other

biosurfactants, including phospholipids and rhamnolipids [5, 29, 30], which can also

be attributed to a reduction in electrostatic repulsion at low pH values. In contrast,

protein-coated oil droplets tend to be unstable to aggregation around their isoelectric

points because this is where they have the lowest surface potential, whereas

polysaccharide-coated ones are stable across a wide range of pH values because they

are mainly stabilized by steric repulsion [5].

In summary, these results show that emulsions stabilized by tea saponins are

stable across most of the pH range found in food products, but that they become

unstable to flocculation and/or coalescence under highly acidic conditions.

3.4 Influence of thermal processing

The emulsions stabilized by the saponins (1wt% TS or QS) remained stable to

thermal processing across the whole range of temperatures studied, i.e., there was little

change in mean particle diameter (around 200 nm) and no visible creaming or oiling off

(Fig. 5a). This may have been because the Zeta-potential remained relatively high and

constant across the entire temperature range (≈ -50 mV), leading to a powerful

electrostatic repulsion between the droplets (Fig. 5b). These results are consistent with

those reported previously for the thermal stability of QS-coated droplets [30]. The

T80-emulsions were stable to droplet aggregation from 30 to 70℃, but exhibited

appreciable droplet growth at higher temperatures (90℃). This effect can be explained

by the increased coalescence rate of the oil droplets when the temperature approaches

the phase inversion temperature of this non-ionic surfactant [31]. The

temperature-dependence of the saponins is therefore quite different from that of other

natural emulsifiers. Polysaccharide-coated oil droplets are often stable to heating

because the biopolymers remain attached to the droplet surfaces and generate a strong

electrostatic repulsion [5]. In contrast, globular protein-coated oil droplets often

aggregate when heated above their thermal denaturation temperature, particularly when

12
the electrostatic repulsion is relatively weak [5]. This is because the adsorbed globular

proteins unfold and expose non-polar groups to the surrounding water, which increases

the hydrophobic attraction between the droplets.

3.5 Influence of ionic strength

The impact of ionic strength was investigated by adding different levels of salt (0

to 500 mM NaCl) to the emulsions and then measuring their physical stability after 24

h storage (Fig. 6a). There was a slight increase in mean particle dimensions for the

Tween 80-emulsions from 0 to 100 mM NaCl, but little further change at higher salt

levels. This effect may have been due to some dehydration of the hydrophilic

polymeric head groups of the non-ionic surfactant by salt addition [32]. The

T80-emulsions exhibited no visible evidence of phase separation (data not shown),

and no change in microstructure, across the entire salt range studied. These results

emphasize the fact that the droplets in the Tween 80 emulsions are mainly stabilized

against aggregation by steric (rather than electrostatic) repulsion [15].

Visual observations and optical microscopy indicated that the emulsions

stabilized by the saponins were stable at low salt levels (≤ 200 mM NaCl), but

unstable to creaming and aggregation at higher levels (Fig. 6c). When measured by

static light scattering the emulsions still had relatively small particle dimensions (Fig.

6a). This effect is due to dissociation of the flocs when the emulsions are diluted and

stirred within the measurement chamber of the light scattering instrument. The fact

that the oil droplets remained relatively small at all salt levels (0-500mM NaCl)

indicates that the aggregation observed at higher salt levels was due to flocculation

rather than coalescence. Overall, these results suggest that a combination of

electrostatic and steric repulsion is responsible for stabilizing saponin-coated droplets

against aggregation (Fig. 6b). At low salt levels, the long-range electrostatic repulsion

between the droplets is enough to prevent aggregation, but at high salt levels the van

der Waals attraction becomes stronger than the electrostatic repulsion, leading to

13
flocculation. However, the short-range steric repulsion at high salt concentrations still

appears to be sufficient to prevent the droplets from merging together and coalescing

[6]. It should be noted that anionic non-saponin ingredients have been identified in

crude quillaja extracts, such as (+)-piscidic acid [27]. Therefore, the stability of

saponin-coated lipid droplets is highly pH dependent and sensitive to increases in

ionic strength, which is different from the behavior of synthetic non-ionic surfactants,

such as Tween 80.

The impact of salt addition on the stability of the saponin-coated oil droplets is

different from that of other natural emulsifiers. Polysaccharide-coated oil droplets

are often stable to aggregation, even at relatively high ionic strengths, because of the

strong steric repulsion between them [5]. In contrast, protein-, phospholipid-and

rhamnolipid-coated oil droplets often aggregate when salt is added because this

reduces the electrostatic repulsion acting between the droplets [5].

In summary, the emulsions stabilized with the saponins have much worse stability

to high ionic strengths than those stabilized by T80, which may limit their application

in commercial products containing high salt levels.

3.6 Effect of long-term storage

The stabilities of TS-, QS-, and T80-emulsions were also compared during

storage at three temperatures (5, 37, and 55 ℃) for 30 days. These experiments were

performed using emulsions at neutral pH containing no added salts. There was no

change in the appearance of emulsions prepared using TS and QS throughout the

entire period and little change in their mean particle diameters (Table 1). These

results indicate that the saponins were fairly efficient at preventing droplet

aggregation. We did not observe any changes in the appearance or mean particle size

of the emulsions stabilized by T80 when they were stored at 5 or 37℃ for a month.

Conversely, there was a small increase in the mean droplet diameter (Table 1) after

one-month storage at 55℃. These results suggest that Tween 80 may be unsuitable for

14
application in emulsions that may be stored at elevated temperatures for appreciable

periods. This effect may have occurred due to a change in the optimum curvature or

hydrolysis of the surfactant headgroups at high temperatures [33].

4 Conclusions
The purpose of this paper was to address the pressing need for effective

plant-based surfactants to replace the animal-based or synthetic ones currently used in

many commercial emulsified products [5, 8, 11, 34]. Recent research has shown that a

saponin extract derived from the bark of the Quillaja Saponaria Molina tree is a highly

effective emulsifier [6, 7, 8, 13, 34]. However, the overexploitation of this natural

resource may not be sustainable or environmentally-friendly [35]. Tea saponins

isolated from waste materials produced during tea manufacture may offer a more

sustainable and economically viable source of natural surfactants [2, 4, 10].

We have shown that tea saponins can be used to successfully form and stabilize

oil-in-water emulsions. We found that nanoemulsions (d < 200 nm) could be

fabricated from tea saponins at relatively low surfactant-to-oil ratios (SOR≤1:10)

using a high-pressure homogenizer. Oil droplets coated with these surfactants were

stable over a range of temperatures (30-90 ℃), salt concentrations (≤200 mM NaCl),

and pH values (pH 3-8). Moreover, they had good long-term stability when stored at

different temperatures (5, 37, and 55℃). However, droplet flocculation occurred when

they were stored under highly acidic (pH 2) or high ionic strength (300-500 mM NaCl)

conditions. We hypothesize that the saponins formed a thin layer around the lipid

droplets that mainly stabilized them by electrostatic repulsion. Droplet aggregation

therefore occurred under conditions where there was a reduction in the electrostatic

repulsion between the saponin-coated oil droplets, such as low pH or high ionic

strength [13,27]. The tea saponins behaved fairly similarly to commercially available

quillaja saponins [4,6,34]. The tea saponins may have advantages over other natural

emulsifiers, such as proteins, polysaccharides, or phospholipids, for certain

commercial applications. Overall, our results show that tea saponins are effective

15
natural surfactants that can be used to form and stabilize emulsions suitable for

application in the food and other industries. Nevertheless, further studies are

important to investigate the flavor profile, toxicity, reliability of source, ease of

utilization, and cost for specific applications before they will find more widespread

use.

Acknowledgements
We would like to thank Ingredion Incorporated for donating Q-Naturale®200 used

in this research. This work was supported by the National Natural Science Foundation

of China (No. 31671888 and 31501443), and Doctoral Research Foundation of Shaanxi

University of Science and Technology (No. 2017BJ-21). This material was also partly

based upon research supported by the National Institute of Food and Agriculture,

USDA, Massachusetts Agricultural Experiment Station (MAS00491).

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Figure Captions

Fig. 1 Proposed molecular structures of the two natural surfactants used:

Tea saponin (a) and quillaja saponin (b).

Fig. 2 Influence of tea saponin, quillaja saponin and Tween 80

concentration on the interfacial tension measured at the MCT oil-water
interface. Note: the surfactant concentration for tea and quillaja saponins is
the active component.

Fig. 3 Influence of surfactant concentration on (a) the mean particle

diameter (d32) and (b) the ζ-potential of 10 wt% MCT oil-in-water
emulsions produced using a high-pressure homogenizer under
standardized homogenization conditions (50 MPa, 3 cycles)

Fig.4 The influence of pH on (a) the mean particle diameter (d32) and (b)
the ζ-potential of oil-in-water emulsions (1% tea saponin, 1% quillaja
saponin or 1% Tween 80) produced using fixed homogenization conditions
(50 MPa, 3cycles) and stored at room temperature for 24 h before analysis.
(c)Eeffect of pH on appearance of Tea saponin, quillaja saponin, and
Tween 80 emulsions after storage of 24h at room temperature; Confocal
micrographs of Tea saponin-, quillaja saponin-, and Tween 80-stabilized
emulsions at pH 2.

Fig.5 Influence of thermal processing (30-90℃, 30 min) on (a) the mean

particle diameter (d32) and (b) the ζ-potential of oil-in-water emulsions

produced using homogenization conditions (50 MPa, 3 cycles) and stored
at room temperature for 24 h before analysis.

20
Fig.6 Influence of salt concentration on (a) the mean particle diameter (d32)
and (b) the ζ-potential of 10 wt% MCT oil-in-water emulsions stabilized
by 1% Tween 80, tea saponin or quillaja saponin after 24 h. (c) Confocal
micrographs of tea saponin-, quillaja saponin- or Tween 80-stabilized
emulsions at different salt concentrations (100-500 mM, NaCl).

21
Figure 1a
H3 C H

O CH 3
O
H
OCOCH3
CH 2OH
COOH OH
CH2OH H H
O O O O
OH
H H
OH H H R1 R H
2
H CH 3 OH H
H OH HH O
OH O
H
OH H
H H
H H O
H O
H
OH H
HO H
H OH

Figure 1b

O
COOH O
O
OH O OO O
O OH OH
O OH
OH
OHOH
CH2OH OHC O O O
O O
OH OO O
OH O
OH
HO OHOH OH
OHOH
OH

O O
OH
HO
OH

22
Figure 2

25
Tea Saponin
Interfacial Tension (mN m-1)

Quillja Saponin
20
Tween 80

15

10

0
0.0001 0.001 0.01 0.1 1
Surfactant Concentration (wt%)

23
Figure 3a

2000
1800 Tea Saponin
Mean Particle Diameter (nm)

Quillaja Saponin
1600
Tween 80
1400
1200
1000
800
600
400
200
0
0 0.5 1 1.5 2
Surfactant Concentration (wt%)

24
Figure 3b

0 0.5 1 1.5 2
0

-10
Zeta-Potential (mV)

-20
Tea Saponin
-30 Quillaja Saponin
Tween 80
-40

-50

-60

Surfactant Concentration (wt%)

25
Figure 4a

pH
2 3 4 5 6 7 8 9 10
0

-10
Zeta-Potential (mV)

-20

-30

-40
Tea Saponin
-50 Quillaja Saponin

Tween 80
-60

26
Figure 4b

400
Tea Saponin Quillaja Saponin Tween 80
Mean Particle Diameter (nm)

300

200

100

0
2 3 4 5 6 7 8 9
pH

27
Figure 4c

28
Figure 5a

29
Figure 5b

30
Figure 6a

600
Tea Saponin
Mean Droplet Diameter (nm)

Quillaja Saponin
500
Tween 80

400

300

200

100

0
0 100 200 300 400 500
Ionic strength (mM)

31
Figure 6b

-10
Zeta-Potential (mV）

-20

-30

-40
Tea Saponin
-50 Quillaja Saponin
Tween 80
-60
0 100 200 300 400 500

Ionic strength (mM)

32
Figure 6c

33
Tables

Table 1. Mean particle diameters of oil-in-water emulsions formed using Tween 80,

Quillaja saponin or Tea saponin as emulsifiers after 30 days storage.

0 Day 15 Day 30 Days

Tea Saponin 5℃ 200 nm 198 nm 199 nm

37℃ 201 nm 203 nm

55℃ 200 nm 207 nm

Quillaja 5℃ 220 nm 221 nm 224 nm

Saponin 37℃ 220 nm 226 nm

55℃ 222 nm 221 nm

Tween 80 5℃ 196 nm 200 nm 205 nm

37℃ 206 nm 211 nm

55℃ 232 nm 248 nm

34
Graphical Abstract