Formation and Stability of Emulsions Prepared with a Water-Soluble

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Formation and Stability of Emulsions prepared with a Water Soluble Extract from the Microalga Chlorella protothecoides Lutz Grossmann, Sandra Ebert, Jorg Hinrichs, and Jochen Weiss J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05337 • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 20, 2019

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Journal of Agricultural and Food Chemistry

Formation and Stability of Emulsions prepared with a Water-Soluble Extract from the Microalga Chlorella protothecoides

Lutz Grossmanna, Sandra Eberta, Jörg Hinrichsb, Jochen Weissa

a

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

b

Department of Soft Matter Science and Dairy Technology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21, 70599 Stuttgart, Germany

Re-Submitted to Journal of Agricultural and Food Chemistry March 2019

*

Correspondence should be addressed to: Phone +49 711 459 24415, Fax: +49 711 459 24446, Email: [email protected]

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ABSTRACT

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Unicellular microalgae are a valuable source of macro- and micronutrients. They contain for

3

example proteins that are potentially useful as novel emulsifiers. The aim of this study was to

4

investigate the emulsifying properties of a less-refined lyophilized crude water-soluble extract

5

(WSE), obtained from the heterotrophically cultivated microalga Chlorella protothecoides.

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Interfacial activity measurements indicated that mainly the proteins in the extract showed

7

interfacial activity. O/W-emulsions were thus prepared by high-pressure homogenization

8

(1,000 bar, 3 passes) with 5.0 wt% of oil and 2.5 wt% of protein from Chlorella protothecoides,

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resulting in emulsions having a volume based mean droplet diameter d43 ≤ 1 µm, and being

10

stable for at least 7 days. Two different stress tests showed that (i) protein-stabilized emulsions

11

were resistant to very high salt concentrations (up to 500 mM NaCl); (ii) emulsions were stable

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over a very broad pH-range of 2-9, with only minor changes in the particle size d43 (increase of

13

300 nm when pH was lowered from 5 to 4) compared to whey protein stabilized emulsions. All

14

WSE emulsions had monomodal particle size distributions and were macro- and

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microscopically stable during a storage of up to 1 week. The results indicate that the WSE of

16

Chlorella protothecoides has remarkably good emulsifying properties and may thus be of use

17

as novel emulsifier in various applications where emulsions are exposed to a broad range of

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ionic strengths and pH-values.

19 20

Keywords: Microalgae; Chlorella; Emulsions; Protein; Stability


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1 INTRODUCTION

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The use of unicellular microalgae in supplementing human diets is well established in many

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Asian and African countries where algae have a long tradition of being consumed and are

24

known to being highly nutritious1. Yet, they have only recently been introduced in western

25

countries, and only few macroalgae as well as microalgae fortified foods currently exist1-2. This

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is mainly caused by a low consumer acceptance, since the incorporation of microalgae biomass

27

may alter taste, odor, and color of the end product. Additionally, the thick and rigid cell walls

28

of most microalgae result in a low digestibility and thus the nutritional benefits of microalgae-

29

based foods are limited3. This fact also makes it difficult to take advantage of the potential

30

beneficial techno-functional properties of various internal cell compounds4-7.

31

Various approaches have therefore been investigated to disrupt the rigid cells and facilitate a

32

liberation of functional compounds8-12. Besides lipids and carotenoids, microalgae proteins

33

have been identified as particularly attractive cell constituents, since they may not only be used

34

as an important source of essential and non-essential amino acids, but also might possess useful

35

techno-functionalities such as emulsifying or foaming properties.

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One of the most heavily investigated microalgae is Chlorella; a unicellular, small, spherical,

37

and non-motile genus. It comprises some species that are highly tolerant to a range of

38

environmental and nutrient states and can accumulate high amounts (up to 70 %) of proteins1.

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In this study, the promising heterotrophically cultivated microalga Chlorella protothecoides

40

was used. It is cultivated in a common bioreactor without light, employing a reduced carbon

41

source as feeding material. It was previously shown that heterotrophic cultivation may be

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superior in terms of environmental sustainability compared to phototrophic cultivation and that

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the final biomass contains less pigments, such as chlorophyll13. This particular species has an

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initial protein content in the cells of 48.2 wt%14. To extract proteins from this microalga, a

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process scheme for the production of protein extracts was previously developed. The method


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uses high pressure-homogenization to disrupt the cells, followed by a protein fractionation by

47

centrifugation and further processing to obtain less-refined lyophilized water-soluble proteins14.

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The process was developed with four intentions: (i) to maintain protein functionality since the

49

use of organic solvents may lead to unwanted configurational changes that may limit interfacial

50

activity, (ii) to minimize downstream efforts thereby keeping costs and protein losses low, (iii)

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to decrease environmental impacts through the use of water as a solvent, and (iv) to retain a

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lower refinement character of the extracts since presence of e.g. polysaccharide moieties has

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shown to be beneficial to certain technofunctionalities, such as the steric stabilization of oil-in-

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water emulsion droplets.

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It was shown that the water-soluble proteins from Chlorella protothecoides obtained by this

56

method retained a high solubility (≥ 84.3±2.2 %) at a pH of 2-6, making them potentially useful

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to stabilize acidified emulsions15. Similarly, emulsions prepared with soluble protein fractions

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from Tetraselmis sp. remained charged at pH values of 3-7, that is they displayed no discernable

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charge transition in that range16. Such emulsions are for example of interest to beverage

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manufactures that ensure microbial safety through a combination of acidification, thermal

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treatments and/or low-pH active antimicrobials.

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In general though, data on emulsifying properties of microalgae compounds is limited and has

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only been reported for a few microalgae so far16-17. The observed high solubility of Chlorella

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protothecoides proteins might enable the stabilization of emulsions at varying environmental

65

conditions such as at different pH and ionic strengths. For this reason, the aim of this work was

66

to study the interfacial activity and emulsifying properties of lyophilized water-soluble extracts

67

from Chlorella protothecoides.


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2 MATERIALS & METHODS

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2.1

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Heterotrophically cultivated Chlorella protothecoides (AlgilityTM HP) was purchased from

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Roquette Frères (Lestrem, France). Medium chain triglyceride oil (Miglyol812, MCT) was

72

bought from Cremer Oleo GmbH & Co. KG (Hamburg, Germany). Whey protein isolate (WPI

73

895) was obtained from Fonterra (Auckland, New Zealand) and contained 93.9% protein. Citrus

74

pectin classic CU-L 002/15 with a degree of esterification of 71% was kindly donated by

75

Herbstreith & Fox KG (Neuenburg, Germany). Tween 20 (Ph. Eur.) was purchased from Carl

76

Roth GmbH & Co. KG (Karlsruhe, Germany). ProclinTM 950 was obtained from Sigma-Aldrich

77

(Missouri, United States). Other chemicals, reagents, and solvents were obtained from Carl

78

Roth GmbH & Co. KG (Karlsruhe, Germany) and were of analytical grade, unless otherwise

79

stated.

80

2.2

81

Protein extracts from Chlorella protothecoides were prepared according to a reported method

82

by Grossmann, et al. 14 with slight modifications. Cells were first disrupted in a high-pressure

83

homogenizer (M110-EH-30, Microfluidics International Cooperation, Newton, MA). A total of

84

6 passes was used to disintegrate cells, and the interaction chamber was cooled to 22 °C to

85

minimize heat denaturation. The resulting disrupted cell suspension was centrifuged (20,000 g,

86

30 min, 25 °C) to separate the water-soluble proteins from the water-insoluble protein fraction.

87

After centrifugation, the supernatant (water-soluble protein fraction) was carefully decanted

88

from the pellet and vacuum filtered (Sartorius AG, Göttingen Germany) using a filter with a

89

pore size of 8-12 µm (FT-3-303-045, Sartorius AG, Göttingen Germany). The permeate

90

containing the water-soluble protein fraction was then lyophilized (L10, WKF-Gesellschaft für

91

elektrophysikalischen Apparatebau, Brandau, Germany) to obtain a dry water-soluble extract

92

(WSE). Lyophilization was carried out in two regimes, whereas tray temperature and pressure

Materials

Fabrication of water-soluble protein extracts


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were -20 °C and 10-1 mbar during first and 20 °C, 10-2 mbar during secondary drying. The

94

obtained lyophilized WSE (refer to product number 3 in Grossmann, et al. 14) was ground with

95

a mortar and pestle, to obtain a homogeneous product, and transferred into air-tight glass vials

96

to keep samples from absorbing moisture. Glass vials were stored at room temperature prior to

97

further analysis.

98

2.2.1 Proximate composition of lyophilized WSE

99

Nitrogen and protein content. Total nitrogen content was determined by using the Dumas

100

method18. In short, the sample was weighed accurately in tin capsules and combusted with

101

oxygen at 950 °C. The resulting release of nitrogen was detected with a thermal conductivity

102

detector (FP-528, LECO Corporation, St. Joseph, MI, USA). Calibration was done with EDTA

103

with a known nitrogen content of 9.58 %. A nitrogen-to-protein conversion factor of 4.24 was

104

used to calculate the total protein content (wt%)14-15.

105

Total lipid content. To assess the total lipid content, the powder was analyzed gravimetrically

106

according to the method of Weibull-Stoldt with subsequent Soxhlet extraction19. Briefly, 10 g

107

of powder was weighed into Merck reaction tubes (NS 29/32, Merck KGaA, Darmstadt,

108

Germany) and topped with 4 M hydrochloric acid. The sample was boiled for 60 min at 115 °C

109

in a thermoreactor (TR 105, Merck KGaA, Darmstadt, Germany), cooled down and transferred

110

into ash-free folded filters (MN 615 ff ¼, Macherey-Nagel GmbH & Co. KG, Düren, Germany).

111

After washing, folded filters were dried overnight and extracted with petroleum ether in a

112

Soxhlet extractor (Büchi 810, Büchi Labortechnik GmbH, Essen, Germany) for 6 hours.

113

Extracted lipids were collected and weight to obtain the lipid content. The employed Weibull-

114

Stoldt method is a certified method according to the German food legislation to determine the

115

total fat content of food products. Commonly, 1-2 g are used for the extraction. Due to the low

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intrinsic fat content of the microalgae extract, 10 g of sample was used to extract a sufficient

117

amount of fat to calculate the lipid concentration with a high accuracy. ACS Paragon Plus Environment

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Water content. The water content was determined by Karl-Fischer titration20. Measurements

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were carried out with an 841 Titrando titrator system with an 800 Dosino dosing system

120

(Metrohm AG, Herisau, Switzerland) using a two-component reagent (titrant: iodine and

121

methanol; solvent: methanol imidazole and sulfur dioxide). Analysis was performed at room

122

temperature. Samples were titrated for at least 300 s, using a bipotentiometric titration method.

123

The end voltage was set to 100 mV, and an additional drift stop of 15 µL min-1 was applied.

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Mineral content. The total mineral content was analyzed by pre-incineration, followed by a

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total combustion in a muffle furnace at 600 °C21. About 2 g of sample were weighed into

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ceramic bins. Magnesium acetate was added to help disperse the powder and samples were pre-

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incinerated by slowly increasing the temperature from 300 to 550 °C. Ceramic bins were

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subsequently put into the muffle furnace for 3 days to complete the combustion. Differential

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weighing yielded the total mineral content.

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Carbohydrate content. The carbohydrate content was calculated as the difference of all

131

components from 100 % (Eq. 1):

132

Carbohydrate (%) = 100 % - Protein (%) – Lipids (%) – Ash (%) – Water (%)

(1)

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2.3

134

Emulsions were prepared using a two-step homogenization process. The lyophilized WSE was

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re-dispersed in bi-distilled water overnight and a coarse primary emulsion was prepared by

136

mixing the dispersed WSE and the oil phase (5 wt% MCT Miglyol 812) with a high shear

137

blender at 24,000 rpm for 2 min (Silent Crusher M with dispersing element 22 G, Heidolph

138

Instruments GmbH & Co. KG, Schwabach, Germany). Primary emulsions were subsequently

139

passed 3 times through a high-pressure homogenizer (LM10 equipped with a G10Z interaction

140

chamber, Microfluidics International Cooperation, Newton, MA) at a process pressure of 1,000

141

bar to obtain a mean droplet size of ~1 µm. A refrigerated ice-water bath was connected to keep

142

process temperature as low as 22 °C in order to prevent heat denaturation of proteins.

Emulsifying properties of lyophilized WSE from C. protothecoides


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Optimum protein concentration. To establish an optimum protein concentration to produce

144

stable emulsions, lyophilized WSE was dissolved in bi-distilled water to obtain final total

145

protein concentrations (i.e. not extract concentration) in the emulsion of 1.7-3.3 wt%. 0.5 wt%

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of whey protein isolate (WPI) was used as a reference protein. All emulsions were prepared

147

with 5 wt% of a standardized MCT.

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Stress test I: pH-shift. O/W-emulsions were adjusted to pH values ranging from 2 to 9 (integer

149

steps) by adding HCl and NaOH (0.1 M, 0.25 M, 1 M, 2 M and 6 M) after emulsion formation.

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Samples were stirred for 30 min. During this time, the pH was regularly adjusted to compensate

151

for the buffer capacity of the proteins and was finally set to the desired value with an accuracy

152

of ±0.05 and checked again after 24 h.

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Stress test II: ionic strength. Various concentrations of sodium chloride (0 mM, 25 mM, 50

154

mM, 100 mM, 250 mM, 500 mM) were achieved by mixing the O/W-emulsions at their native

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pH with an appropriately concentrated stock solution at a ratio of 9:1 (v/v).

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All samples were stored at 4 °C and analyzed after 24 h and again after 7 days. 50 ppm of

157

ProclinTM 950 was added to inhibit any microbial growth.

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2.4

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Surface charge measurement. The surface charge was measured as the ζ-potential of the

160

droplets using a particle electrophoresis instrument (Nano ZS, Malvern Instruments, Malvern,

161

UK). Samples were diluted 1:50 with pH-adjusted bi-distilled water to obtain the required

162

particle concentration and transferred into disposable folded capillary cells (DTS1070, Malvern

163

Instruments, Malvern, UK). All measurements were taken at 25 °C. The Smoluchowski model,

164

which is used for particles larger than 200 nm and salt concentrations above 1 mM, was used

165

to calculate the ζ-potential from the electrophoretic mobility of droplets that are moving in the

166

applied electric field.

Analysis of O/W-emulsions


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Particle size measurement. Particle size distributions of O/W-emulsions were determined using

168

a static light scattering device (Horiba LA-950, Retsch Technology GmbH, Haan, Germany).

169

O/W-emulsions were diluted 1:1000 with pH-adjusted bidistilled water to prevent multiple

170

scattering. The measurement principle is based on the angular dependence of the intensity of a

171

laser beam, which is scattered by the particles in the diluted emulsion. The Mie theory was used

172

to calculate the particle size distribution from the scattering pattern, and the volume based mean

173

diameter d43 was determined according to Equation 2. A refractive index of 1.45 (related to the

174

oil phase MCT) was used for all measurements.

175

d4,3 =

∑ d4i ni ∑ d3i ni

(2)

176

d43 = volume based mean diameter (m), di = diameter of ith particle related to size of

177

measurement channel, ni = number of particles in ith measurement channel

178

Visualization of microstructure. An optical light microscope was used to characterize the

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emulsions’ microstructure. After gently mixing the samples, about 10 µl of O/W-emulsion were

180

transferred on an objective slide and covered with a cover slip. An axial mounted Canon

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Powershot G10 digital camera (Canon, Tokyo, Japan) was used to take pictures of the samples

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under an Axio Scope optical microscope (A1, Carl Zeiss Microimaging GmbH, Göttingen,

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Germany) at a 400-fold magnification.

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2.5

185

The interfacial tension was measured for four different systems at a fixed protein concentration

186

of 0.04 wt%: (i) lyophilized WSE of C. protothecoides, (ii) whey protein isolate, (iii) citrus

187

pectin (c = 0.04 wt%), and (iv) tween 20 (c = 0.04 wt%).

188

The interfacial tension was determined for each system with a drop-shape analyzer (DSA-G10,

189

MK2, Krüss, USA) at the oil-water interface. In this method, a pendant drop of the sample

190

solution is formed at the tip of a syringe and the interfacial tension is then calculated from the

Interfacial tension


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shape of the drop. The shape is determined by the balance of forces acting on the drop as

192

described by the Young-Laplace equation. The software performs a numerical iterative method

193

to fit the calculated drop shape to the recorded shape of the drop. To measure the interfacial

194

tension, the respective sample solution was filled into a syringe with a narrow tip (d = 0.90 mm)

195

and submerged into a cuvette with the oil phase (MCT, Miglyol 812). The syringe/cuvette

196

system was positioned on an optical bench between a light source and a high-speed charge

197

coupled device camera. The required densities for the calculation of the interfacial tension were

198

measured with a digital density meter (DMA 35N, Anton Paar Physica, Ostfildern-

199

Scharnhausen, Germany).

200

2.6

201

All experiments were done at least in duplicate from freshly prepared samples and all

202

measurements were done at least in twice. A one-way analysis of variance with a Tukey-test

203

was carried out to test statistically significant differences among samples. An α-level of 0.05

204

was used for all tests (SPSS statistics V23, IBM Corp., Armonk, USA). Results are reported as

205

mean ± standard deviation.

Statistics


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3 RESULTS & DISCUSSION

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3.1

208

Before emulsion preparation, the composition of lyophilized WSE from Chlorella

209

protothecoides was analyzed in terms of its macro components (Table 1). As expected, the

210

powder mainly consisted of proteins and carbohydrates, with a low inherent color, compared to

211

whole spray dried microalgae cells of Chlorella protothecoides. This is in accordance with

212

previous results, which showed that microalgae protein fractions contained a high amount of

213

proteins, but also a considerable amount of other components11, 14.

214

3.2

215

The aim of the first experiments was to investigate whether the obtained extract shows

216

interfacial activity and thus might be suitable as potential emulsifier. Moreover, because the

217

extract contains a mixture of carbohydrates, proteins, and residual lipids, the adsorption

218

behavior could indicate which component is mainly adsorbing to the interface. The interfacial

219

tension at the oil/water interface was measured over the course of 25 min for: whey proteins

220

(pure protein), citrus pectin (pure polysaccharide), tween 20 (water-soluble small molecular

221

weight surfactant), and the WSE (Figure 1). These different components were chosen to reveal

222

general patterns in adsorption behavior and subsequently compare their behavior with that of

223

WSE.

224

First, the WSE from Chlorella protothecoides was able to decrease the interfacial tension and

225

the final interfacial tension obtained was lower compared to the final interfacial tension of whey

226

protein isolate at pH 7 (Figure 1). However, overall adsorption behavior at pH 7 was

227

comparable to that of whey protein, indicating the presence of surface-active proteins in the

228

WSE. Citrus pectin did not decrease the interfacial tension to a great extent (interfacial tension

229

of the pure MCT/water interface was 25.9±0.5 mN m-1) even though it had a high degree of

230

esterification (DE 71%)22. Thus, it can be concluded from the profile that pure unaltered

Proximate composition of lyophilized powder

Stabilizing component: interfacial tension profiles


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surface-active polysaccharides in the WSE do not play a major role in decreasing the interfacial

232

tension23-24. Additionally, surface-active polysaccharides are large molecules that increase the

233

viscosity of the solution and thereby enhance the stability of the interface. The extract used in

234

the study can be dissolved at concentrations of more than 35 wt% without incurring any

235

pronounced thickening, indicating that larger polysaccharides are not present to a great extent.

236

Finally, tween 20 as a water-soluble small-molecular weight surfactant decreased the interfacial

237

tension faster and to a greater extent, which was different from the profile obtained for the WSE.

238

Second, the pH of the solution was varied to study how the adsorption behavior changes with

239

varying pH. Here, the adsorption behavior of whey protein changed with pH (after 25 min: pH

240

3: 19.2±0.5 mN m-1, pH 5: 12.3±0.3 mN m-1, pH 7: 17.9±0.3 mN m-1) whereas the changes

241

observed for the WSE C. protothecoides were rather small (after 25 min: pH 3: 15.3±0.3 mN

242

m-1, pH 5: 14.4±0.2 mN m-1, pH 7: 16.7±0.2 mN m-1). A faster decrease in interfacial tension

243

near the isoelectric point of the proteins is commonly attributed to a decrease in electrostatic

244

repulsion between the protein and the interface, which increases the adsorption at the

245

interface25. Such strong change in adsorption behavior with changing pH was not observed for

246

the WSE of C. protothecoides, which might be related to the previously detected high degree

247

of hydrophilic amino acids and the presence of glycoproteins in this extract that can alter the

248

adsorption behavior15, 26.

249

Taken together, the presented results gave a first implication about which component in the

250

WSE of Chlorella protothecoides is adsorbing to the oil/water-interface. The measurements

251

indicated that mainly proteins adsorb to the interface and that some of these proteins might be

252

glycosylated. Nonetheless, even though the initial composition at the interface might be mainly

253

proteins, slower adsorbing surface-active polysaccharides could alter the interface during

254

interfacial aging, resulting in a mixed interface. More studies will be necessary to further

255

provide evidence for the specific role of each component at the interface.


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3.3

257

The observed interfacial adsorption behavior of the proteins in the lyophilized WSE might

258

enable the formulation of stable food emulsions. The preparation of a stable emulsion is related

259

to an optimal concentration of the emulsifier in the oil-water system27. Because the results from

260

the previous chapter indicated that mainly proteins adsorb to the interface, three different

261

protein concentrations of 1.7, 2.5, and 3.3 wt% (i.e. not extract concentration) were tested to

262

find the minimal needed amount of proteins from Chlorella protothecoides to form and stabilize

263

a 5 wt% O/W-emulsion obtained by high-pressure homogenization (Figure 2). The optimal

264

protein concentration was determined by the following criteria: (i) a protein concentration,

265

where a further increase does not lead to a significant decrease (p ≤ 0.05) in volume based mean

266

diameter d43, (ii) a sufficiently small d43 to prevent O/W-emulsions from destabilization by

267

gravitational separation (d43 ≤ 1 µm), and (iii) no significant increase (p > 0.05) in d43

268

throughout 7 days storage.

269

All concentrations resulted in a macroscopically stable O/W-emulsion. Yet, a storage test of 7

270

days revealed destabilization at a protein concentration of 1.7 wt% with a significant (p ≤ 0.05)

271

increase in d43 and final values of ≥ 1 µm (Figure 2). A protein concentration of 3.3 wt%

272

resulted in overall smallest droplet sizes. However, this concentration showed a bimodal

273

particle size distribution, which indicates a droplet destabilization possibly through a depletion

274

mechanism or is the result of non-attached proteins. This concentration also led to an occasional

275

blockage of the high-pressure homogenizer. Therefore, a protein concentration of 2.5 wt% was

276

used for emulsion preparation, which resulted in a d43 ≤ 1 µm and no significant increase during

277

the 7 d storage period. The displayed performance was comparable to other mixed biopolymer

278

systems, such as lentil proteins and gum arabic28-29. However, a Tetraselmis sp. microalgae

279

protein extract concentration of 6 mg mL-1 (which equals approximately a concentration of 0.6

280

wt%) was previously reported to yield in a saturated interface and thereby in a stable emulsion

Emulsifying properties of water-soluble proteins of Chlorella protothecoides


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at a given energy input, because the oil droplets are covered with emulsifier and coalescence is

282

prevented16. This reported concentration is lower compared to the concentration in the present

283

study, which might be related to the particular nature of each respective extract, because the

284

protein composition varies from microalga to microalga. Thus, specific emulsifying properties,

285

such as particle size obtained at a certain energy input and concentration can differ.

286

3.4

287

The presented results demonstrate that the WSE from C. protothecoides can facilitate the

288

formation of emulsions, leading to dispersions that are stable for at least 7 days. Another

289

important emulsifier quality criterion is the behavior in changing environmental conditions,

290

which commonly occur during food formulation and processing. Therefore, the behavior of

291

model emulsions (native pH of 6.4) was investigated over a pH-range of 2-9.

292

Initially, samples were analyzed in terms of their particle size distribution (Figure 3A,B).

293

Emulsions exhibited only a minor increase in particle size with decreasing pH. Remarkably, the

294

mean particle size increased only by 300 nm when the pH was decreased from 5 to 4. Emulsions

295

stabilized with other proteins, such as soy protein or WPI, exhibit a stronger increase in the

296

mean particle size when the pH is lowered30. For example, a much stronger increase in mean

297

particle diameter in emulsions stabilized with WPI was found between pH 6 to 5 with an

298

increase of 15.50 µm (results not shown). These results indicate a high emulsion stability of

299

emulsions prepared with proteins of C. protothecoides at low pH values where isoelectric points

300

are typically located. All distributions remained monomodal across the entire tested pH-range.

301

Similar observations were made in emulsions prepared with extracts from the microalga

302

Tetraselmis sp., which were also stable in slight acidic conditions (pH 5)16. On the other hand

303

the results are in strong contrast to emulsions prepared with common food proteins such as

304

casein31, soy32 and non-native whey33, which show poor emulsion stability at low pH-values

305

close to their isoelectric point. However, mixed biopolymer systems such as gum arabic, soy

Stability of O/W-emulsion (stress test I): pH-shift


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bean soluble polysaccharides, and electrostatically stabilized complexes containing proteins

307

and polysaccharides are known to stabilize emulsions over a broader pH-range28, 34-37 and the

308

results further suggest that the remaining carbohydrate content in the microalgae extract plays

309

a role in the stabilization of the oil droplets, as suggested in the previous chapter 3.2.

310

To further elucidate the stabilization mechanism, ζ-potential measurements were carried out

311

(Figure 4A), since one of the major droplet stabilization mechanisms in protein-stabilized

312

emulsions are electrostatic repulsion forces that prevent droplet-droplet collisions. The

313

emulsions showed an increase in net ζ-potential with decreasing pH from 9 to 2 and a shift to a

314

slightly positive ζ-potential (0.63±0.07 mV at pH 2) between pH 3 and 2, which was in contrast

315

to WPI stabilized emulsions with a shift from a negative to a positive ζ-potential between pH 5

316

and 4 and a low emulsion stability around this pH (Figure 5). Nevertheless, these observations

317

are in accordance with previous results, where the proteins from Chlorella protothecoides

318

possessed a high solubility throughout a broad pH-range and where soluble extracts from

319

Tetraselmis spp. revealed no visual isoelectric point and a negative ζ-potential between pH 3 to

320

7, which was linked to presence of charged polysaccharides11, 14, 16, 38. The observed negative

321

ζ-potential and high solubility might explain the high emulsion stability in the pH-range 2-9,

322

which facilitates droplet repulsions. As the pH decreased, the ζ-potential decreased as well,

323

which resulted in slightly larger droplet sizes. Nevertheless, emulsions were still stable, since

324

micro- and macro-appearance of emulsions showed neither visual destabilization (Figure 5),

325

nor distinct changes in microstructure (Figure 3), even after a storage of 7 days (all microscopy

326

images available as supplementary data).

327

3.5

328

After the characterization of pH-induced changes, an assessment of the impact of ionic strength

329

can give further insights in the emulsifying properties of proteins. This is relevant since food

330

systems often contain high amounts of salts, which can destabilize protein stabilized emulsion

Stability of O/W-emulsion (stress test II): ionic strength


14


Page 16 of 34

331

droplets due to electrostatic screening of charges. Therefore, the stability of model emulsions

332

prepared with proteins from Chlorella protothecoides were tested at increasing sodium chloride

333

concentrations (0-500 mM). Again, samples were analyzed in terms of particle size (Figure

334

3C,D), ζ-potential (Figure 4B), microstructure (Figure 3), and macroscopic appearance

335

(Figure 5) after 24 h and 7 days (all microscopy images available as supplementary data).

336

In general, emulsions displayed a high stability even at high ionic strengths with no significant

337

(p > 0.05) change in particle size when salt was added, and only minor increases in particle size

338

being observed throughout storage. The high stability was also confirmed by visual and

339

microscopic methods; i.e. no change in microscopic structure and macroscopic appearance was

340

found over the storage period (Figure 3&5). Mixed biopolymer systems can retain a stability

341

due to protein-polysaccharide electrostatic complexation or covalent glycosylation, even at high

342

ionic strengths and pH-values near the isoelectric point, where purely protein stabilized systems

343

are generally failing due to ionic shielding not only among adsorbed proteins but in between

344

droplets39-40. This is related to the increase in repulsive steric interactions, because the

345

hydrophilic polysaccharides increase the layer thickness of the emulsifier compared to the pure

346

unaltered protein

347

and polysaccharides in the WSE. That other stabilization mechanisms play a role during salt

348

addition was also supported by ζ-potential measurements, which showed that O/W-emulsions

349

retained a decreasing, but negative ζ-potential up to 500 mM of NaCl for both whey protein and

350

WSE. This can be linked to positively charged sodium ions accumulating around the negatively

351

charged droplet surfaces (electrostatic screening, which results in a decrease in electrostatic

352

repulsion) and resulted in whey protein stabilized emulsions in emulsion break-up at NaCl

353

concentrations of > 100 mM.

354

All in all, the reported functionalities are in many respect superior to that of other common food

355

proteins, which often possess poor emulsifying properties in between pH 4-6 where isoelectric

22

. These results further indicate that there is an interplay between proteins


15

Page 17 of 34


356

precipitation occur, and at high ionic strengths where droplets often flocculate. Therefore, this

357

study should be of substantial interest to food and beverage manufacturer that are in need of

358

formulating protein-based acidified emulsions or emulsions containing high amounts of

359

minerals.

360


16


Page 18 of 34

361

4 ACKNOWLEDGMENT

362

This work by L. Grossmann, S. Ebert, J. Hinrichs, J. Weiss was supported by a grant from the

363

Ministry of Science, Research and the Arts of Baden-Württemberg Az: 7533-10-5-87 as part of

364

the BBW ForWerts Graduate Program.

365 366

The authors declare no competing financial interest.


17

Page 19 of 34


367

5 REFERENCES

368 369

1. Szabo, N. J.; Matulka, R. A.; Chan, T., Safety evaluation of Whole Algalin Protein (WAP) from Chlorella protothecoides. Food and Chemical Toxicology 2013, 59, 34-45.

370 371 372 373

2. Enzing, C.; Ploeg, M.; Barbarosa, M.; Sijtsma, L., Microalgae-based products for the food and feed sector: an outlook for Europe. In JRC Scientific and Policy Reports, Vigani, M.; Parisi, C.; Rodríguez Cerezo, E., Eds. Publications Office of the European Union: Luxembourg, 2014; pp 1-78.

374 375

3. Bleakley, S.; Hayes, M., Algal Proteins: Extraction, Application, and Challenges Concerning Production. Foods 2017, 6 (5).

376 377 378

4. Yamamoto, M.; Kurihara, I.; Kawano, S., Late type of daughter cell wall synthesis in one of the Chlorellaceae, Parachlorella kessleri (Chlorophyta, Trebouxiophyceae). Planta 2005, 221 (6), 766-775.

379 380 381

5. Zeeb, B.; Grossmann, L.; Weiss, J., Accessibility of Transglutaminase to Induce Protein Crosslinking in Gelled Food Matrices - Impact of Membrane Structure. Food Biophysics 2016, 11 (2), 176-183.

382 383 384

6. Beacham, T. A.; Bradley, C.; White, D. A.; Bond, P.; Ali, S. T., Lipid productivity and cell wall ultrastructure of six strains of Nannochloropsis: Implications for biofuel production and downstream processing. Algal Research 2014, 6, 64-69.

385 386

7. Becker, E. W., Micro-algae as a source of protein. Biotechnology Advances 2007, 25 (2), 207-210.

387 388 389

8. Gerken, H. G.; Donohoe, B.; Knoshaug, E. P., Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta 2013, 237 (1), 239253.

390 391

9. Halim, R.; Harun, R.; Danquah, M. K.; Webley, P. A., Microalgal cell disruption for biofuel development. Applied Energy 2012, 91 (1), 116-121.

392 393 394

10. Safi, C.; Charton, M.; Pignolet, O.; Silvestre, F.; Vaca-Garcia, C.; Pontalier, P.-Y., Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors. Journal of Applied Phycology 2013, 25 (2), 523-529.

395 396 397

11. Schwenzfeier, A.; Wierenga, P. A.; Gruppen, H., Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp. Bioresource Technology 2011, 102 (19), 9121-9127.

398 399 400 401

12. Shene, C.; Monsalve, M. T.; Vergara, D.; Lienqueo, M. E.; Rubilar, M., High pressure homogenization of Nannochloropsis oculata for the extraction of intracellular components: Effect of process conditions and culture age. European Journal of Lipid Science and Technology 2016, 118 (4), 631-639.

402 403 404

13. Smetana, S.; Sandmann, M.; Rohn, S.; Pleissner, D.; Heinz, V., Autotrophic and heterotrophic microalgae and cyanobacteria cultivation for food and feed: life cycle assessment. Bioresource Technology 2017, 245, 162-170.

405 406 407

14. Grossmann, L.; Ebert, S.; Hinrichs, J.; Weiss, J., Effect of precipitation, lyophilization, and organic solvent extraction on preparation of protein-rich powders from the microalgae Chlorella protothecoides. Algal Research 2018, 29, 266-276.


18


Page 20 of 34

408 409 410

15. Grossmann, L.; Hinrichs, J.; Weiss, J., Solubility and aggregation behavior of protein fractions from the heterotrophically cultivated microalga Chlorella protothecoides. Food Research International 2019, 116, 283-290.

411 412

16. Schwenzfeier, A.; Helbig, A.; Wierenga, P. A.; Gruppen, H., Emulsion properties of algae soluble protein isolate from Tetraselmis sp. Food Hydrocolloids 2013, 30 (1), 258-263.

413 414 415

17. Ursu, A.-V.; Marcati, A.; Sayd, T.; Sante-Lhoutellier, V.; Djelveh, G.; Michaud, P., Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris. Bioresource Technology 2014, 157, 134-139.

416

18.

417 418

19. Matissek, R.; Steiner, G.; Fischer, M., Lebensmittelanalytik. 5 ed.; Springer Spektrum: Berlin, 2014.

419 420

20. Fischer, K., Neues Verfahren zur maßanalytischen Bestimmung des Wassergehaltes von Flüssigkeiten und festen Körpern. Angewandte Chemie 1935, 48 (26), 394-396.

421 422

21. Grau, R., Die Bestimmung von Asche und Kochsalz in Fleisch und Fleischerzeugnissen. Zeitschrift für Untersuchung der Lebensmittel 1942, 84 (5), 397-408.

423 424 425

22. Schmidt, U. S.; Schütz, L.; Schuchmann, H. P., Interfacial and emulsifying properties of citrus pectin: Interaction of pH, ionic strength and degree of esterification. Food Hydrocolloids 2017, 62, 288-298.

426 427 428 429

23. Cuevas-Bernardino, J. C.; Lobato-Calleros, C.; Román-Guerrero, A.; Alvarez-Ramirez, J.; Vernon-Carter, E. J., Physicochemical characterisation of hawthorn pectins and their performing in stabilising oil-in-water emulsions. Reactive and Functional Polymers 2016, 103, 63-71.

430 431 432

24. Arancibia, C.; Riquelme, N.; Zúñiga, R.; Matiacevich, S., Comparing the effectiveness of natural and synthetic emulsifiers on oxidative and physical stability of avocado oil-based nanoemulsions. Innovative Food Science & Emerging Technologies 2017, 44, 159-166.

433 434

25. Davis, J. P.; Foegeding, E. A.; Hansen, F. K., Electrostatic effects on the yield stress of whey protein isolate foams. Colloids and Surfaces B: Biointerfaces 2004, 34 (1), 13-23.

435 436 437

26. Rangsansarid, J.; Cheetangdee, N.; Kinoshita, N.; Fukuda, K., Bovine Serum AlbuminSugar Conjugates through the Maillard Reaction: Effects on Interfacial Behavior and Emulsifying Ability. Journal of Oleo Science 2008, 57 (10), 539-547.

438 439 440

27. Ralla, T.; Salminen, H.; Edelmann, M.; Dawid, C.; Hofmann, T.; Weiss, J., Sugar Beet Extract (Beta vulgaris L.) as a New Natural Emulsifier: Emulsion Formation. Journal of Agricultural and Food Chemistry 2017, 65 (20), 4153-4160.

441 442 443 444

28. Charoen, R.; Jangchud, A.; Jangchud, K.; Harnsilawat, T.; Naivikul, O.; McClements, D. J., Influence of biopolymer emulsifier type on formation and stability of rice bran oil-inwater emulsions: whey protein, gum arabic, and modified starch. Journal of Food Science 2011, 76 (1), 165-172.

445 446 447

29. Gumus, C. E.; Decker, E. A.; McClements, D. J., Formation and stability of ω-3 oil emulsion-based delivery systems using plant proteins as emulsifiers: lentil, pea, and faba bean proteins. Food Biophysics 2017, 12 (2), 186-197.

448 449 450

30. Kong, X.; Jia, C.; Zhang, C.; Hua, Y.; Chen, Y., Characteristics of soy protein isolate/gum arabic-stabilized oil-in-water emulsions: influence of different preparation routes and pH. RSC Advances 2017, 7 (51), 31875-31885.

Dumas, J.-B., Procédés de l’analyse organique. 47 ed.; Ann. Chem Phys.: 1831.


19

Page 21 of 34


451 452 453

31. Biasutti, E. A. R.; Vieira, C. R.; Capobiango, M.; Silva, V. D. M.; Silvestre, M. P. C., Study of Some Functional Properties of Casein: Effect of pH and Tryptic Hydrolysis. International Journal of Food Properties 2007, 10 (1), 173-183.

454 455 456

32. Bengoechea, C.; Romero, A.; Aguilar, J. M.; Cordobés, F.; Guerrero, A., Temperature and pH as factors influencing droplet size distribution and linear viscoelasticity of O/W emulsions stabilised by soy and gluten proteins. Food Hydrocolloids 2010, 24 (8), 783-791.

457 458 459

33. Demetriades, K.; Coupland, J. N.; McClements, D. J., Physical Properties of Whey Protein Stabilized Emulsions as Related to pH and NaCl. Journal of Food Science 1997, 62 (2), 342-347.

460 461 462

34. Reichert, C. L.; Salminen, H.; Leuenberger, B. H.; Hinrichs, J.; Weiss, J., Miscibility of Quillaja Saponins with other Co-surfactants under Different pH Values. Journal of Food Science 2015, 80 (11), E2495-503.

463 464 465

35. Grossmann, L.; Wefers, D.; Bunzel, M.; Weiss, J.; Zeeb, B., Accessibility of transglutaminase to induce protein crosslinking in gelled food matrices - Influence of network structure. LWT - Food Science and Technology 2017, 75, 271-278.

466 467 468 469

36. Nakauma, M.; Funami, T.; Noda, S.; Ishihara, S.; Al-Assaf, S.; Nishinari, K.; Phillips, G. O., Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids 2008, 22 (7), 1254-1267.

470 471 472

37. Zhang, J.-B.; Wu, N.-N.; Yang, X.-Q.; He, X.-T.; Wang, L.-J., Improvement of emulsifying properties of Maillard reaction products from β-conglycinin and dextran using controlled enzymatic hydrolysis. Food Hydrocolloids 2012, 28 (2), 301-312.

473 474 475

38. Schwenzfeier, A.; Wierenga, P. A.; Eppink, M. H. M.; Gruppen, H., Effect of charged polysaccharides on the techno-functional properties of fractions obtained from algae soluble protein isolate. Food Hydrocolloids 2014, 35, 9-18.

476 477 478

39. Gu, Y. S.; Regnier, L.; McClements, D. J., Influence of environmental stresses on stability of oil-in-water emulsions containing droplets stabilized by β-lactoglobulin–ιcarrageenan membranes. J Colloid Interface Sci 2005, 286 (2), 551-558.

479 480

40. Ho, Y.-T.; Ishizaki, S.; Tanaka, M., Improving emulsifying activity of ε-polylysine by conjugation with dextran through the Maillard reaction. Food Chemistry 2000, 68 (4), 449-455.

481


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Page 22 of 34

482

6 TABLES

483

Table 1:

484 485

Proximate composition and appearance of a lyophilized water-soluble extract (WSE) obtained from C. protothecoides.

486 compound

content (wt%)

protein (N x 4.24)

33.2±0.4

minerals

9.6±0.1

lipids

0.4±0.1

water

3.3±0.2

carbohydrates

53.5±1.2

appearance

487 488


21

Page 23 of 34


489

7 FIGURES & CAPTIONS

490 491 492 493

Figure 1: Oil-water interfacial tension profiles at pH 3, 5, and 7 of lyophilized water-soluble extract (WSE) of C. protothecoides (cprotein = 0.04 wt%), whey protein isolate (WPI) (cprotein = 0.04 wt%), citrus pectin (CP, c = 0.04 wt%), and Tween 20 (T20, c = 0.04 wt%) at pH 7; standard deviation of samples ≤ 9.1 %.

494 495 496 497 498 499

Figure 2: Influence of protein concentration and storage time on particle size distribution and d43 of O/W-emulsions prepared with a lyophilized water-soluble extract (WSE) from Chlorella protothecoides. Different small superscripts indicate significant difference (p ≤ 0.05) between concentrations. Different capital superscripts indicate significant difference between t = 0 d and t = 7 d at same concentrations (p ≤ 0.05), n = 2.

500 501 502 503 504

Figure 3: Influence of pH (A, B) and sodium chloride concentration (C, D) on particle size distributions, d43, and microstructure (after 24 h) of model O/W emulsions (2.5 wt% protein, 5 wt% oil, 1,000 bar, 3 passes) throughout 7 days of storage; particle size measurements were carried out after 24 h and 7 days. Scale bar = 10 µm.

505 506 507 508

Figure 4: Influence of pH (A) and sodium chloride concentration (B) on ζ-potential of model O/W-emulsions prepared with 2.5 wt% of proteins from C. protothecoides (1,000 bar, 3 passes), 0.5 wt% WPI and 5 wt% oil. WPI was used as a reference protein.

509 510 511 512 513

Figure 5: Influence of pH and increasing sodium chloride concentration on appearance of model O/W emulsions (2.5 wt% protein of C. protothecoides, 0.5 % WPI and 5 wt% oil) throughout 7 days of storage; pictures taken after 24 h and 7 days. WPI was used as a reference protein.

514


22


515

Page 24 of 34

Figure 1.

30

interfacial tension (mN m-1)

CP pH5 CP pH3

25

CP pH7 WPI pH3

20

WPI pH7 WSE pH7 WSE pH3

15

WSE pH5 WPI pH5

10 5

T20 pH7

0 0

250

500

750

1000 1250 1500

time (s) 516 517


23

Page 25 of 34

Figure 2.

relative volume frequency (%)

518


105 3.3 wt%, t = 7 d

90 75

0.640.06 µmcA 3.3 wt%, t = 0 d

0.670.06 µmbA

60

2.5 wt%, t = 7 d

1.020.08 µmbA

45

2.5 wt%, t = 0 d

0.880.02 µmbA

30

1.7 wt%, t = 7 d

1.790.01 µmaB

15

1.7 wt%, t = 0 d

1.170.10 µmaA

0 0.01

0.1

1

10

100

droplet diameter (µm) 519 520 521


24


522

Page 26 of 34

Figure 3.

523 524


25

Page 27 of 34

525


Figure 4. A 80

B

60

40

-potential (mV)

-potential (mV)

60 WPI

20 0 -20

WSE C. protothecoides

-40 -60

40 20 WSE C. protothecoides

0 -20 WPI

-40 -60

-80

-80 2

526

80

3

4

5

6

pH

7

8

9

0

100

200

300

400

500

NaCl concentration (mM)

527


26


528

Page 28 of 34

Figure 5. WSE of C. protothecoides

WPI

529 WSE of C. protothecoides

WPI

530 531


27

Page 29 of 34

532


Supplemental Figure

533 534

Figure: Microstructure (after 24 h) of model O/W emulsions (2.5 wt% protein, 5 wt% oil, 1,000

535

bar, 3 passes) at different pH and NaCl concentrations. Scale bar 20 µm.


28


Figure 1: Oil-water interfacial tension profiles at pH 3, 5, and 7 of lyophilized water-soluble extract (WSE) of C. protothecoides (cprotein = 0.04 wt%), whey protein isolate (WPI) (cprotein = 0.04 wt%), citrus pectin (CP, c = 0.04 wt%), and Tween 20 (T20, c = 0.04 wt%) at pH 7; standard deviation of samples ≤ 9.1 %. 185x156mm (300 x 300 DPI)


Page 30 of 34

Page 31 of 34


Figure 2: Influence of protein concentration and storage time on particle size distribution and d43 of O/Wemulsions prepared with a lyophilized water-soluble extract (WSE) from Chlorella protothecoides. Different small superscripts indicate significant difference (p ≤ 0.05) between concentrations. Different capital superscripts indicate significant difference between t = 0 d and t = 7 d at same concentrations (p ≤ 0.05), n = 2. 160x152mm (300 x 300 DPI)



Figure 3: Influence of pH (A, B) and sodium chloride concentration (C, D) on particle size distributions, d43, and microstructure (after 24 h) of model O/W emulsions (2.5 wt% protein, 5 wt% oil, 1,000 bar, 3 passes) throughout 7 days of storage; particle size measurements were carried out after 24 h and 7 days. Scale bar = 10 µm. 1401x1115mm (96 x 96 DPI)


Page 32 of 34

Page 33 of 34


Figure 4: Influence of pH (A) and sodium chloride concentration (B) on ζ-potential of model O/W-emulsions prepared with 2.5 wt% of proteins from C. protothecoides (1,000 bar, 3 passes), 0.5 wt% WPI and 5 wt% oil. WPI was used as a reference protein. 207x100mm (300 x 300 DPI)



Figure 5: Influence of pH and increasing sodium chloride concentration on appearance of model O/W emulsions (2.5 wt% protein of C. protothecoides, 0.5 % WPI and 5 wt% oil) throughout 7 days of storage; pictures taken after 24 h and 7 days. WPI was used as a reference protein. 162x179mm (300 x 300 DPI)


Page 34 of 34

Formation and Stability of Emulsions Prepared with a Water-Soluble

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