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Sugar Beet Extract (Beta vulgaris L.) as New Natural Emulsifier: Emulsion Formation Theo Ralla, Hanna Salminen, Matthias Edelmann, Corinna Dawid, Thomas Hofmann, and Jochen Weiss J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 30, 2017

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Journal of Agricultural and Food Chemistry

Sugar Beet Extract (Beta Vulgaris L.) as New Natural Emulsifier: Emulsion Formation Theo Ralla1, Hanna Salminen1, Matthias Edelmann2, Corinna Dawid2, Thomas Hofmann2, Jochen Weiss1*

1

Department of Food Physics and Meat Science, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany 2

Food Chemistry and Molecular Sensory Science, Technical University Munich, LiseMeitner-Strasse 34, 85354 Freising, Germany

Submitted to Journal of Agricultural and Food Chemistry April 2017

____________________ * Corresponding author: Tel: +49 711 459 24415; E-mail address: [email protected]; (J. Weiss)

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ABSTRACT

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The interfacial and emulsion forming properties of sugar beet extract (Beta vulgaris

3

L.) were examined and compared to a Quillaja extract that is widely used within the

4

food industry. We investigated the influence of extract concentration on surface

5

activity at water and air-water interfaces, and on the formation of oil-in-water

6

emulsions (10% w/w, pH 7). Sugar beet extract reduced the interfacial tension up to

7

38% at oil-water interface, and the surface tension up to 33% at air-water surface. The

8

generated emulsions were negatively charged (ζ ≈ −46 mV) and had the smallest

9

particle sizes (d43) of ~1.3 µm at a low emulsifier-to-oil ratio of 0.75:10. Applying

10

lower or higher extract concentrations increased the mean particle sizes. The smallest

11

emulsions were formed at an optimum homogenization pressure of 69 MPa. Higher

12

homogenization pressures led to increased particle sizes. Overall, sugar beet extract

13

showed high surface activity. Furthermore, the formation of small emulsion droplets

14

was successful, however, the droplets were bigger compared to Quillaja extract.

15

These results indicate sugar beet as an effective natural emulsifier that may be utilized

16

for a variety of food and beverage applications.

17 18

Keywords:

19

Sugar beet, Quillaja, natural emulsifier, protein, saponin, oil-in-water emulsion.

1 ACS Paragon Plus Environment

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INTRODUCTION

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Rational design of emulsion-based food is largely influenced by the emulsifiers that

22

stabilize the dispersed phase by adsorbing onto the freshly formed interface during

23

homogenization and forming a protective layer around the droplets to prevent

24

aggregation.1-3 Emulsifiers differ in their emulsifying properties and can conveniently

25

be characterized by their functional characteristics such as adsorption kinetics as well

26

as by their stability towards external stresses including ionic strength, pH and

27

temperature.4

28

There is a recent demand of consumers towards ‘natural’ products, thus, ‘natural

29

emulsifiers’ have become more attractive for the food industry.5 Typical ‘natural’

30

food-grade emulsifiers are polysaccharides, proteins and phospholipids.6 However,

31

polysaccharides usually exhibit a low surface activity and therefore require a high

32

emulsifier-to-oil ratio,7 whereas phospholipids build up only a thin membrane making

33

the emulsions prone to coalescence during storage.8, 9 The emulsifying properties of

34

proteins strongly depend on pH, temperature and ionic strength due to their

35

polyelectrolyte properties and their three-dimensional structures.10, 11 Consequently,

36

the food and beverage manufacturers are in need of suitable natural emulsifiers with

37

excellent emulsifying properties to stabilize food dispersions.6 Recent studies focus

38

on another potential highly surface-active group, the saponins.6, 12-14 These secondary

39

plant metabolites consist of a highly hydrophobic triterpene or steroid aglycone (such

40

as quillaic acid) with hydrophilic sugar moieties attached such as fucose, glucose, and

41

rhamnose.15,

42

soapbark tree (Quillaja saponaria Molina) is naturally rich in surface-active saponins

43

and that diminutive amounts of this extract (~0.2% w/w) could form nano-sized

16

Recent studies have shown that the extract of the South American



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emulsion droplets at a low emulsifier-to-oil ratio.12, 17-20 Saponins are common in a

45

variety of plants such as sugar beet, oat, red beet, and green pea that serve as

46

antifeedants.21, 22 New sources for ‘natural’ saponin extracts would therefore increase

47

the available options for the food and beverage industry. Sugar beets (e.g.

48

~115 million tons of sugar beets per year in the EU)23 are a promising source for

49

saponins as they contain significant amounts that may be used as natural emulsifier.

50

For example, saponins found in sugar beet are mainly of triterpenoidal nature such as

51

betavulgaroside I with an oleanane sapogenin moiety backbone but with different

52

sugar moieties (Fig. 1).24,

53

combination of saponin and protein exhibited synergistic foaming properties due to

54

the highly viscous interface formed.26, 27 Consequently, only small amounts of highly-

55

surface active saponins in combination with protein may be sufficient to form oil-in-

56

water emulsions.

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In this study, the interfacial properties and emulsifying ability of sugar beet extract

58

(Beta vulgaris L.) were investigated. First, we characterized the interfacial properties

59

by measuring the surface-tension at different extract concentrations at oil-water and

60

air-water interfaces. Second, we studied the influence of sugar beet extract

61

concentration and homogenization pressure on the mean particle size and ζ-potential

62

of 10% (w/w) oil-in-water emulsions, which were produced by high-pressure

63

homogenization. The capability of sugar beet extract was compared to commercially

64

available Quillaja extract that is already in use as natural emulsifier within the food

65

and beverage industry.5,

66

extracts, their chemical composition was characterized.

12

25

In addition, recent studies also reported that the

Third, to gain more insights into the behavior of the


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MATERIALS AND METHODS

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Materials

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Sugar beet roots were provided by Pfeifer & Langen GmbH Co. KG (Köln, Germany).

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Spray dried Quillaja extract (Andean QDP Ultra Organic) from Desert King Intl. (San

71

Diego, California) was obtained from PERA GmbH (Springe-Eldagsen, Germany).

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The medium chain triglyceride oil Miglyol 812N was purchased from Cremer Oleo

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GmbH & Co. KG (Hamburg, Germany). Methanol (HPLC grade) was purchased

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from J.T. Baker (Deventer, Netherlands). Water for the sugar beet extract was

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obtained from a Milli-Q Advantage A10 Water Purification System made by

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Millipore S.A.S (Molsheim, France). Sodium phosphate monobasic monohydrate

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(≥98.0%) and sodium phosphate dibasic (≥99.0%) were purchased from Sigma-

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Aldrich (Steinheim, Germany). Round filter paper, type 111A, Ø 110 mm,

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hydrochloric acid (2 M), and sodium hydroxide (≥98%) were purchased from Carl

80

Roth GmbH & Co. KG (Karlsruhe, Germany). Folin-Ciocalteu’s phenol reagent,

81

gallic acid (≥98.0%), sodium carbonate (≥99.9%) and di-sodium tetraborate

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decahydrate (≥99.0%), and sulphuric acid (≥98.0%) were obtained from Merck KGaA

83

(Darmstadt, Germany). m-Hydroxydiphenyl was obtained from Eastman Kodak

84

Company (Rochester, NY). Double distilled water was used throughout the study.

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Methods

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Sugar beet solvent extraction

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Fresh sugar beet roots (Beta vulgaris L. ssp. vulgaris var. altissima [Doell

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Annemaria] KWS, 100 g) were washed, chopped, frozen in liquid nitrogen, ground in

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a blender (Grindomix GM 300, Retsch GmbH, Haan, Germany) at 4.000 rpm for 45 s

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and then extracted with methanol (3 x 500 mL) at room temperature for 30 min at 3 ACS Paragon Plus Environment


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minimum. After filtration by means of a Buechner funnel lined with filter paper (Carl

92

Roth, 111A, Ø 110 mm) the filtrates were collected and the residue was extracted two

93

times (500 mL each) with a mixture of methanol/water (70/30, v/v). After filtration,

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the combined filtrates were separated from methanol in vacuum at 40 °C, freeze-dried

95

and stored at −20 °C until use. No additional preservatives were added.

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Protein, fat and mineral quantification

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The protein content of the extracts was measured according to the Dumas method

98

with a LECO FP-528 Nitrogen Determinator (Leco Corporation, St. Joseph, USA)

99

using a protein factor of Nx6.25.28 The fat content was determined according to

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procedure B of the determination of crude oils and fats of the Commission Regulation

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(EC) No. 152/2009 III H.29 The mineral content was analyzed by inductively coupled

102

plasma optical emission spectroscopy.30

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Saponin, polyphenol and pectin quantification

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The saponin content was determined through separation by means of gradient flash

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chromatography using a Buechi sepacore system (Buechi, Flawil, Switzerland)

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equipped with a 150 × 40 mm i.d. column filled with RP-18 material (LiChroprep, 25-

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40 µm, Merck KGaA Darmstadt, Germany) and subsequent identification of the

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saponin containing fractions by UPLC-HDMS analysis performed on an acquity

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UPLC core system equipped with a BEH C18 column (150 × 2.1 mm, 1.7 µm,

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Waters, Eschborn, Germany) and coupled to a Synapt G2S HDMS mass spectrometer

111

(Waters, Manchester, UK). The polyphenol content was measured according to the

112

method of Singleton and Rossi.31 In short, the extract solution was oxidized with the

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Folin-Ciocalteu reagent and the reaction was neutralized after 3 min with sodium

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carbonate solution. The absorbance was measured at 720 nm with a Lambda 750


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spectrophotometer (Perkin Elmer, MA, USA) after 60 min of incubation at room

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temperature. Results are expressed as milligram of gallic acid equivalent (mg

117

GAE/mg extract). The pectin content of the extracts was measured according to the

118

m-hydroxydiphenyl method of Sirisakulwat, et al. 32. In short, the extract solution was

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hydrolysed using H2SO4 (72%) before tetraborate (dissolved in H2SO4) was added

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and the solution heated to 100 °C for 10 min. Thereby, uronic acid formed mucic acid.

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Mucic acid reacted with the added m-hydroxydiphenyl, giving a red-violet color. The

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absorbance was measured at 520 nm with a Lambda 750 spectrophotometer (Perkin

123

Elmer, MA, USA) 20 min after m-hydroxyphenyl addition. The results are expressed

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as anhydrouronic acid (M=176.13 g mol-1) content (AUA, g/100g extract).

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Solution and emulsion preparation

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Aqueous extract stock solutions were prepared by dissolving different amounts of

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sugar beet or Quillaja extract in 10 mM sodium phosphate buffer with subsequent

128

stirring overnight. The pH was adjusted to the appropriate pH using 0.1 and 1 M HCl

129

or NaOH. Oil-in-water emulsions were prepared by blending 10% (w/w) lipid phase

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(Miglyol oil 812) with 90% (w/w) aqueous phase that contained 0.1 to 5% (w/w)

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sugar beet or Quillaja extract. Coarse emulsions were prepared by blending the lipid

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and aqueous phases for 2 min using an ultra turrax at 15,000 rpm (Silent Crusher M,

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Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) at room temperature.

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The coarse emulsions were passed through a high-pressure homogenizer (Avestin

135

Emulsiflex C-3, Ottawa, Canada) for 4 passes at various homogenization pressures

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(5000 to 25,000 psi / 34 to 172 MPa) and the pH was adjusted to pH 7 afterwards if

137

necessary. All samples were stored for at least 24 h at 5 °C prior further analysis.

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Particle characterization



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The particle size distributions of emulsions were determined by static light scattering

140

(Horiba LA-950, Retsch Technology GmbH, Haan, Germany) in which the angular

141

dependence of the scattered light is measured and the particle size calculated based on

142

the Mie theory. Emulsions were diluted using pH-adjusted 10 mM sodium phosphate

143

buffer to a final droplet concentration of approximately 0.005% (w/w) to prevent

144

multiple scattering effects. The refractive indices were set to 1.33 for the aqueous

145

phase and 1.54 for the dispersed phase. Particle sizes are expressed as mean surface-

146

based (d32) or volume-based (d43) diameter. The ζ-potential of emulsifier solutions

147

and emulsions (diluted 1:50) was determined using a particle electrophoresis

148

instrument with dynamic light scattering (Nano ZS, Malvern Instruments, Malvern,

149

UK) that measured the velocity of the droplets that move in the applied electric field

150

with the calculations based on the Smoluchowski equation.

151

Interfacial and surface tension

152

The interfacial tension at an oil-water interface was determined using a Krüss drop

153

shape analyzer (DSA 10, Krüss GmbH, Hamburg, Germany) and calculated using the

154

Young-Laplace equation according to the recorded drop shape. Miglyol oil was

155

poured in a glass cuvette and the shape of a drop generated by a needle immersed into

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the oil was recorded with a frame rate of 30 per minute for an incubation time of

157

5 min. The surface tension at a water-air interface was determined using a DCAT 11

158

tensiometer equipped with a Wilhelmy plate (DataPhysics, Filderstadt, Germany) and

159

a water bath to maintain a constant temperature of 25 °C. Prior to the analysis, the

160

Wilhelmy plate and glass beakers were flushed with ethanol and rinsed with distilled

161

water several times. The Wilhelmy plate was heated to light red glow to remove

162

possible organic compounds. Before each measurement, the surface tension of

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purified water was determined to ensure that no contamination was present 6 ACS Paragon Plus Environment

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(γH2O = 72.0 ± 0.5 mN m-1).33 The surface tension as a function of extract

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concentration was determined by titrating different extract concentrations (0.0003 to

166

5.5% w/w) into the beaker, slowly mixing it for 30 s, and measuring the surface

167

tension after 300 s of incubation time.

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Optical microscopy

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All emulsion samples were mixed using a vortex mixer and one drop of undiluted

170

sample was placed on an objective slide and covered by a cover slip. To visualize

171

two-dimensional structures of the samples an optical microscope (Axiocam ICc3, Carl

172

Zeiss Microimaging GmbH, Goettingen, Germany) equipped with 20x and 40x

173

objectives was used.

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Statistical analysis

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All values reported represent means and standard deviations from a minimum of three

176

measurements from two fresh independently prepared samples that were calculated

177

using Excel (Microsoft, Redmond, WA). Linear regression was also performed using

178

Excel.

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RESULTS AND DISCUSSION

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Characterization of extracts

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Natural emulsifiers are typically a mixture of different amphiphilic constituents that

182

exhibit different surface-active and emulsion forming properties. For example,

183

saponins as well as proteins comprise hydrophilic (e.g. sugar groups; serine) and

184

hydrophobic regions (e.g. steroid or triterpene aglycone; alanine) within the same

185

molecule, thus making them surface-active.5 In addition, other components such as

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polyphenols that are present in plant extracts can exhibit emulsifying properties that 7 ACS Paragon Plus Environment


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also play a significant role as antioxidants and in browning reactions and therefore

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contribute to product characteristics such as the color.34

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Sugar beet extract had a much lower saponin and polyphenol content but a higher

190

protein content compared to Quillaja extract (Tab. 1). The saponin content in the

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sugar beet extract (>0.5%, w/w) is slightly higher than previously reported amounts

192

found in roots (~0.3%, w/w)35, but reported protein contents are lower (~0.8%,

193

w/w),36 which may be attributed to the accumulation of surface-active materials

194

during the solvent extraction. In addition, sugar beet extract had a lower mineral

195

content compared to Quillaja extract (Tab. 1) with the following minerals present: K

196

0.323%, Ca 0.004%, Mg 0.049%, Na 0.001%, S 0.009%, P 0.053%, Mn 0.001%, Fe

197

0.0001%, Al 0.001%, Zn 0.001%, Se 0.0005‰. The mineral composition of the

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Quillaja extract was determined in a previous study.13 The presence of polyphenolic

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compounds can also be observed in the color of the powdered extracts, where

200

Quillaja extract had a dark brown color and the sugar beet extract a white appearance

201

(data not shown). However, the used Folin-Ciocalteu reagent detects all phenolic

202

groups and is easily interfered by reducing substances such as ascorbic acid and

203

should therefore only be considered as a first approximation instead of an exact

204

quantification. Sugar beet is commonly known for its relatively high pectin content

205

that is widely used as biopolymer-based emulsifier (E440) within the food and

206

beverage industry. Consequently, we determined the anhydrouronic acid content

207

calorimetrically to be ~1.7 AUAc (g/100g extract), which is much lower compared to

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commercially available sugar beet pectin products with typical galacturonic acid

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contents of ~66%.37

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Interfacial and surface tension


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The purpose of conducting the interfacial and surface tension measurements was to

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compare the adsorption properties of both extracts at oil-water and water-air surfaces.

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An ideal emulsifier is capable of rapidly adsorbing to the interface and reducing the

214

interfacial tension between the thermodynamically unfavorable contacts of the

215

different kinds of molecules. Therefore, a high and fast decrease in interfacial tension

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is a good indicator for the ability to form and stabilize emulsions because the energy

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required to deform and disrupt droplets is reduced.4

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For both extracts, the interfacial tension decreased with increasing extract

219

concentration, indicating the adsorption of surface-active material to the oil-water

220

interface (Fig. 1a). The data showed that sugar beet extract reduced the interfacial

221

tension by up to 38% to minimal values of 14.5 ± 0.3 mN m-1, whereas Quillaja

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extract reduced the interfacial tension by up to 82% to 4.2 ± 0.1 mN m-1 at the highest

223

extract concentration of 5% (w/w) (calculated from the plain oil-water interface of

224

23.3 ± 0.2 mN m-1), indicating that both emulsifiers adsorbed to the oil-water

225

interface. In comparison, commercially available emulsifiers such as polysorbates

226

(Tween 20), polysaccharides (gum arabic), or proteins (β-casein) reduce the

227

interfacial tension at saturation to minimal values of ~2, 7-47, and ~20,38 respectively,

228

indicating that the efficiency for interfacial tension reduction of sugar beet extract is

229

higher compared to those of proteins. Additionally, we measured the surface tension

230

at an air-water surface as a function of extract concentration (Fig.1b). Sugar beet

231

extract reduced the surface tension by up to 33% to 48.3 ± 0.8 mN m-1, whereas

232

Quillaja extract reduced the surface tension by up to 50% to 35.9 ± 0.4 mN m-1

233

(calculated from the plain air-water interface of 72.0 ± 0.5 mN m-1,33 Fig. 1b). The

234

surface tension of Quillaja extract is in agreement with earlier reported surface



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tension values (~35 mN m-1).12, 38 In addition, the obtained surface tension values for

236

sugar beet extract correspond well with proteins such as β-casein (~50 mN m-1).38

237

The different interfacial properties may be attributed to the different chemical

238

compositions of the extracts, namely the different protein and saponin contents

239

(Tab. 1). Previous studies investigating the foaming properties of purified saponin

240

and protein have suggested synergistic effects for saponin and protein stabilized

241

foams

242

that in binary systems, saponins and proteins can interact by hydrogen bonds and/or

243

hydrophobic interactions, forming biogenic complexes. These surface-active

244

complexes formed a highly viscoelastic interface in foams that could withstand

245

dilatational and shear deformation. The authors demonstrated that the foam stabilizing

246

mechanisms of saponins and proteins are not antagonistic but rather synergistic.

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Consequently, the surface-activity of the sugar beet extract may mainly be attributed

248

to the presence of complexes of surface-active materials such as proteins and saponins,

249

as shown by its higher reduction of interfacial tension compared to commercially

250

available proteins. Overall, both extracts were effective at reducing the interfacial and

251

surface tension at an oil-water and air-water interfaces, respectively, which is a first

252

indicator for emulsion forming and stabilizing properties.

253

Influence of extract concentration on emulsion formation

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Influence on the mean particle size

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The purpose of these experiments was to characterize the influence of extract

256

concentration on the formation of emulsions as the mean droplet size is strongly

257

influenced by the ratio of emulsifier to dispersed phase (emulsifier-to-oil ratio).10 For

258

this, 10% (w/w) oil-in-water emulsions stabilized by sugar beet and Quillaja extract at

18, 20, 26, 39

and emulsions.40 Böttcher, Scampicchio and Drusch

26

hypothesized


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different

concentrations

were

prepared

under

standardized

high-pressure

260

homogenization conditions (10,000 psi, 4 passes).

261

Quillaja extract stabilized emulsions showed a continuous decrease in their mean

262

droplet sizes upon increasing the concentration: At 0.75% (w/w) Quillaja extract, the

263

emulsions had the lowest d43-value of 0.18 ± 0.00 µm. In addition, the droplet sizes

264

stayed relatively constant >1% (w/w) with d32-values ranging between 0.19 and

265

0.15 µm (Fig. 2a) and d43-values ranging from 0.20 to 0.23 µm (Fig. 2b), indicating

266

no larger emulsion droplets were present, which is in good agreement with

267

microscopic images (data not shown). For sugar beet extract, the smallest emulsion

268

droplet size (d32) of 0.91 ± 1.02 µm was obtained at 1% (w/w) (Fig. 2a), whereas the

269

volume-based mean droplet size (d43) decreased only up to extract concentrations of

270

0.75% (w/w) with the smallest d43-value of 1.29 ± 0.12 µm (Fig. 2b). Sugar beet

271

extract concentrations ≥1% (w/w) led to the formation of emulsions with mean sizes

272

of 22-37 µm and a broad particle size distribution (Fig. 2c, d). This was also

273

corroborated by the optical microscopy images (Fig. 2b). It should be noted, that

274

larger droplets are neglected in the surface-based diameter (d32), whereas they are

275

emphasized in the volume-based diameter (d43). The overall particle size distribution,

276

rather than just the mean particle diameter, is a good indicator of the stability of

277

emulsions. We therefore plotted the change in particle size distribution (d43) for all

278

emulsions. The width of the particle size distribution of Quillaja extract stabilized

279

emulsions decreased with increasing concentration leading to a lower mean droplet

280

diameter (Fig. 2c). Sugar beet extract stabilized emulsions (Fig. 2d) showed a shift in

281

the particle size distribution to smaller values up to 0.75% (w/w), whereas higher

282

concentrations led to the formation of larger particles. This is in good agreement with

283

the visual appearance of the emulsions and the corresponding microscopic images: 11 ACS Paragon Plus Environment


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We observed phase separation with dense flocs at the top and a few precipitates at the

285

bottom of the test tubes above 0.75% (w/w) sugar beet extract (Fig. 2b).

286

The emulsifying behavior of Quillaja extract may be attributed to two main factors:

287

The increased emulsifier concentration can stabilize a larger amount of oil-water

288

interfaces and therefore protect droplets more efficiently against physical

289

destabilization mechanism such as gravitational separation, flocculation, and

290

coalescence.4,

291

larger (d43 1.29 µm vs. 0.18 µm), than the ones stabilized by Quillaja extract (Fig. 2b),

292

which corresponds well with the microscopic images. This behavior might be

293

attributed to a lower surface activity of the sugar beet extract with a lower ability to

294

decrease the interfacial tension as illustrated in Fig. 1a, possibly due to the lower

295

saponin content in the extracts (Tab. 1). However, Böttcher, Scampicchio and Drusch

296

26

297

sufficient to form stable foams. The authors hypothesized the formation of saponin-

298

protein complexes due to hydrophobic and/or electrostatic interactions.26 These

299

complexes may contribute to the formation of larger emulsion droplets due to the

300

modified surface-activity compared to the individual groups. It is also possible that

301

the non-adsorbing surface active components in the sugar beet extract may induce

302

depletion flocculation of the emulsions droplets when added at higher concentrations.4

303

Based on the above mentioned results (Fig. 2), we propose that the emulsion droplets

304

are mainly stabilized by proteins when using sugar beet extract as it has a lower

305

saponin-to-protein ratio compared to Quillaja extract (Tab. 1). Nevertheless, it is

306

likely that also some saponins are complexed with these proteins. Typical native

307

proteins found in sugar beet roots range between 25 and 100 kDa, with the main

308

fractions comprising proteins between 32 and 61 kDa.43 During methanol extraction

41, 42

The droplets stabilized by sugar beet extract were appreciably

showed that even diminutive amounts of saponins in combination with proteins are


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of sugar beet, larger proteins (>20–30 kDa) should have been removed, leading to the

310

accumulation of smaller protein fractions. In addition, larger subunits may have

311

broken down during the lyophilization process.43 This suggests that the concentration

312

of low molecular weight fractions of proteins in sugar beet extract are mainly

313

responsible for the stabilization of the emulsions. This is also in agreement with

314

earlier studies reporting that low molecular weight proteins such as β-lactoglobulins

315

(~18 kDa) at low concentrations (~0.5% w/w) can form oil-in-water emulsions (10%

316

w/w) with d32 values of >0.15 µm44 using ~0.5% (w/w), whereas much higher

317

concentrations (~5% w/w) were needed for higher molecular weight proteins

318

(lactoferrin, ~80 kDa).44 For comparison, other commercially available natural

319

emulsifiers such as polysaccharides (gum arabic), phospholipids (lecithin), and

320

proteins (whey protein) can form submicron sized oil-in-water emulsion droplets (10%

321

w/w) at concentrations of ~3%, ~1 %, and ~0.5% (w/w).9, 45 This indicates that the

322

used sugar beet extract is effective at forming emulsions. In contrast, Quillaja extract

323

had a higher saponin-to-protein ratio (Tab. 1), and therefore the interfaces of the

324

emulsions may mainly be covered by saponins.12, 46

325

Influence on the ζ-potential

326

The

327

−49.4 ± 2.7 mV at 0.1% (w/w) up to −69.8 ± 3.7 mV at 1% (w/w), and stayed fairly

328

constant even at higher concentrations (Fig. 3). This electrical charge is in agreement

329

with literature values of around –60 mV for saponins at pH 7.38 For the sugar beet

330

extract stabilized emulsions, the ζ-potential of the droplets decreased from

331

−38.7 ± 1.5 mV at 0.1% (w/w) to almost constant values of ~−46 mV >1% (w/w).

332

The differences in ζ-potential between both extracts may be explained by several

333

factors, including the different extract compositions. The negative ζ-potential values

ζ-potential

of

Quillaja

extract

stabilized

emulsions

decreased

from



Page 16 of 31

334

of emulsion droplets stabilized by either Quillaja or sugar beet extract may be

335

attributed to carboxylic acid groups with typical pKa values of ~3.5.12 At pH 7, this

336

group would be fully charged (-COO-), thus leading to a strong negative droplet

337

charge. On the other hand, sugar beet extract had a higher protein content

338

(4.00 ± 0.10%, w/w, Tab. 1) compared to Quillaja extract (1.61 ± 0.22%, w/w) that

339

may influence the ζ-potential as proteins have acidic and basic groups. The

340

commercially available whey, soy and chickpea proteins typically exhibit negative ζ-

341

potentials around –40 mV at pH 7,

342

by proteins than saponins. Other natural emulsifiers such as gum arabic (a complex

343

mixture of glycoproteins and polysaccharides) and lecithin typically have negative ζ-

344

potentials around –30, and –60 mV, respectively.38

345

In general, this large negative ζ-potential can have a significant role for the

346

application in foods and beverages for two main reasons: First, cationic multivalent

347

ions may induce bridging due to the opposite electrical charge leading to flocculation3,

348

12

349

opposite charge leading to lipid oxidation.12,

350

extract formed larger emulsion droplets and showed a lower effectiveness at

351

generating

352

destabilization mechanisms than Quillaja extract as depicted in Fig. 2 and Fig. 3.2

353

Overall, both extracts were capable of forming highly negatively charged emulsions,

354

where the emulsion size depended on the concentration and the used homogenization

355

conditions. Nevertheless, the sugar beet extract was not as efficient as the Quillaja

356

extract.

357

Influence of homogenization conditions on emulsion formation

38, 47, 48

which also suggests a stabilization rather

. Second, cationic pro-oxidants may be attracted to the droplet surface due to the

repulsive

interactions

to

47

Based on these results, sugar beet

stabilize

emulsions

against

physical


Page 17 of 31


358

The mean droplet size is mainly influenced by the high-pressure homogenization

359

conditions such as the homogenization pressure when sufficient emulsifier is present.4,

360

10, 49

361

homogenization pressure on the formation of emulsions (10% w/w) using both

362

extracts at 0.75% (w/w).

363

Sugar beet extract stabilized emulsions showed an initial decrease in both d32 and d43-

364

values up to a homogenization pressure of 69 MPa (Fig. 4). At 86 MPa and above, the

365

d32-values decreased, whereas the d43-values increased, indicating that larger droplets

366

were formed at higher homogenization pressures. Similar behavior with increasing

367

mean particle sizes at higher homogenization pressures has been observed for

368

biopolymer stabilized emulsions, which is referred to as ‘over-processing’.10,

369

This happens if the shear forces dissipating during the homogenization step lead to a

370

change in the protein conformation due to the rupture of non-covalent interactions.

371

This modifies the emulsifying properties, as intra- and intermolecular bonds between

372

and within proteins are re-formed, which may again lead to larger emulsion

373

droplets.51, 52

374

For Quillaja extract stabilized emulsions, the mean droplet size (both d32 and d43)

375

decreased with increasing homogenization pressure (Fig. 4a, d43: 0.094 ± 0.004 µm),

376

which can be attributed to the increase in disruptive energy input.53 Moreover, we

377

observed an almost linear relationship (R2: 0.94) between log(d) and log(P) for

378

Quillaja extract stabilized emulsions. The equation for this linear relationship was

379

Log(d43) = −0.522 Log(P) + 4.38. The slope of −0.52 was slightly lower compared to

380

previously reported values between −0.6 and −0.8 for high-pressure homogenizers

381

using synthetic (Tween 80) and natural emulsifiers (Quillaja extract, Q-Naturale®)12,

382

but close to the value reported for turbulent-inertial breakup.10, 54 We attributed the

Consequently, the objective of these experiments was to evaluate the influence of

41, 50



Page 18 of 31

383

lower slope to a slightly lower adsorption rate of the used Quillaja extract onto the

384

interface as seen in Fig. 1 and described by previous studies.19, 20 It should be noted,

385

that differences in the emulsifier composition as well as in homogenizer designs and

386

sample preparation may lead to different results. The increase in homogenization

387

pressure led to a reduction of the width in particle size distribution for Quillaja extract

388

stabilized emulsions (Fig. 4c). In contrast, a broader particle size distribution with

389

higher homogenization pressures was obtained for sugar beet extract stabilized

390

emulsions (Fig. 4d) that showed larger droplets at higher homogenization pressure.

391

No similar linear dependence between droplet size and pressure was observed for

392

sugar beet extract (data not shown), which may also be explained by ‘over-

393

processing’.

394

In summary, this study showed for the first time that a natural extract obtained from

395

sugar beet can be used as a natural emulsifier. This offers great opportunities for food,

396

feed and pharmaceutical industries to stabilize dispersions. Additional investigations

397

focusing on the influence of saponins and proteins on the stability of the prepared

398

emulsions regarding external stresses are underway. In addition, in order to gain a

399

better understanding of the structure-function relationship between the saponins and

400

proteins, purified compounds with determined molecular structures need to be further

401

analyzed.

402

ABBREVIATIONS USED

403

GAE

Gallic acid equivalent


Page 19 of 31


404

ACKNOWLEDGEMENTS

405

We acknowledge Pfeifer & Langen GmbH Co. KG for generously providing us sugar

406

beet samples. We would like to thank Sonja Schlosser and Holger Hrenn

407

(Landesanstalt für Landwirtschaftliche Chemie, University of Hohenheim) for

408

conducting the mineral and fat analysis, respectively, Barbara Maier (University of

409

Hohenheim) for taking some of the microscopic images and Hanna Bogner for

410

numerous fruitful discussions.

411

SUPPORTING INFORMATION DESCRIPTION

412

No supporting information is provided.



Page 20 of 31

413

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414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461

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20. Wojciechowski, K.; Piotrowski, M.; Popielarz, W.; Sosnowski, T. R., Shortand mid-term adsorption behaviour of Quillaja bark saponin and its mixtures with lysozyme. Food Hydrocolloid 2011, 25, 687-693. 21. Vincken, J.-P.; Heng, L.; de Groot, A.; Gruppen, H., Saponins, classification and occurrence in the plant kingdom. Phytochem 2007, 68, 275-297. 22. Chapagain, B. P.; Wiesman, Z.; Tsror, L., In vitro study of the antifungal activity of saponin-rich extracts against prevalent phytopathogenic fungi. Ind Crop Prod 2007, 26, 109-115. 23. FAO Production of sugarbeet in the EU. http://faostat3.fao.org/ 24. Yoshikawa, M.; Murakami, T.; Kadoya, M.; Yamahara, J.; Matsuda, H., Medicinal Foodstuffs. XV. Sugar Beet.(2): Structures of Betavulgarosides V, VI, VII, VIII, IX, and X from the Roots and Leaves of Sugar Beet (Beta vulgaris L., Chenopodiaceae). Chem Pharm Bull 1998, 46, 1758-1763. 25. Yoshikawa, M.; Murakami, T.; Kadoya, M.; Yamahara, J.; Matsuda, H., Medicinal Foodstuffs. XV. Sugar Beet.(1): Hypoglycemic Oleanolic Acid Oligoglycosides, Betavulgarosides I, II, III, and IV, from the Root of Beta vulgaris L. (Chenopodiaceae). Chem Pharm Bull 1996, 44, 1212-1217. 26. Böttcher, S.; Scampicchio, M.; Drusch, S., Mixtures of saponins and betalactoglobulin differ from classical protein/surfactant-systems at the air-water interface. Colloid Surface A 2016, 506, 765-773. 27. Stanimirova, R.; Marinova, K.; Tcholakova, S.; Denkov, N. D.; Stoyanov, S.; Pelan, E., Surface rheology of saponin adsorption layers. Langmuir 2011, 27, 1248698. 28. Bremner, J. M.; Mulvaney, C. S., Nitrogen—total. In Methods of soil analysis. Part 2. Chemical and microbiological properties, Page, A. L.; Miller, R. H., Eds. ASA and SSSA: Madison, WI, 1982. 29. Regulation, E. C., No. 152/2009, Annex III, Part H. 2009. 30. VDLUFA, VDLUFA-Methodenbuch Band VII „Umweltanalytik “. In VDLUFA Verlag: Darmstadt, Germany, 2011. 31. Singleton, V.; Rossi, J. A., Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Eenol Viticult 1965, 16, 144158. 32. Sirisakulwat, S.; Nagel, A.; Sruamsiri, P.; Carle, R.; Neidhart, S., Yield and quality of pectins extractable from the peels of Thai mango cultivars depending on fruit ripeness. J Agr Food Chem 2008, 56, 10727-10738. 33. Vargaftik, N. B.; Volkov, B. N.; Voljak, L. D., International tables of the surface tension of water. J Phys Chem Ref Data 1983, 12, 817-820. 34. Maier, C.; Conrad, J.; Steingass, C. B.; Beifuss, U.; Carle, R.; Schweiggert, R. M., Quillajasides A and B: New phenylpropanoid sucrose esters from the inner bark of Quillaja saponaria molina. J Agr Food Chem 2015, 63, 8905-11. 35. Brehneva, T. A.; Nikolaevskii, V. A.; Selemenev, V. F.; Slivkin, A. I.; Muad, A. A.; Khind, T.; Safonova, E. F., Isolation of saponins from sugar beet roots and preliminary characterization of their adaptogen properties. Pharm Chem J-USSR 2001, 35, 159-161. 36. Parpineuo, G.; Versari, A.; Riponi, C.; Parpinello, P., Characterization of sugar beet (Beta Vulgaris L.) protein. J Sugar Beet Res 2004. 37. Salminen, H.; Weiss, J., Effect of Pectin Type on Association and pH Stability of Whey Protein—Pectin Complexes. Food Biophys 2013, 9, 29-38.



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38. McClements, D. J.; Gumus, C. E., Natural emulsifiers - biosurfactants, phospholipids, biopolymers, and colloidal particles: molecular and physicochemical basis of functional performance. Adv Colloid Interfac 2016, 234, 3-26. 39. Kezwon, A.; Wojciechowski, K., Interaction of Quillaja bark saponins with food-relevant proteins. Adv Colloid Interfac 2014, 209, 185-95. 40. de Faria, J. T.; de Oliveira, E. B.; Minim, V. P. R.; Minim, L. A., Performance of Quillaja bark saponin and β-lactoglobulin mixtures on emulsion formation and stability. Food Hydrocolloid 2017, 67, 178-188. 41. Jafari, S. M.; Assadpoor, E.; He, Y.; Bhandari, B., Re-coalescence of emulsion droplets during high-energy emulsification. Food Hydrocolloid 2008, 22, 1191-1202. 42. Uluata, S.; McClements, D. J.; Decker, E. A., Physical stability, autoxidation, and photosensitized oxidation of ω-3 oils in nanoemulsions prepared with natural and synthetic surfactants. J Agr Food Chem 2015, 63, 9333-40. 43. Parpineuo, G.; Versari, A.; Riponi, C.; Parpinello, P., Characterization of sugarbeet (Beta vulgaris, L.) protein. 2004. 44. Mao, Y.; McClements, D. J., Modulation of bulk physicochemical properties of emulsions by hetero-aggregation of oppositely charged protein-coated lipid droplets. Food Hydrocolloids 2011, 25, 1201-1209. 45. Ozturk, B.; Argin, S.; Ozilgen, M.; McClements, D. J., Formation and stabilization of nanoemulsion-based vitamin E delivery systems using natural biopolymers: Whey protein isolate and gum arabic. Food Chem 2015, 188, 256-63. 46. Chung, C.; Sher, A.; Rousset, P.; McClements, D. J., Use of natural emulsifiers in model coffee creamers: Physical properties of Quillaja saponinstabilized emulsions. Food Hydrocolloid 2017. 47. Hu, M.; McClements, D. J.; Decker, E. A., Lipid oxidation in corn oil-in-water emulsions stabilized by casein, whey protein isolate, and soy protein isolate. J Agr Food Chem 2003, 51, 1696-1700. 48. Can Karaca, A.; Nickerson, M. T.; Low, N. H., Lentil and chickpea proteinstabilized emulsions: optimization of emulsion formulation. J Agric Food Chem 2011, 59, 13203-11. 49. Walstra, P., Formation of emulsions. In Encyclopedia of Emulsion Technology, Becher, P.; Dekker, M., Eds. Ney York, NY, 1983; Vol. 1 - Basic Theory. 50. Jafari, S. M.; He, Y.; Bhandari, B., Production of sub-micron emulsions by ultrasound and microfluidization techniques. J Food Eng 2007, 82, 478-488. 51. Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C., Formation and stability of nano-emulsions. Adv Colloid Interfac 2004, 108, 303-318. 52. Messens, W.; Van Camp, J.; Huyghebaert, A., The use of high pressure to modify the functionality of food proteins. Trends in Food Science & Technology 1997, 8. 53. Floury, J.; Desrumaux, A.; Lardieres, J., Effect of high-pressure homogenization on droplet size distributions and rheological properties of model oilin-water emulsions. Innov Food Sci Emerg 2000, 1, 127-134. 54. Walstra, P., Physical chemistry of foods. CRC Press: New York, NY, 2002.

553


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554

FUNDING SOURCE

555

This work was supported by the FEI (Forschungskreis der Ernährungsindustrie e.V.),

556

Bonn, Germany via AiF/BMWi (AiF 18815N).



557

FIGURE CAPTIONS

558 559

Fig. 1: Influence of the extract concentration (pH 7) on the interfacial tension at an oil-water interface (a) and surface tension at an air-water interface (b) at 25 °C.

Page 24 of 31

560 561 562 563 564 565 566

Fig. 2: Influence of the extract concentration on the mean particle size d32 (a) and d43 (b) and the corresponding particle size distribution (d43) of Quillaja (c) and sugar beet extract (d) stabilized 10% (w/w) oil-in-water emulsions (pH 7) produced under standardized homogenization conditions (69 MPa, 4 cycles) after 24 h storage at 5 °C. Optical microscopy image insets of emulsions stabilized by 0.75 and 5.0% (w/w) sugar beet extract in (b).

567 568 569 570

Fig. 3: Influence of extract concentration on the ζ-potential of 10% (w/w) oil-in-water emulsions (pH 7) stabilized by Quillaja or sugar beet extract produced under standardized homogenization conditions (69 MPa, 4 cycles) after 24 h storage at 5 °C.

571 572 573 574 575

Fig. 4: Influence of the homogenization pressure (4 cycles) on the mean particle size d32 (a) and d43 (b) as well as the corresponding particle size distributions (d43) of Quillaja (c) and sugar beet extract (d) stabilized 10% (w/w) oil-in-water emulsions produced using 0.75% (w/w) sugar beet and Quillaja extract after 24 h storage at 5 °C.


Page 25 of 31


576

Tab. 1: Chemical composition of the used plant extracts in % (w/w). Extract

Saponin

Mineral

Protein

Lipid

Quillaja

>68.6a

3.94 ± 0.00b

1.61 ± 0.22

< 0.6c

Polyphenol (mg GAE/g) 4.52 ± 0.05

Sugar beet

>0.5

0.44 ± 0.00

4.00 ± 0.10

< 0.6c

0.07 ± 0.04

a

stated by the manufacturer

b

reference 13

c

below the detection limit (< 0.6%)

577



Page 26 of 31

Fig. 1


Page 27 of 31


Fig. 2



Page 28 of 31

Fig. 3


Page 29 of 31


Fig. 4

578



Page 30 of 31

579


Page 31 of 31


580

TABLE OF CONTENTS

581


Sugar Beet Extract - American Chemical Society

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