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Emulsifying stability of enzymatically hydrolyzed egg yolk granules and structural

analysis

Qin Li, Shitao Tang, Fayez Khalaf Mourad, Wenjie Zou, Lizhi Lu, Zhaoxia Cai

PII: S0268-005X(19)31648-0
DOI: https://doi.org/10.1016/j.foodhyd.2019.105521
Reference: FOOHYD 105521

To appear in: Food Hydrocolloids

Received Date: 28 July 2019

Revised Date: 10 November 2019
Accepted Date: 14 November 2019

Please cite this article as: Li, Q., Tang, S., Mourad, F.K., Zou, W., Lu, L., Cai, Z., Emulsifying stability
of enzymatically hydrolyzed egg yolk granules and structural analysis, Food Hydrocolloids (2019), doi:
https://doi.org/10.1016/j.foodhyd.2019.105521.

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 1 Emulsifying Stability of Enzymatically Hydrolyzed Egg Yolk
2 Granules and Structural Analysis

3 Qin Li1, Shitao Tang1, Fayez Khalaf Mourad1, Wenjie Zou1, Lizhi Lu2,

4 Zhaoxia Cai1*

5 1. College of Food Science and Technology, Huazhong Agricultural University,

6 National Research and Development Center for Egg Processing, Wuhan, Hubei

7 430070, PR China

8 2. Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of

9 Agricultural Sciences 310000, Zhejiang, PR China

10

11

12

13

14
*
Corresponding author: Zhaoxia Cai

15 College of Food Science and Technology of Huazhong Agricultural University,

16 Wuhan, Hubei Province, P. R. China

17 Tel.: +86 27 87283177

18 Fax: +86 27 87283177

19 E-mail address: [email protected]

20

1
21 Abstract: Egg yolk granules (EYG) are natural and unique protein-lipid complexes,

22 but their application in the food industry is limited because of their poor solubility and

23 emulsifying properties. In this paper, the structural characteristics, emulsifying

24 properties and interfacial adsorption were compared between the untreated granules

25 (UG) and the enzymatically hydrolyzed granules (EG) (treated with subtilisin).

26 EG-stabilized emulsion did not form cream at 0.2% to 1.0% protein concentrations,

27 while UG-stabilized emulsion formed a cream layer quickly after emulsification. The

28 emulsion stability index of EG increased by nearly 8 times of UG at the protein

29 concentration of 1.0%. The scanning electron microscope, confocal laser scanning

30 microscopy (CLSM) and optical microscopy indicated that enzymatic hydrolysis

31 changed the aggregation state of the EYG in powder, suspension and emulsions. Also,

32 it was proven that enzymatic hydrolysis strengthened the internal hydrogen bonding

33 and increased surface wettability and surface charge. The CLSM images confirmed

34 that EYG are Pickering particles. The interfacial tension dropped to 8.4 mN/m after

35 enzymatic hydrolysis. The SDS-PAGE analysis of the protein showed that enzymatic

36 hydrolysis induced changes in the granules and interface, resulting in a strong

37 adsorption capacity for EG at the interface. The enzymatic modifications of EYG

38 promoted their emulsifying properties and showed that EYG have the potential to be

39 used as novel food-grade particles through structural modification.

40

41 Keywords: Enzymatically hydrolyzed granules; Protease hydrolysis; Interfacial

42 adsorption; Emulsion stability

2
43 1. Introduction

44 Egg yolk is an efficient source of many essential nutrients, such as proteins of

45 high nutritional value, vitamins, minerals, essential fatty acids and phospholipids. It is

46 widely used as an ingredient in the food industry due to its excellent emulsifying

47 properties (Gouda, Zu, Ma, Sheng, & Ma, 2018). Since the 1970s, egg yolk has been

48 easily separated into egg yolk granules (EYG) and yellow plasma using mild

49 centrifugation (Bee & Cotterill, 1979; Laca, Paredes, Rendueles, & Díaz, 2014).

50 EYG are natural and unique protein-lipid complexes, which have a potential for

51 use as a novel food-grade Pickering emulsifier (Anton, Le Denmat, Beaumal, & Pilet,

52 2001). EYG consist of 70% high-density lipoproteins (HDLs), 16% phosvitin, and 12%

53 low-density lipoproteins (LDLs) (Anton, 2013). Numerous phosphocalcic bridges,

54 which link HDL and phosvitin, endow EYG with a compact structure to defend

55 against thermal denaturation (Le Denmat, Anton, & Beaumal, 2000) and iron

56 chelation (Gautron et al., 2007). Also, EYG contain significantly lower amounts of

57 phospholipids and cholesterol and higher amounts of proteins than whole egg yolk

58 (Rayner et al., 2014); thus, EYG could be used as a substitute to create healthier diets.

59 This unique protein-lipid complex may also increase the possibility of combining

60 active substances and achieving better emulsion stability compared with the use of

61 single proteins or lipids. The Gliadin−phospholipid hybrid nanoparticle has been

62 proven to have better foam-formation and stability characteristics than single gliadin

63 nanoparticles (Chen et al., 2019). In multiple studies, researchers have attributed the

64 improved emulsion properties of modified egg yolks to the “partial destruction” of

3
65 EYG due to the release of some active components from the granules into the

66 interface (Daimer & Kulozik, 2009). However, no targeted study has ever focused on

67 the characteristics of partially destructed EYG. The vague concept of “partial

68 destruction” of EYG and their associations with emulsion properties require

69 clarification. Previous studies have proven that if a particle is too hydrophobic, it will

70 completely immerse in oil instead of being adsorbed at the oil-water interface (Sorbier,

71 Aimable, & Pagnoux, 2015; Yuan, Baalen, Yang, & Scholten, 2018). Therefore, we

72 hypothesize that the higher surface hydrophobicity is one of the possible surface

73 characteristics of EYG that lead to their poor solubility and emulsifying properties.

74 Their relatively higher surface hydrophobicity prevents EYG from being adsorbed

75 better at the interface, which limits their emulsifying properties. Additionally, the

76 complexity of the protein-lipid structure of EYG also makes it difficult to understand

77 the interactions between their constituents, the exact role each plays, and the

78 proportion each contributes to the emulsifying process.

79 At present, most studies about EYG have been focused mainly on their functional

80 properties under different environmental situations such as different ionic strengths

81 (Causeret, Matringe, & Lorient, 1991; Le Denmat, Anton, & Beaumal, 2000), pH

82 levels (Anton, et al., 2001), freeze-drying processing (Amanda, Benjamín, & Mario,

83 2010), heat treatments (Le Denmat, Anton, & Gandemer, 1999), and mechanical

84 treatments (Sirvente et al., 2007). However, few studies have paid attention to the

85 hydrophilic modification of native EYG to improve their emulsifying properties.

86 Enzymatic hydrolysis has been regarded as an effective and environmentally friendly

4
 87 method to improve the functional properties of proteins (Chang et al., 2017;

88 Kristinsson & Rasco, 2000). Enzymatic modification of egg yolk by phospholipase is

89 favored and has been proven to be an effective method to improve emulsifying

90 properties (Maria, Vind, Oxenbøll, Svendsen, & Patkar, 2007). The most commonly

91 used enzymes, phospholipase D and phospholipase A2, were reported to significantly

92 increase the emulsifying properties and heat stability of EYG-stabilized emulsions by

93 converting phospholipids into lysophospholipids (Buxmann, Bindrich, Heinz, Knorr,

94 & Franke, 2010; Jin, Huang, Ding, Ma, & Oh, 2013; Strixner, Würth, & Kulozik,

95 2013b). Additionally, the structural changes in EYG and LDL micelles induced by

96 phospholipase A2 were also thought to be the decisive factors affecting the

97 emulsifying properties of egg yolk (Daimer et al., 2009). However, the modification

98 of EYG by proteases is relatively less explored. Notably, Bao, Zhao, Wang, and Chi

99 (2017) used alcalase, neutrase and flavourzyme to hydrolyze egg yolk, which induced

100 a significant decrease in the creaming index (CI) of emulsions. Similarly, Tang, Zhou,

101 Gouda, Cai, and Jin (2019) found that subtilisin-hydrolyzed egg yolk significantly

102 increased the solubility of egg yolk powder. It is assumed that the proteases could

103 probably have enhanced the emulsifying properties of the EYG by changing the

104 protein constituents through affecting the cleavage sites at aromatic amino acid

105 residues. The structural changes in EYG would produce a series of surface

106 characteristics that benefit emulsification.

107 Until recently, EYG were believed to have poor emulsifying properties until being

108 considered from the perspective of Pickering-type particles (Rayner et al., 2014).

5
109 Compared with the traditional chemical emulsifiers, the biomass-based food-grade

110 particles have better biodegradability, higher security and excellent emulsifying

111 properties. EYG, even with their naturally distinguished advantages, have been

112 limited in application due to their improper surface properties. Therefore, to expand

113 the applications of EYG in the food industry, it is necessary to explore an effective

114 modification method for EYG and understand the mechanism that promotes their

115 emulsifying stability.

116 This paper aims to investigate the emulsifying properties, structural characteristics

117 and interfacial adsorption of enzymatically hydrolyzed EYG treated with subtilisin

118 based on the former work of our lab (Tang et al., 2019). The structural description of

119 the granules and interface will help understand how enzymatic hydrolysis works on

120 EYG to improve the emulsifying properties. The emulsifying performances of

121 untreated granules (UG) and enzymatically hydrolyzed granules (EG) were compared

122 from both macro and micro perspectives. Structural characteristics were investigated

123 to analyze the effect of enzymatic hydrolysis on the structure of EYG. Also, changes

124 in the interfacial adsorption were explored to determine the structural changes

125 associated with the emulsifying performance changes. This will provide a basis for

126 the later modulation of emulsifying properties through the targeted structural

127 modification of EYG. Modified EYG hold promise to be used in delivery systems, as

128 Pickering emulsifiers or as food ingredients at the industrial scale.

129

130 2. Material and methods

6
131 2.1 Materials

132 Fresh eggs were purchased from the Hubei Institute of Animal Husbandry and

133 Veterinary (Wuhan, China). Soybean oil was produced by Yihai Kerry Co., Ltd.

134 (Wuhan, China). Subtilisin (150 U/mg) was purchased from Yuanye Biological Co.,

135 (Shanghai, China). Nile Red and fluorescein isothiocyanate (FITC) were obtained

136 from Sigma-Aldrich (St. Louis, MO, USA). The common chemicals used were of

137 analytical grade and were purchased from Sinopharm Chemical Regent Co., Ltd.

138 (Shanghai, China). The SDS-PAGE gel quick preparation kit was produced by

139 Servicebio Technology Co., Ltd. (Wuhan China). Deionized water (DW) purified by a

140 Clever-S30 (Shanghai, China) was used to prepare all solutions.

141 2.2 Preparation of EYG

142 The hydrolyzed egg yolk powder was prepared according to Tang et al. (2019).

143 The EYG were obtained according to Lei and Wu (2012) with some modifications.

144 The obtained enzymatically hydrolyzed egg yolk powder was diluted (1:10, w/v) with

145 DW and stirred using a magnetic stirrer for 1 h before centrifugation at 1,000 x g for

146 45 min at 4°C. The precipitated granules were then washed carefully with DW to

147 remove all the adhering plasma. EYG were obtained using an ALPHA 1-4/2-4 LD

148 plus vacuum freeze dryer (Martin Christ, Osterode, Germany). Untreated granules

149 extracted from natural egg yolk powder were served as a control.

150 2.3 Emulsion preparation

151 Oil-in-water emulsions were prepared with EYG and soybean oil. The granules

152 were diluted with DW. Then, they were homogenized for 10 s at 10,600 g at pH 7.0.

7
153 The protein content of the EYG was measured using a K9840 automatic nitrogen

154 analyzer (Hanon Instrument Ltd., Dezhou, China), which was of 305.5 mg/g for UG

155 and 282.4 mg/g for EG. Then, 9 mL soybean oil (the oil volume fraction was 0.3) was

156 then added to 21 mL of granule solutions with four different protein concentrations.

157 The emulsion mixtures were homogenized for 2 min at 10,600 g. The final protein

158 concentrations of the emulsion were 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (w/v),

159 respectively.

160 2.4 Determination of the CI (spell out)

161 The CI was measured based on the method of Niu et al. (2016) with some

162 modifications. Immediately after preparation, the emulsion was transferred into a

163 glass bottle and stored at ambient temperature for 30 min. Both the height of the

164 creamed emulsion at the top and the height of the transparent water phase at the

165 bottom were recorded. The CI was calculated as follows:

He
166 CI= (1)
Ht

167 where He (cm) is the height of the creamed emulsion and Ht (cm) is the height of

168 creamed emulsion and transparent aqueous phase.

169 2.5 Determination of emulsion stability index (ESI)

170 The ESI was measured according to the method of Pearce and Kinsella (1978)

171 with modifications. After emulsification, 20 µL emulsion was sampled from the

172 bottom and diluted into 8 mL 0.1% (w/v) sodium dodecyl sulphate solution (SDS).

173 The absorbance A0 of the dilution was measured by a Nanodrop 2000 C ultraviolet

174 visible spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) at 500 nm,

8
175 with 0.1% (w/v) SDS solution as a blank. After 30 minutes, the bottom emulsion was

176 sampled again for dilution, and its absorbance At was redetermined. The ESI was

177 calculated by the following equation:

A0 × ∆T
178 ESI= (2)
A0 − At

179 where A0 and At are the absorbance of the diluted emulsion at the beginning and 30

180 min later and ∆ T was chosen as 30 min.

181 2.6 Determination of the droplet size distribution

182 The droplet size distribution was estimated by a Mastersizer 2000 laser particle

183 size analyzer (Malvern Instruments Ltd., Worcestershire, UK) according to the

184 method of Le Denmat et al. (2000) with some modifications. First, 0.4 mL fresh

185 emulsion was diluted with 5.0 mL DW and 5.0 mL1% SDS, respectively, before

186 loading into a cuvette. The mean diameter d4,3 was used to describe the average

187 droplet size. The refractive indices of the granule dispersion and soybean oil were set

188 to 1.330 and 1.476, respectively. Triplicate samples were used from each group.

189 2.7 Microscopic observation of the emulsion

190 The microscopic morphology of the emulsion was investigated using a DM3000

191 optical microscope (Leica Instruments Ltd., Weztlar, Germany). First, 5 µL emulsion

192 and 5 µL aqueous phase from the bottom of the emulsion were dropped onto the slide,

193 respectively. The coverslip was carefully added to ensure no bubbles were trapped. A

194 representative field of view was sought under different lenses, and a camera was used

195 to take images.

196 The adsorption at the oil-water interface was further observed with a

9
197 FV12000MPE confocal laser scanning microscope (CLSM, Olympus Optical Co. Ltd.,

198 Tokyo, Japan) according to the method of Tang et al. (2017) with modifications. The

199 emulsions were dyed with a mixed fluorescent dye solution consisting of 1 mg/mL

200 Nile Red and 1 mg/mL FITC at ratios of 100:3 (v/v) and 100:1 (v/v), respectively.

201 Then, 5 µL of the stained emulsion was placed on the center of the slide and covered

202 with a coverslip. Each sample was observed using a laser with excitation values of

203 519 nm and 617 nm.

204 2.8 Morphology observation of granules

205 The morphology of the lyophilized EYG was imaged by a JM-6390LV scanning

206 electron microscope (SEM, NTC, Fukuyama, Japan). The granules were loosely glued

207 onto an adhesive tape attached to an aluminum stub and sputter-coated with a thin

208 conductive gold layer. The EYG samples were then observed with a SEM. The

209 dispersed EYG in water 0.2% (w/v) after homogenization at 10,600 g for 2 min was

210 imaged using a CLSM.

211 2.9 Internal forces between EYG

212 The internal forces between EYG were investigated according to the method

213 described by Zhang, Liang, Tian, Chen, and Subirade (2012) with modifications. The

214 particle sizes of EYG under four different protein-disturbing agents (SDS, urea,

215 EDTA and DTT) were measured using a Mastersizer 2000 laser particle size analyzer

216 (Malvern Instruments Ltd., Worcestershire, UK).

217 2.10 Fourier-transform infrared spectroscopy (FTIR)

218 The granules were mixed with KBr at a mass ratio of 100:1 and then compressed

10
219 into thin tablets. The FTIR (Nexus-470, Thermo Nicolet Corporation, Madison, WI,

220 USA) spectra of the EYG were recorded at 400-4000 cm-1. The background of each

221 sample was scanned using air.

222 2.11 Three-phase contact angles

223 The three-phase contact angles were measured by a DSA25 100W video optical

224 contact angle measurement instrument (Kruss, Hamburg, Germany) according to Li,

225 Li, Sun, and Yang (2013) with modifications. The EYG were compressed into pellets

226 of 10 mm in diameter and 2 mm in thickness. The pellets were then immersed and

227 placed at the bottom of an optical glass container containing soybean oil. Then, a 5 µL

228 drop of DW was deposited at 5 mm above the pellet using a high-precision syringe.

229 After the droplet was dripped from the syringe, the droplet image was captured and

230 collected using the video camera. The profiles of the droplet were numerically solved

231 and fitted to the Laplace-Young equation.

232 2.12 Determination of protein solubility

233 Protein solubility was determined according to the method of Le Denmat et al.

234 (2000) with some modifications. The protein concentration was first adjusted to 1

235 mg/ml with DW. The solutions were then equilibrated at pH 7.0 stirring and

236 centrifuged at 10,000 g for 20 min at 4°C. The protein concentration of the

237 supernatant was determined using the Coomassie brilliant blue method. The protein

238 solubility was calculated as follow:

   
  
× 100 (3)

      

239 2.13 Measurement of zeta potential

11
240 The zeta potential was measured using a Zetasizer Nano ZS zeta potential

241 analyzer (Malvern Instruments Ltd., Worcestershire, UK). EYG were diluted to 0.1

242 mg/mL protein concentration with DW before loading to a cuvette. A refractive index

243 of 1.460 was used for the EYG suspension, and 1.330 was used for water. Each

244 sample was examined at 25°C in triplicate.

245 2.14 Interfacial tension

246 The interfacial tension between EYG and soybean oil was measured by a K100

247 surface tension meter (Kruss, Hamburg, Germany) according to the Wilhelmy plate

248 method. The test time was 1 h.

249 2.15 SDS-PAGE

250 The protein composition of EYG, as well as unadsorbed proteins and adsorbed

251 proteins at the interface, was investigated by SDS-PAGE. Fresh emulsions were

252 centrifuged at 9,310 g for 30 min. The cream layer (adsorbed proteins) and the

253 aqueous phase (unadsorbed proteins) were carefully separated using a syringe. The

254 aqueous phase was passed through a 0.45 µm filter. The EYG suspension, cream layer

255 and aqueous layer were diluted 1:1 (v/v) with a buffer solution. The mixtures were

256 then heated in boiling water for 5 min and loaded (10 µL) onto a polyacrylamide gel

257 (stacking, 3.5%, and resolving, 12%). After electrophoresis, the gel was stained

258 (Coomassie blue G-250 0.1%) and then destained (ethanol: glacial acetic acid: water

259 = 25: 8: 67, v/v/v.).

260 2.16 Statistic analysis

261 All data analysis was performed using an analysis of variance (ANOVA)

12
262 procedure (SPSS 17.0 statistical analysis program), and a least significant difference

263 (LSD) test (P < 0.05) was conducted. The images were drawn using the Origin 8.5

264 software.

265 3. Results and discussion

266 3.1 Emulsifying properties of EYG

267 3.1.1 Emulsion stability

268

269 Fig. 1 CI (A), creaming images (inset of A) and ESI (B) of emulsions stabilized by UG and

270 EG at varying protein concentrations of 0.2-1.0%. Mean values ± standard deviations of at

271 least triplicate measurements. Bars denoted with different lowercase letters are significantly

272 different (P < 0.05).

273 The CI is a critical parameter used to evaluate the physical stability of emulsions.

274 From Fig. 1A, it can be seen that the CI value of EG remained stable with the increase

275 of protein concentration from 0.2% to 1.0%, while that of the UG-stabilized emulsion

276 dropped steadily from 48% to 9%. The images of fresh emulsions (within 30 min) can

277 more intuitively reflect the differences in the emulsifying performance of UG and EG

278 (inset of Fig. 1A). The results indicated that the emulsions stabilized by EG exhibited

13
279 much better stability against creaming than those by UG.

280 The ESI was further measured to illustrate the differences between UG and EG in

281 terms of emulsifying performance. As seen in Fig. 1B, the ESI values of UG showed

282 no significant difference (P < 0.05) and remained constant at 50 over the increase of

283 protein concentrations. However, the ESI values of EG increased from 50 to 442 as

284 the protein concentration increased from 0.2% to 1.0%, which was an increase of 8

285 folds. These results indicated that the emulsion stability of EG was greatly promoted

286 compared with that of UG. The emulsion stability also significantly improved with

287 the increase of protein concentrations.

288 3.1.2 Particle size distribution

289

290 Fig. 2 Particle size distributions of emulsions stabilized by UG (A) and EG (B) at varying

291 protein concentrations of 0.2 -1.0%.

292 The difference in emulsion stability between UG and EG was also reflected by

293 the droplet size distribution (Fig. 2). The bridged droplets were separated when

294 diluted in 1% SDS. The d4,3 (in 1% SDS) reflects the real particle size of single

295 droplets (Liang & Tang, 2014). The UG-stabilized emulsion showed an obvious

14
296 bimodal size distribution at lower protein concentrations (0.2%-0.6%) and a unimodal

297 distribution at higher concentrations (0.8%-1.0%) (Fig. 2A). This suggested that the

298 UG-stabilized emulsions at lower concentrations were inclined to be unevenly

299 distributed. Increasing the protein concentration of UG significantly improved the

300 inhomogeneity of the emulsion droplets. In contrast, the EG-stabilized emulsion

301 showed better emulsifying properties, with the peak value decreased from 15.9 µm to

302 12.6 µm (Fig. 2B). These results indicated that increasing protein concentration and

303 employing enzymatic hydrolysis can reduce the droplet size. Modified maize starch

304 (Ye et al., 2017), chitin nanocrystals (Tzoumaki, Moschakis, Kiosseoglou, &

305 Biliaderis, 2011) and soy protein nanoparticle aggregates (Liu & Tang, 2013) have

306 been reported to have a similar association between the protein concentration and

307 droplet size. This is probably because increasing protein concentration provides more

308 proteins to saturate the interface and decrease interfacial tension more efficiently.

309 Another positive effect of increasing protein concentration is that it might strengthen

310 the interactions between adsorbed proteins and unadsorbed proteins, trapping the

311 droplets in a weak network structure. The network structure will not only weaken the

312 flocculation between adjacent droplets (Liang & Tang, 2014) but also will hinder the

313 creaming of emulsions. The particle size distribution of the emulsions in the optical

314 microscopic analysis also showed similar trends (Supplemental Fig. S1) and in the

315 mean droplet diameter measurements (Supplemental Table. S1).

316 3.2 Structural characterization of EYG

317 3.2.1 Aggregation state

15
318

319 Fig. 3 SEM micrographs (A) of UG (1) and EG (2) with a magnification of 1000× (the image

320 on the lower right was taken at a magnification of 3000×), CLSM images (B) of the UG

321 dispersion (1) and EG dispersion (2) with a magnification of 20× and optical microscope

322 images (C) of the aqueous phase at the bottom of the creaming emulsion stabilized by UG (1,

323 2) with a magnification of 40×.

324 The aggregation states of EYG in the powder, suspension and emulsions were

325 studied to explore the morphological change of EYG after enzymatic hydrolysis. Fig.

326 3A shows the SEM micrographs of UG and EG. Numerous globular particles with a

327 diameter of approximately 2 µm were considered to be EYG (amplified and

328 wireframed in red), and dense aggregations of EYG were also observed (Fig. 3A).

329 These results agreed with those of a previous study indicating that EYG varied from

330 0.3-2 µm in diameter (Chang, Powrie, & Fennema, 1977). Although the size of a

331 single granule showed no significant change after hydrolysis, the aggregation status of

332 UG and EG appeared slightly different. While evident aggregations could be seen in

333 UG (Fig. 3A-1), the aggregation of EG was relatively looser and closely packed into

16
334 clusters with less connectivity (Fig. 3A-2). Similarly, Jin et al. (2013) used

335 phospholipase A1 to hydrolyzed EYG and found no significant changes between the

336 structure of enzymatically modified EYG and the control. The SEM results showed

337 that EYG were prone to aggregation due to their structural characteristics. The

338 granular structure of EG still existed with slight changes in the aggregation state after

339 limited enzymatic hydrolysis.

340 The dispersity of EYG in water was further investigated (Fig. 3B). More tiny

341 aggregates of granules were observed in the UG dispersion (Fig. 3B-1), while EG

342 mainly existed in the form of single granules in the dispersion (Fig. 3B-2). These

343 results showed that hydrolytic modification improved the dispersibility of EYG and

344 reduced the large-scale granule aggregation deposition in water. This was probably

345 due to the relatively less-aggregated EG in Fig. 3A, which were easier to disperse in

346 water.

347 The optical micrographs of the aqueous phase at the bottom of the UG-stabilized

348 emulsion after creaming are shown in Fig. 3C. Small UG aggregates were stuck to the

349 surface of the emulsion droplet (Fig. 3C-2) and did not truly participate in the

350 emulsifying process. Additionally, many extra EYG aggregates that failed to adsorb at

351 the interface were noted in the aqueous phase (Fig. 3C-1). This phenomenon has

352 probably resulted from the dispersal inhomogeneity of UG (Fig. 3B-2). The granule

353 aggregates in the aqueous phase (water) not only reduced the emulsifying efficiency

354 of the granules but also dragged the droplets down to the bottom (Fig. 3C-2). Also,

355 the poor uniformity of the granule dispersion led to the acceleration of creaming.

17
356 Therefore, the dispersal homogeneity of EYG in water may further affect the physical

357 stability of the emulsion.

358 3.2.2 Internal forces, wettability and zeta potential

359

360 Fig. 4 Effects of various protein-perturbing solvents on the particle size (A), FTIR spectra (B),

361 oil-in-water three-phase contact angles (C) and zeta potential (D) of UG and EG. Mean values

362 ± standard deviations of at least triplicate measurements. Bars denoted with different

363 lowercase letters are significantly different (P < 0.05).

364 The internal forces, wettability and zeta potential of EYG were speculated to

365 have close associations with the aggregation state of the granules. Fig. 4A shows the

366 changes of the particle size between UG and EG when treated with SDS, urea, EDTA

367 and DTT, which were used to destroy hydrophobic interactions, hydrogen bonds,

368 coordination bonds and disulfide bonds, respectively (Gezimati, Creamer, & Singh,

18
369 1997; Schmitt et al., 2010). Compared with the control group (using water as the

370 dispersant), both UG and EG showed a decrease in particle size. This suggested that

371 these four internal forces exist in egg yolk granules in different proportions. In the

372 presence of 0.5% SDS, the particle size of UG showed the most significant plunge

373 from 21.09 µm to 1.08 µm, which was a decrease of 94.9% (Fig. 4A). Similarly, the

374 particle sizes of EG dropped the most significantly from 11.96 µm to 0.74 µm when

375 treated with 0.5% SDS, with a decrease of 93.8% (Fig. 4A). This indicated that

376 hydrophobic interactions were the main force among the granules. However, the

377 particle size of EG also showed a significant decline in the presence of urea, with a

378 decrease of 88.7%, while that of UG merely declined by 4% (Fig. 4A). The results

379 showed that the hydrogen bonding between EG was significantly enhanced after

380 enzymatic hydrolysis. The main forces maintaining the EG were hydrophobic

381 interactions and hydrogen bonds.

382 The results of the FTIR spectra confirmed the above assumption (Fig. 4B). The

383 vibrational frequencies of the hydrogen bonds of UG and EG were 3410 cm-1 and

384 3290 cm-1, respectively (Fig. 4B). They shifted slightly to lower frequencies,

385 indicating that the hydrogen bonding between the granules was enhanced after

386 hydrolysis. This indicated that hydrolysis did not have a fundamental destruction

387 effect on the internal structure of EYG. Instead, the hydrogen bond was merely

388 strengthened. These results were in line with the significant changes in the size of the

389 EG aggregates when treated with 6 M urea to break the hydrogen bonds (Fig. 4A).

390 The wettability of solid particles can reflect the hydrophobicity and the

19
391 interfacial adsorption capacity of the particles (Duan, et al., 2014). It is generally

392 evaluated using the three-phase contact angle of particles at the oil/water interface

393 (Paunov et al., 2007). Proper wettability enhances the adsorption efficiency and

394 broadens the coverage at the droplet surface to form a physical barrier (Binks, 2002).

395 Fig. 4C shows the three-phase contact angle of UG, which were highly hydrophobic,

396 with a contact angle of 106.3°. After enzymatic modification, the hydrophilicity of

397 EG was greatly enhanced, and the contact angle was reduced to 93.4°. The strong

398 hydrophobicity of native EYG can also be reflected in its extreme insolubility

399 (Supplemental Fig. S2). The near-neutral wettability with a contact angle of

400 approximately 90° endows the particles with the largest desorption energy, which

401 would facilitate interfacial adsorption and thereby produce steric hindrance against the

402 aggregation of oil droplets (Dai, Sun, Wei, Mao, & Gao, 2018; Yuan et al., 2018).

403 Similarly, octenyl succinic anhydride-modified gliadin nanoparticles have been

404 reported to show an increase of the contact angle from 55.1° to 99.4°, better

405 adsorption efficiency and excellent properties against the aggregation of oil droplets

406 (Li et al., 2019). The decreased hydrophobicity of EYG after hydrolysis could be

407 attributed to several reasons. Both hydrophilic and hydrophobic groups were exposed

408 after hydrolysis. However, further hydrophobic interactions occurred due to the weak

409 steric hindrance between the peptide chains, which in turn reduced the surface

410 hydrophobicity (Tang et al., 2019). Additionally, subtilisin has preferred cleavage

411 sites next to Trp, Tyr and Phe at aromatic amino acid residues (Tamm et al., 2016).

412 After hydrolysis, the newly produced polar groups (-COOH, -NH2) increased the

20
413 charge density, producing stronger hydrophilicity of the granules. Additionally,

414 enzymatic hydrolysis by subtilisin produced many small-molecule peptides which

415 were of better hydrophilicity. The above-combined effects led to a decrease in the

416 surface hydrophobicity of EYG. The modified EG had a suitable surface wettability

417 and were relatively more hydrophilic than UG, which explained their better dispersity

418 in water in Fig. 3B.

419 The zeta potential of EYG is shown in Fig. 4D. The zeta potential of EYG

420 decreased from -19.63 mV to -22.97 mV after enzymatic hydrolysis. It was speculated

421 that more phosphocalcic bridges were exposed after enzymatic hydrolysis, increasing

422 the unit charge density on the surface of EG. The increase of surface charge could

423 increase the electrostatic repulsive force between granules, effectively inhibiting the

424 formation of EG aggregates in water (Fig. 3B) (Gouda, Zhang, Liu, Sheng, & Ma,

425 2017).

426 3.2.3 Protein composition of EYG

427

21
428 Fig. 5 SDS-PAGE profiles of UG, EG, untreated plasma (UP) and enzymatic plasma (EP).

429 The changes in the protein composition of EYG were characterized by

430 SDS-PAGE analysis (Fig. 5). In total, 15 major bands with molecular weights varying

431 from 10 to 221 kDa were observed in UG (Fig. 5-UG). Five of these 15 bands were

432 assumed to be the main apo-HDL from UG (marked with a line), with molecular

433 weights of 110 kDa, 100 kDa, 78k Da, 47 kDa and 32 kDa, respectively (Freschi,

434 Razafindralambo, Danthine, & Blecker, 2011; Orcajo, Marcet, Paredes, & Díaz, 2013;

435 Strixner & Kulozik, 2013a). Enzymatic hydrolysis by subtilisin seemed to mainly

436 exert a great impact on polypeptides with large molecular weights. The 38 kDa, 78

437 kDa and 110 kDa peptides from apo-HDL and two others with higher molecular

438 weights distinctly disappeared after enzymatic hydrolysis. Accordingly, five new

439 bands neighboring the area are seen in Fig. 5-EG, which might be attributed to the

440 aggregation of low-molecular-weight peptides produced during the hydrolysis.

441 Notably, the 100 kDa, 47 kDa and 32 kDa apo-HDL remained in the EG, which

442 suggested their excellent adsorption capacities. Also, several peptides with small

443 molecular weights of less than 35 kDa appeared (Fig. 5-EG). Tang et al. (2019)

444 attributed the new protein groups to the combination of the hydrophobic groups and

445 lipoproteins. Interestingly, similar peptides of 110 kDa in the hydrolyzed plasma and

446 UG were observed (Fig. 5-EG, EP). We assumed that the apo-HDL in UG might fall

447 off during the hydrolysis process and move into the hydrolyzed plasma. The

448 compositional changes in the granules are likely to further affect their adsorption at

449 the oil-water interface.

22
450 3.3 Interfacial adsorption of EYG

451

452 Fig. 6 CLSM images of emulsions stabilized by EYG (A) with a magnification of 20×,

453 oil-water interfacial tension (B) of UG and EG, and SDS-PAGE profiles (C) of unadsorbed (u)

454 and adsorbed (a) proteins in emulsions stabilized by UG and EG.

455 The CLSM images were used to investigate how EYG adsorbed onto the droplets

456 (Fig. 6A). The red area is soybean oil stained with Nile Red. The green distributed

457 area is EYG stained with FITC. EYG can be seen on the surface of the oil droplets

458 (Fig. 6A). They formed a solid interface film with high spatial hindrance, which could

459 prevent the infiltration of oil and water into each other. This result showed that EYG

460 could adsorb onto the oil-water interface. The EYG-stabilized emulsion was likely to

461 be a kind of solid-stabilized Pickering emulsion. Additionally, many free, unadsorbed

462 EYG were observed in the continuous phase (Fig. 6A). It was assumed that the EYG

463 in the continuous phase formed a solid three-dimensional network structure through

23
464 hydrophobic interactions and hydrogen bonds. The droplets were trapped in the

465 network, preventing further accumulation and creaming. Similarly, photocatalytic

466 soybean protein has been reported to form static hindrance and electrostatic effects of

467 particle-based networks at the oil-water interface through flocculation (Jin et al.,

468 2019). The waxy cassava starch modified by acid hydrolysis and esterification could

469 form a stable network over time through granule-granule and granule-interface

470 interactions (Fonseca-Florido et al., 2019).

471 The oil-water interfacial tension of UG and EG was then measured to compare

472 their ability to adsorb at the interface (Fig. 6B). Colloidal particles with stronger

473 interfacial adsorption properties usually yield lower interfacial values when balanced

474 (Soltani & Madadlou, 2015). In our experiment, a low protein concentration was

475 employed to reduce the effects of aggregation. The interfacial tension of both UG and

476 EG were reduced (Fig. 6B), further indicating the capacity of EYG to adsorb at the

477 oil-water interface. After enzymatic hydrolysis, the interfacial tension of EG tended to

478 equilibrate at a much lower value of 8.4 mN/m compared with the equilibrium value

479 of 15.5 mN/m of UG (Fig. 6B). This result proved that EG had a stronger interfacial

480 adsorption capacity than that of the UG, which explained the better emulsifying

481 properties of EG.

482 To further understand how enzymatic hydrolysis led to the difference in the

483 adsorption capacity, the adsorbed and unadsorbed proteins at the interface were

484 detected by SDS-PAGE analysis (Fig. 6C). Before enzymatic hydrolysis, four main

485 peptides in UG at approximately 82 kDa, 70 kDa, 64 kDa and 38 kDa (Fig. 6C-UG-u)

24
486 did not fully adsorb onto the interface. Almost all the apo-HDL in UG participated in

487 the adsorption of the interfacial film (Fig. 6C-UG-a). Le Denmat et al. (2000) also

488 reported that the 105 kDa peptide from apo-HDL became the major peptide in the

489 interfacial film. After hydrolysis, there was no such great difference between the

490 adsorbed and unadsorbed proteins in EG. However, the 38 kDa peptide in EYG,

491 which failed to adsorb at the interface, seemed to be completely hydrolyzed into

492 smaller peptides (Fig. 6C-EG-u/a). However, more newly produced peptides

493 (approximately 100 kDa, 95 kDa and 74 kDa), which were probably the hydrolysates

494 of large-molecular weight peptides induced by subtilisin (Fig. 6C-EG-a), appeared

495 among both unadsorbed and adsorbed proteins in EG. Notably, the new peptide with

496 an approximate 100 kDa molecular weight became the major peptide at the interface.

497 However, the main apo-HDL that was previously adsorbed at the interface

498 disappeared or became less prominent. Thus, enzymatic hydrolysis improved the

499 adsorption of EYG by modification of their protein constitutes. Subtilisin mainly

500 hydrolyzed peptides in the granules with poor adsorption capacities and generated

501 new peptides with stronger interfacial adsorption capacities. However, the core

502 peptides with excellent adsorption capacities, especially the 32 kDa peptide from

503 HDL and those between 55 kDa to 85 kDa, remained intact after hydrolysis.

504 Hence, it can be concluded that enzymatic hydrolysis led to a significant change

505 in the protein composition of the granules as well as of the interfacial film, which

506 made the proteins more easily adsorb at the interface in the emulsion. Enzymatic

507 hydrolysis significantly (P < 0.05) strengthened the hydrogen bonds between EG,

25
508 which may have enhanced the interaction between the granules adsorbed on the

509 interface layer and that with unadsorbed granules in the continuous phase. Then, the

510 improvement of the wettability made the EG adsorb onto the oil-water interface faster

511 and significantly reduced the interfacial tension to maintain their stability.

512 Comparatively, UG were easily desorbed from the interface due to their strong

513 surface hydrophobicity. The surface charge enhanced the electrostatic repulsive force

514 to allow adsorption onto the interface (Gao et al., 2017).

515 4. Conclusion

516 Enzymatically hydrolyzed EYG are potential Pickering particles with improved

517 emulsifying stability compared with untreated EYG. This effect is probably due to the

518 changes in the protein constituents of EYG after subtilisin treatment. The structural

519 changes in EYG subsequently induced a series of positive changes in the surface

520 characteristics. The internal hydrogen bonding was strengthened, the surface

521 wettability was improved and the surface charge was increased, which benefitted the

522 emulsification. Further work is recommended to examine the interactions between

523 proteins from different sources (HDLs, LDLs and the other proteins), as well as those

524 of proteins and lipids at the interface using a better nondestructive detection method.

525

526 Acknowledgments
527 This research was supported by Excellent Youth Foundation of Hubei Province

528 Natural Science Foundation (No. 2018CFA073).

529

26
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689

690

691

692

30

Egg yolk granules (EYG) were enzymatically modified and structurally analyzed.

The aggregation states were imaged by SEM, CLSM and OM.

The protein composition of EYG and the interface was measured by SDS-PAGE.

Hydrogen bonding was enhanced and the wettability and charge were increased

after hydrolysis.
 We declare that this work has not been published elsewhere and is not under

consideration by another journal. The manuscript is approved by all authors to be

submitted to Food Hydrocolloids. The authors have no conflicts of interest to declare.