Chemical Lipophilization of Bovine Î±-Lactalbumin with Saturated Fatty

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Functional Structure/Activity Relationships

Chemical lipophilization of bovine #-lactalbumin with saturated fatty acyl residues: effect on structure and functional properties Liliana Gabriela Mendoza-Sanchez, Maribel Jimenez-Fernandez, Guiomar Melgar-Lalanne, Gustavo F. Gutiérrez-López, Andres Hernandez-Arana, Francisco Reyes-Espinosa, and Humberto Hernandez-Sanchez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05174 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Journal of Agricultural and Food Chemistry

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Chemical lipophilization of bovine α-lactalbumin with saturated fatty acyl residues: effect on

2

structure and functional properties

3

Liliana G. Mendoza-Sánchez†, Maribel Jiménez-Fernández‡, Guiomar Melgar-Lalanne‡, Gustavo F.

4

Gutiérrez-López†, Andrés Hernández-Arana§, Francisco Reyes-Espinosa§, Humberto Hernández-

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Sánchez†*

6

† Depto.

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Nacional, Unidad Adolfo López Mateos, Av. Wilfrido Massieu esq. Cda. Manuel L. Stampa, CP.

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07738, Mexico City, México

9

‡Instituto

de Ingeniería Bioquímica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico

de Ciencias Básicas, Universidad Veracruzana, Av. Dr. Luis Castelazo Ayala s/n, Col.

10

Industrial Animas, CP. 91190, Xalapa, Veracruz, México

11

§Área

12

Iztapalapa, Av. San Rafael Atlixco 186, Col. Vicentina, CP. 09340, Mexico City, México

de Biofisicoquimica, Depto. de Química, Universidad Autónoma Metropolitana Unidad

13 14

1

ACS Paragon Plus Environment


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Abstract

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Bovine α-lactalbumin (α-LA) was chemically modified by the covalent attachment of fatty acid

17

residues of different length (lauroyl, palmitoyl, and stearoyl) to modify its functional and antioxidant

18

properties. Structural changes, functional properties and antioxidant capacity in the pH interval

19

between 3 and 10 were analyzed. Surface properties were improved. The esterification increased the

20

hydrophobic interactions leading to a reduction in the solubility dependent on the incorporation ratio of

21

the fatty acid residues. Improvement in emulsifying, foaming, and antioxidant properties were observed

22

when the length of the fatty acid chains was short and mostly at a basic pH. With these results in mind,

23

experiments could be conducted for the technological applications of these derivatives in the food,

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pharmaceutical and cosmetic industries.

25 26

Keywords: lipophilization, α-lactalbumin, structural analysis, functional properties, antioxidant

27

activity

28 29

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30

INTRODUCTION

31

Bovine α-lactalbumin (α-LA) is the second most abundant protein in bovine whey concentrates where

32

it accounts for 15 to 20% of the total proteins in bovine whey. It has a molecular weight of 14.2 kDa

33

and possesses a high-quality protein profile being particularly rich in tryptophan, lysine and cysteine.

34

Among its functional and technological properties, its high-water solubility along a wide range of pH

35

values (2.0-9.0), its calcium binding capacity and its emulsifying and foaming capacities stand out

36

(1,2). These characteristics make α-LA a useful additive in the formulation of emulsions, foams and

37

gels, providing flexibility in product formulations including infant formulas, protein-fortified

38

beverages, lactose free and reduced-carbohydrate foods and pharmaceutical and cosmetic products (3).

39

The structure of the protein reveals its biological and technological functions (4,5) which are explained

40

both by their solubility and the hydrophobic interactions with the aqueous phase (6). When a protein is

41

modified by physical or chemical methods, the most changing structures are the secondary and the

42

tertiary which alter the surface exposure of amino acids (7). Thus, any change in the protein

43

hydrophobicity might lead to an improvement on the surface properties (8), and this increase in

44

hydrophobicity may enhance their application in lipophilic systems (9).

45

Protein functionality can be determined by the length scale of structural elements which define its

46

functionality. These functionalities can be classified based on their scale properties into molecular

47

(hydrophobicity), molecular and mesostructure (solubility), and mesostructure (related to creation and

48

stabilization of colloidal mesostructures such as foams, emulsions, gels, etc.). This capacity to form and

49

stabilize colloidal mesostructures is a good method to compare relative functionality among proteins

50

which have undergone a chemical modification (10).

51

Proteins can be modified by the covalent union of specific molecules; these molecules are able to

52

change the protein behavior and are the main factors responsible of the foaming capacity, aggregation 3



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inhibition and surface properties improvement (11). A relatively simple way to improve the protein

54

hydrophobicity is through lipophilization, which involves the modification of the

55

hydrophobic/hydrophilic protein characteristics by the covalent attachment of lipophilic groups to the

56

protein. Chemical lipophilization of proteins with hydrophobic groups can be done with N-

57

hydroxysuccinimide ester (12), succinic and acetic anhydrides (13), and fatty acid acyl chlorides (6) to

58

produce significant alterations in the structure, and so in the functional properties (14).

59

Lipophilization could be defined as the chemical or enzymatic esterification of different substrates

60

(proteins, polyphenols, carbohydrates, etc.) with a lipophilic moiety (fatty acid or fatty alcohol)

61

resulting in molecules with an enhanced hydrophobicity (15). This process has been recently proven to

62

increase the antioxidant activity of different molecules such as ascorbic acid, resveratrol, ferulic acid,

63

vanillyl alcohol and rutin (9, 16, 17, 18). Moreover, lipophilized molecules can extend the stability of

64

oil-soluble dyes (19) and improve the quality of camelia seed oils (20).

65

Some chemically lipophilized proteins (wheat and soybean) have shown an improvement in functional

66

properties such as emulsion stabilizing properties (6, 21). This improvement has also been

67

demonstrated in milk proteins such as αs1 casein (22) in which its ability to form and stabilize

68

emulsions increased significantly and β-lactoglobulin (8) in which an increase in its emulsifying and

69

foaming properties was observed. In all cases, the authors indicated that a decrease in water solubility

70

caused by the lipophilization process was responsible for these improvements.

71

However, the modification of α-LA, a protein with important technological and bioactive properties

72

(23), by chemical lipophilization has not previously been studied.

73

Therefore, the objective of the present research was to evaluate the effect of lipophilization on bovine

74

α-LA and to determine if this chemical modification can generate an improvement in some of its

75

functional and bioactive properties. 4


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MATERIALS AND METHODS

78

Materials

79

Bio PURETM α-lactalbumin (α-LA) from bovine milk was kindly provided by Davisco Foods

80

International (Eden Prairie, MN, USA). It was isolated from fresh, sweet dairy whey and was in native

81

form and fully soluble in a wide pH range. It contains 6.0% moisture, 95.0 % protein (dry basis) (N

82

factor 6.25; 90.0 % α-lactalbumin), 0.5% fat, 3.5% ash, 0.2% lactose according to the supplier. The use

83

of a purified protein, such as α-LA, has the additional advantage that it can be used to study variations

84

in its secondary structure by circular dichroism. Corn oil (MazolaTM, Mexico) used in the present

85

research was purchased in a local supermarket (Mexico City, Mexico). Lauroyl, palmitoyl, and stearoyl

86

chlorides and the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from

87

Sigma Aldrich (St. Louis, MO, USA).

88

Sample preparation

89

Native and chemically lipophilized α-LA were stored in hermetically sealed containers under

90

refrigeration (4 °C) until further use. All the analyses were performed by dissolving the α-LA protein

91

(native or modified) with magnetic agitation for 30 min at room temperature (∼ 25 °C) in 0.1 M

92

phosphate buffer at different pH values (3, 5, 7, and 10).

93

Chemical lipophilization

94

Chemical lipophilization was done following the methodology of Roussel-Philippe et al. (6) with slight

95

modifications. The protein was dispersed in water in a 1/5 (w/v) protein/water ratio and pH was

96

adjusted to pH 9.0 with 4M NaOH. The reaction was completed with dropwise addition of the acyl

97

chlorides at pH 9.0, 30°C and under magnetic agitation. The protein / acyl chloride ratio was 1/0.5

98

(w/w). The acyl chlorides used were lauroyl chloride, palmitoyl chloride, and stearoyl chloride, 5



99

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respectively. The reaction mixtures were adjusted to pH 4.3 with the addition of 6M HCl for

100

precipitation and centrifuged (Sorvall Legend RT, Germany) at 18000xg for 20 min and then washed

101

twice and centrifuged as above with acidified water at pH 4.3 to eliminate residues. The dispersion was

102

dried at 38°C. The excess of fatty acid present in the lipophilized proteins was eliminated by extraction

103

with n-hexane.

104

Structural assays

105

Determination of the degree of modification

106

The degree of modification was determined by using the OPA assay (o-phtalaldehyde assay) as

107

previously described (24). A 50 µL sample with 0.1% protein was mixed with 1 ml of OPA reagent

108

prepared in sodium tetraborate buffer (pH 9.5) and incubated at room temperature for 2 min.

109

Absorbance was read at 340 nm (spectrophotometer Genesys 10S UV-Vis, Waltham, MA, USA). The

110

degree of modification (%) was calculated based on the absorbance decrease of the acylated samples

111

compared with the native one.

112

Circular dichroism (CD)

113

Conformational changes in the secondary structure of the protein were evaluated by CD spectra as

114

described by Rodiles-López et al (1), with slight modifications, in the range of 185 to 245 nm (far UV)

115

in a J715 spectropolarimeter (Jasco Inc., Easton, MD, USA) equipped with a PTV-348WI type peltier-

116

cell holder for temperature control; The measurements were made in cuvettes with a 1-mm pathlength

117

at 25°C using 0.1 mg/ml protein solutions in 5 mM phosphate buffer. The results were expressed as

118

molar ellipticity (degree cm2 / dmol). The secondary structure estimation of both native and

119

lipophilized samples (α-helix, β-sheet, and random conformation percentages) was assessed according

120

to Greenfield (25).

121

Surface hydrophobicity by extrinsic fluorescence 6


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122

Surface hydrophobicity was determined with a hydrophobic fluorescence probe 1-anilino-8-

123

naphtalenesulfonate (ANS) following the method described by Nakai and Kato (26) with some

124

modifications. Each protein sample was prepared at 1 µM in 5mM phosphate buffer, pH 7.0. A 1:100

125

protein:ANS (M/M) mixture was prepared. The interaction with the ANS was analyzed with an ISS K2

126

spectrofluorometer (ISS Inc., Champaign, IL, USA) equipped with a water-jacketed cell holder for

127

temperature control. The ANS excitation was performed at 380 nm and emission was measured from

128

400 to 600 nm at 25 °C. Both bandwidths were set at 1 nm.

129

Surface hydrophobicity by Intrinsic fluorescence

130

The intrinsic fluorescence spectra were measured with a Spectrofluorometer ISS K2 (ISS Inc.,

131

Champaign, IL) according to the method of Edwin and Jagannadham (27). Protein samples

132

concentration was 1 µM of protein in 5 mM phosphate buffer, pH 7. The excitation wavelength was at

133

280 nm and the emission was measured at a wavelength range of 300 to 450 nm (slit width1 nm).

134

Potential ζ

135

Potential ζ was measured following the method described by Arroyo-Maya et al. (28). Titration

136

experiments were performed over a pH range between 3 and 10 at 25 °C with a Malvern Zetasizer

137

Nano S (model MAL1600, Malvern, Worcestershire, United Kingdom). Samples were previously

138

diluted 1:10 in deionized water.

139

Evaluation of functional properties

140

Solubility index (SI)

141

SI was determined by the method of Hou et al. (29) with slight modifications. Diluted protein solutions

142

(1%) were dispersed in 0.1 M phosphate buffers. Suspensions were stirred for 30 min avoiding foam

143

formation and centrifuged at 10000xg for 20 min at 20 °C. The protein content of the supernatants was

144

determined by the Bradford method (30). The solubility index was calculated as in equation (1): 7



145

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(1) SI (%) = (protein in the supernatant x 100)/ protein in the sample

146

Emulsifying activity index (EAI)

147

The EAI was determined according to Diniz et al. (31). A mixture of 30 mL of 0.5 % protein solution

148

in 0.1 M phosphate buffer and 10 mL of corn oil was prepared. The mixture was homogenized for 60 s

149

at 12,000 rpm with a blender (D 130 Wiggen Hauser, Sdn Bhd). Fifty milliliters of emulsion were

150

dispersed into 5 mL of 0.1 % sodium dodecyl sulfate (SDS). Absorbance was measured at 500 nm with

151

a spectrophotometer (Genesys 10S UV-Vis, USA). The EIA was calculated using equations (2) and (3):

152

(2) Tb = 2.303 A / l

153

Where Tb is turbidity, A is the emulsion absorbance at 500 nm, and l is the path length of the cuvette

154

(3) EAI = 2 Tb / ϕC

155

Where EAI is the Emulsifying Activity Index, C is the weight of protein per unit of aqueous phase

156

before the emulsion is formed, and ϕ is the volume fraction of dispersed phase.

157

Emulsion stability

158

The emulsion stability was determined with a Turbiscan Lab Expert (Expert, Formulation Inc., France).

159

This instrument allows the characterization of the instability of concentrated emulsions through the

160

detection of the transmitted and backscattered (BS) light from a near infrared source (32). Briefly, 18

161

mL of sample were transferred to a cylindrical glass cell. The emulsion destabilization was analyzed

162

with the BS profiles obtained by scanning the sample from the bottom to the top with a light beam

163

(λ=880 nm) at different time intervals. The variation of the BS signal (ΔBS) was calculated as the

164

difference between BS at the initial time and the BS at a defined time. Finally, the Turbiscan stability

165

index (TSI), which is a statistical parameter used to estimate the emulsion stability (Wisniewska,

166

2010), was calculated with the software Turbiscan Lab Expert as equation (4):

8


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𝑛

167

(4) 𝑇𝑆𝐼 =

∑𝑖 = 1𝑥𝑖 ― 𝑥𝐵𝑆 𝑛―1

168

Where xi is the backscattering value for each minute; xBS is the average of xi and n is the number of

169

measurements.

170

Emulsions were prepared in a protein dispersion: oil ratio of 3:1; both, the native proteins and the

171

modified proteins were previously dispersed at a 0.5 % concentration in 0.1 M phosphate buffer.

172

Emulsions were monitored each 10 min up to an hour in the equipment.

173

Foaming capacity (FC) and foam stability (FS)

174

The FC and FS were evaluated following the methodology reported by Miedzianka and Pęksa (33) with

175

a few modifications. A 1% protein suspension was blended (D 130 Wiggen Hauser) at 12000 rpm for

176

90 s and the foaming capacity was calculated using equation (5):

177

(5) FC = (B-A) x100 / A

178

Where A is the volume of the sample before blending and B is the volume of the sample after blending.

179

For FS the foam volume was registered for 120 min at 10 min-intervals. FS was calculated as reported

180

by Haque et al. (12) from equation (6):

181

(6) FS = (F2 x 100) / F1

182

Where F1 and F2 are the foam volumes after agitation and after rest, respectively.

183

Surface and interfacial tension

184

The surface and interfacial tension of native and lipophilized proteins were estimated according to

185

Daverey and Pakshirajan (34) with some modifications using a surface tensiometer (DCAT 11,

186

Dataphysics Instruments, Germany) based on the Wilhelmy plate method. The Wilhelmy plate used

187

was 10 mm in length, 19.9 mm in width, and 0.2 mm height. Several 0.25 % (w/v) protein solutions

9



Page 10 of 37

188

were prepared in 0.1 M phosphate buffer and their surface tension measured. Interfacial tension

189

measurements were carried out against corn oil.

190

Antioxidant activity

191

FRAP (Ferric Reducing Antioxidant Power) assay was performed as described by Manzi and Durazzo

192

(35). The method is based on the reduction of a Fe3+ complex tripyridyltriazine (TPTZ) to the Fe2+

193

form at low pH. This reaction is monitored by measuring the absorbance variation at 593 nm. The

194

FRAP reagent included 10 mM TPTZ and 20 mM of FeCl3 in 0.25 M acetate buffer (pH 3.6). Then, 3

195

ml of FRAP reactive were mixed with 100 µL of sample and the absorbance after 30 min of incubation

196

at 37 °C was measured at 593 nm with FRAP reactive diluted in distilled water as the blank. The ABTS

197

(antioxidant activity determined by 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) antioxidant

198

activity assay was performed as described by Oh et al. (9) with slight modifications. The ABTS•+

199

radical was generated when an ABTS solution in water was oxidized by addition of 2.45 mM

200

potassium persulfate. The mixture was allowed to stand in the dark at 20ºC for 12–16 h before use and

201

then diluted in 0.1 M potassium phosphate buffer, pH 7.4 prior to assay. An adequate amount of sample

202

was added to 1 mL of reagent and incubated at 25°C for 10 min. Scavenging of the ABTS•+ radical was

203

followed by the decrease in absorbance at 730 nm measured spectrophotometrically. The results for

204

both methods were expressed in µmol of Trolox equivalents per liter of sample.

205

Statistical analysis

206

All data are representative of at least 3 separate experiments. These results represent the means ±

207

standard deviations of triplicate determinations. A one-way analysis of variance (ANOVA) with a α=

208

0.05 was used to evaluate possible differences between the treated and untreated samples.

209 210

RESULTS AND DISCUSSION 10


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211

Structural properties

212

Degree of modification

213

The union between the carboxylic group of the fatty acid with the amine group of the protein results in

214

a decrease in the number of free amine groups. The modified α-LA with acyl chlorides of lauroyl,

215

palmitoyl, and stearoyl showed a 42.26, 54.54, and 66.09 % free amine groups respectively. The

216

modification degree of α-LA decreased with the size of the fatty acid chain. So, the short chain fatty

217

acid acyl chlorides may react better than long chain fatty acids which might be explained in terms of

218

the steric hindrance caused by the size of the fatty acids and its reactivity as previously described by

219

Milstien and Fife (36).

220

Circular dichroism (CD)

221

CD was used to measure the effect of chemical lipophilization on the secondary structure of α-LA at

222

different pH values. CD spectra in the far ultraviolet (190 to 250 nm) was used to calculate this effect

223

using the K2D2 software (37). The α-helix, β-strand, and random structure contents in α-LA native and

224

modified by chemical lipophilization at different pH values are shown in Table 1. Results showed that

225

pH played an important role in the secondary structure of the protein, as well as the amount of fatty

226

acid incorporated in the α-LA. Robbins and Holmes (38) reported that α-LA native is formed by 25-

227

26% α-helix, 14-15% β-strand y 60% random structure, which is in agreement with the results obtained

228

in this study and shown in Table 1 for native α-LA at pH 7. Similar results were recently obtained by

229

Bi et al. (39). At pH 3 and 5, structural transitions from α-helix to random structure were observed

230

when 16 C and 18 C chains were incorporated. The opposite happens at pH 7 and 10. A different

231

transition occurred at pH 5 when α-helix decreased to 11 % and β-strand increased to 41 % when

232

lauroyl residues were incorporated into the protein. The opposite occurred with the same sample at pH

11



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233

10, where the maximal amount of α-helix (55%) was obtained. These facts could indicate that great

234

changes in several functional properties for the LA-12C samples at pH 5 and 10 could be expected.

235

Surface hydrophobicity by intrinsic fluorescence

236

The fluorescence emission in protein has its origins in the Phe, Tyr, and Trp residues. In proteins that

237

contain residues of these three amino acids, such as α-LA, the fluorescence is usually dominated by Trp

238

residues because its absorbance, excitation length and quantum yield are larger than in Phe and Tyr

239

(40). Fluorescence emission spectra were generated by exciting at 280 nm. The maximum wavelength

240

of intrinsic fluorescence (Table 2) was obtained in the 315 to 322 nm region of the spectrum and

241

variations in the fluorescence intensity could be observed, indicating the existence of conformational or

242

structural changes in the native and modified α-LA and in response to pH variations. The fluorescence

243

intensity observed in Table 2 is normalized with respect to the native α-LA sample at pH 7. An increase

244

in hydrophobicity was observed due to the chemical lipophilization reaction. The highest increases

245

were observed in the α-LA-12C sample at pH 5, 7, and 10. It is quite possible that the structural

246

modification of α-LA or the increase in negative charges on the surface of α-LA upon lipophilization

247

lead to the exposure of buried hydrophobic sites (41).

248

Surface hydrophobicity by extrinsic fluorescence

249

The exposition of some hidden hydrophobic zones in the native and modified proteins was monitored

250

by the ANS probe. The emission data in the presence of ANS were obtained by applying UV light

251

(λ=380 nm) and are shown in Table 2. The maximum wavelength (λ max) registered was between 430

252

to 528 nm.

253

In the case of the native α-LA at pH 5, 7, and 10, no significant changes were observed both in λ max

254

and in fluorescence intensity (close to 484 nm); however, at pH 3, an increase of ~ 29% in the emission

255

fluorescence intensity compared to α-LA at pH 7 (λ max = 484 nm) was observed. In all the emission 12


Page 13 of 37


256

fluorescence intensities of the modified α-LA, magnitudes lower than 32 % with respect to α-LA at pH

257

7 were found. There were three cases (α-LA-12C at pH 5, α-LA-16C at pH 5, and α-LA-12C at pH 7),

258

where decreases in λ max of 20, 54 and 26 nm compared to native α-LA at pH 7 were detected. Also, a

259

redshift in λ max of 44 nm was observed in the case of α-LA-18C at pH 7, compared to the one of

260

native α-LA a pH 7. This decrease in fluorescence may be due to the blocking of the hydrophobic

261

residues of tyrosine by the fatty acid chain (42).

262

ζ Potential

263

Figure 1 shows the changes in ζ potential of the native and chemically lipophilized α-LA with pH. As

264

expected, it was observed that ζ potential decreases when the pH increases. The isoelectric point for the

265

native α-LA was pH 4.1 which is in agreement with previous reports (between 3.5-4.8) (23). However,

266

the isoelectric points for the modified α-LAs with lauroyl, palmitoyl, and stearoyl residues were 3.5,

267

3.7, and 3.8 respectively. This may be due to the fact that chemical lipophilization produced an increase

268

in hydrophobic regions in the surface of α-LA due to the union of aliphatic chains. A reduction in

269

positive surface charges can also be expected due to the formation of amide type covalent unions

270

between the free amino groups of lysine, arginine and histidine and the carbonyl groups of acyl

271

chlorides during the lipophilization (8). In the same figure, it can be observed that the lipophilization

272

reaction with any acyl chloride gives stability to the α-LA against agglomeration phenomena or

273

precipitation when the pH is higher than 6 where the ζ potential is < -30 mV reaching a theoretical

274

range of instability.

275

Functional properties

276

Solubility

277

Table 3 shows the solubility values calculated from Eq. (1) at four different pH values in the native and

278

modified protein. It could be observed that chemical lipophilization produces a significant reduction in 13



Page 14 of 37

279

solubility compared with the native protein. Solubility ranged from 3.66 to 90.63 % for modified

280

proteins and was dependent on pH and fatty acid being incorporated. The lowest solubility was

281

observed for α-LA-12C at pH5 sample, probably because of the reduction in α-helix content and

282

increment in β-sheet conformation probably related to a higher modification degree and with the

283

concomitant increment of surface hydrophobicity. The highest solubilities in lipophilized proteins were

284

observed at alkaline pH values and, as expected, when the pH was close to the isoelectric point, the

285

solubility was lower. This reduction in solubility could be due mainly to a reduction in the negative

286

total charges and to an increase in the hydrophobic interactions related to the fatty acid incorporation

287

which caused a reduction in the solvation degree and in hydrogen bonding in the aqueous medium (8).

288

Emulsifying activity index (EAI)

289

The effect of incorporating fatty acids over the EAI of α–LA at different pH values is shown in Table

290

3. The EAI of α-LA improved with the fatty acid addition, probably because the interfacial tension

291

decreases and the surface layer of the protein over the oil drop increased (43). The highest value of EAI

292

(149.94 m2g-1) was observed for the α-LA modified with lauric acid at pH 10. The EAI improved

293

significantly by chemical lipophilization for every fatty acid tested at pH 10. Similarly, (24) found an

294

improvement in the emulsifying properties in soya protein when it was modified with different fatty

295

acids (6C-18C) at pH 7 with mean values of 110 m2g-1. Nakai and Kato (26) also found that the

296

emulsifying activity increased when linoleate residues were non-covalently bound to soya and

297

sunflower proteins. In this study, the EAI was higher when the length of the incorporated fatty acid was

298

shorter; similar results were reported by Akita and Nakai (8) for β-lactoglobulin lipophilized with

299

stearoyl residues. Meanwhile, Haque and Kito (22) reported that the covalent binding of palmitoyl

300

residues to αs1 casein increased its ability to form and stabilize emulsions. They found that, generally,

301

the less modified protein showed a higher EAI than the more modified ones. At pH 3 there were not 14


Page 15 of 37


302

significant differences (p > 0.05) in EAI between the native and palmitoyl and linoleoyl α-LAs;

303

however, in the case of the α-LA modified with lauric acid, there was an important reduction in EAI of

304

46.51%, probably due to factors affecting hydrophobicity, solubility and conformation such as pH and

305

ionic effects which are directly involved in protein flexibility (44).

306

At the isoelectric point, both the native and the modified protein did not show good emulsifier activity.

307

The emulsifying capacity of the lipophilized proteins at pH 5, for the three tested fatty acids, decreased

308

compared with the native protein (see Table 3) probably because of a reduction in solubility, an

309

increase in the interfacial tension in the lipophilized products and steric effects that hindered the polar

310

segments in the aqueous media (43).

311

Regarding EAI for each fatty acid used to chemically modify the protein at different pH values, α-LA

312

modified with lauric acid showed the best values at pH 7 and 10; while in the case of palmitic acid, the

313

EAI was improved at pH 3, 7 and 10 and in the case of stearic acid, the improvement occurred at pH 3

314

and 10. Rodiles-López et al. (1) obtained similar results with α-LA treated with high hydrostatic

315

pressures. These results are probably related to the fact that the emulsifying properties are minimal at

316

the isoelectric pH and improvements are possible with an increase or decrease in pH (43).

317

Emulsion stability

318

The stability indices of emulsions at pH 3, 5 7 and 10 at 60 min for native α-LA and lipophilized with

319

three different fatty acids are shown in Table 3. The TSI values of emulsions at 60 min decreased when

320

the chain length of the fatty acids increased which implied an improvement in the emulsion stability

321

due to the lipophilization.

322

This is probably due to fact that the stability of the emulsion depended on the balance of hydrophilic

323

and hydrophobic amino acid residues and the unfolding of the lipophilized protein in the oil-water

324

interface (21). 15



Page 16 of 37

325

Haque and Kito (22) reported that the covalent union between palmitoyl residues in lipophilized αs1

326

casein increased the stability of the emulsion. Moreover, Mattarella and Richardson (45) incorporated

327

methyl groups to β-lactoglobulin through esterification and found that the stability of the emulsion

328

prepared with the modified protein was significantly higher than that of the one prepared with the

329

native protein.

330

The stability of the emulsions formulated with the lipophilized protein increased when the pH of the

331

medium in which they were dispersed increased reaching maximal values of stability (minimal values

332

of TSI) at pH 10 (Table 3).

333

Foaming capacity (FC) and foam stability (FS)

334

The effect of lipophilization of α-LA on foaming capacities at different pH values is shown in Table 4.

335

FC of modified α-LA with lauric acid improved at pH 7 and 10. The FS of the proteins was dependent

336

on the pH of the dispersing medium and on the length of the fatty acid residue. FC at pH 3 improved

337

with the number of carbons in the aliphatic chain of the fatty acids. At pH 3, 7, and 10, FS was higher

338

for the lipophilized proteins probably because of the increase in protein-protein interactions and to the

339

intermolecular cohesion which significantly reduced the surface tension. These results were similar to

340

those obtained by Kitabatake and Doi (46) for casein and whey protein. The application of physical

341

treatments such as high hydrostatic pressure, which decreased the solubility of α-LA, also improved the

342

FC and FS of the protein (1).

343

Surface and interfacial tension

344

The surface tension is a physical important property of the foam. Furthermore, it is known that lower

345

surface tensions are related to higher foamability (46). Surface tension values obtained in lipophilized

346

α-LA at different pH values are shown in Figure 2a. Surface tension values were lower for the

347

lipophilized samples at all the pH values tested, suggesting a reduction in the free surface energy in the 16


Page 17 of 37


348

air-protein solution interface in the modified α-LA. It can be observed that the surface tension of the

349

lipophilized samples had a maximum at pH 7 and decreased as the pH turned more acid or basic. The

350

minimum value of surface tension was obtained for the α-LA lipophilized with lauroyl groups at pH 10.

351

This sample, as expected, showed the highest FC (FC = 65.51%). These results agree with those

352

reported by Grahams and Phillips (44) who indicated that the foamability is related to the decrease of

353

the surface tension in β-casein, bovine whey albumin, and lysozyme.

354

The interfacial tension between protein solutions and corn oil was measured as a function of pH (Figure

355

2b). The effect of lipophilization and pH on the interfacial tension was significant (p ≤ 0.05). An

356

increase in the interfacial tension due to lipophilization in the pH range between 3 and 7 could be

357

observed. It is possible that the electrostatic repulsion between lipophilized α-LA molecules in the

358

interface were stronger that the one observed at pH values above 7 and as consequence the unfolding to

359

form a viscoelastic film was more complicated (47).

360

The higher capacity to reduce the interfacial tension was obtained, again, with the α-LA modified with

361

lauric acid at pH 10 and this sample, as anticipated, was the one with the greatest emulsifying activity

362

and stability (Table 3).

363

The decrease in interfacial tension is related to the increase in emulsifying activity, because changes in

364

hydrophobicity and conformation due to lipophilization, could explain the improved ability of the

365

sample α-LA-12C at pH10 to adsorb in the water-oil interface (48). These results agree with those

366

previously reported by Nakai and Kato (26) which showed a direct correlation between surface

367

hydrophobicity and emulsifying activity. In this study, the sample α-LA-12C at pH10 was the one with

368

the greatest surface hydrophobicity as measured by intrinsic fluorescence.

369

Antioxidant activity

17



Page 18 of 37

370

The main contribution of the antioxidant activity of the whey proteins is due to its content of

371

potentially antioxidant amino acids such as proline, tyrosine, tryptophan, histidine, lysine and

372

methionine previously reported by Pihlanto (49) and Rival et al. (50). The antioxidant activity in

373

biological systems usually differs widely and the mechanisms involved in evaluation methods are

374

diverse so two methods based on electron transfer were used in this study. Results obtained with the

375

FRAP and ABTS techniques for the native and lipophilized α-LA dispersed in buffer at different pH

376

values are shown in Table 5. The results from the ABTS assay indicate that all the lipophilized samples

377

have stronger antioxidant activities than the native protein at all pH values. In the case of the FRAP

378

assay, only the lauroylated derivatives have a highly significant increase in antioxidant activity in the

379

whole pH range. It can also be observed that the antioxidant activity decreased with the increase in the

380

carbon chain length of the fatty acids bound to the protein. Antioxidant activity values for α-LA-12C

381

at all the pH values tested were notoriously higher, sometimes up to ten orders or magnitude, than the

382

rest of the samples. These results are, then, in agreement with the cut-off hypothesis for the role of

383

hydrophobicity on the antioxidant activity of lipophilized compounds (51). This hypothesis indicates

384

that the antioxidant capacity increases as the incorporated alkyl chain is elongated until a threshold

385

(denominated the critical chain length) is reached for 12 carbon atoms (lauroylated samples) and that

386

beyond this limit, the antioxidant capacity immediately goes downward. However, the mechanism of

387

how hydrophobicity impacts the antioxidant activity is still unknown (51).

388

The differences between the values for each of the samples were statistically significant (p ≤ 0.05),

389

suggesting that the pH and the type of incorporated fatty acid influenced the antioxidant capacity of the

390

α-LA. These results agree with those obtained by Chen et al. (52) who, when studying the antioxidant

391

capacity of bovine milk using spectrophotometrical methods including ABTS and FRAP, found that

392

antioxidant activity increased when the pH increased. No correlation (R = 0.0477) could be found 18


Page 19 of 37


393

between the ABTS and FRAP assays, however, both results suggest that the lauroylated derivatives of

394

α-LA may have the potential of serving as antioxidants in lipophilic systems. A similar conclusion was

395

reported by Oh et al (9) for lipophilized resveratrol.

396

In conclusion, the chemical modification of α-LA with fatty acid residues improved the functional

397

properties of α-LA, although the effect depended on the length of the fatty acid chain and degree of

398

incorporation. The best results in the emulsifying, antioxidant and foaming properties through

399

lipophilization were obtained with lauric acid binding to α-LA. Moreover, significant differences were

400

observed in the behavior of the pH-dependent functional and bioactive properties, showing improved

401

properties at basic pH values, far from the isoelectric point. Finally, although the best properties were

402

obtained at pH 10, the use of lipophilized protein in foods with pH values close to 7 (such as many

403

leavened Mexican foods containing lime-treated corn flour) could be recommended, with good results

404

in terms of their functional properties and maximum antioxidant capacity. In general, the α-LA and its

405

acylated derivatives could be used as emulsifying or foaming additives for foods.

406

AUTHOR INFORMATION

407

Corresponding Author

408

*Telephone: +52-555-729-6000. E-mail: [email protected]

409

ORCID

410

Humberto Hernandez-Sanchez: 0000-0003-0769-8037

411

Acknowledgement

412

Author Mendoza-Sánchez acknowledges the support of the National Council of Science and

413

Technology (CONACyT) for the scholarship awarded to conduct this research in the Food Science

414

Ph.D. program at the Instituto Politécnico Nacional (Mexico City, Mexico).

415

Funding sources 19



416

This work was supported by SIP-IPN [grant number 20161435].

417

Notes

418

The authors declare no competing financial interest.

Page 20 of 37

419 420

ABBREVIATIONS USED

421

ϕ, volume fraction of dispersed phase; α-LA, α-lactalbumin; A, emulsion absorbence at 500 nm;

422

ABTS, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; ANS, 1-anilino-8-naphtalenesulfonate;

423

BS, backscattered light; C, weight of protein per unit of aqueous phase; CD, circular dichroism; EAI,

424

emulsifying activity index; F, foam volume; FC, foaming capacity; FS, foam stability; FRAP, ferric

425

reducing antioxidant power; l, path length of the cuvette; LA-12C, lauroylated α-lactalbumin; LA-16C,

426

palmitoylated α-lactalbumin; LA-18C, stearoylated α-lactalbumin; OPA, o-phtalaldehyde; SI

427 428

REFERENCES

429

1. Rodiles-López, J. O.; Jaramillo-Flores, M. E.; Gutiérrez-López, G. F.; Hernández-Arana, A.;

430

Fosado-Quiroz, R. E.; Barbosa-Cánovas, G. V.; Hernández-Sánchez, H. Effect of high hydrostatic

431

pressure on bovine α-lactalbumin functional properties. J. Food. Eng. 2008, 87 (3), 363-370.

432

2. Li, Q.; Zhao, Z. Characterization of the Structural and Colloidal Properties of α-

433

Lactalbumin/Chitosan Complexes as a Function of Heating. J. Agric. Food. Chem. 2018, 66, 972–

434

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3. Layman, D. K.; Lönnerdal, B.; Fernstrom, J. D. Applications for α-lactalbumin in human nutrition. Nutr. Rev. 2018, 76 (6), 444–460.

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5. Hussain, R.; Gaiani, C.; Scher, J. From high milk protein powders to the rehydrated dispersions in variable ionic environments: A review. J. Food. Eng. 2012, 113 (3), 486–503. 6. Roussel-Philippe, C.; Pina, M.; Graille, J. Chemical lipophilization of soy protein isolates and wheat gluten. Eur. J. Lipid. Sci. Technol. 2000, 102 (2), 97-101. 7. Foegeding, E. A.; Davis, J. P. Food protein functionality: A comprehensive approach. Food Hydrocoll. 2011, 25 (8), 1853–1864. 8. Akita, E.M.; Nakai, S. Lipophilization of β-lactoglobulin: effect on hydrophobicity, conformation and surface functional properties. J. Food Sci. 1990, 55 (3), 711–717. 9. Oh, W. Y.; Shahidi, F. Lipophilization of resveratrol and effects on antioxidant activities. J. Agric. Food Chem. 2017, 65 (39), 8617–8625. 10. Foegeding, E.A. Food protein functionality—A new model. J. Food Sci. 2015, 80 (12), C2670C2677. 11. Torchilin, V.P.; Omel'Yanenko, V.G.; Klibanov, A.L.; Mikhailov, A.I.; Gol'Danskiib, V.I.;

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18. Kharrat, N.; Aissa, I.; Sghaier, M.; Bouaziz, M.; Sellami, M.; Laouini, D.; Gargouri, Y.

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anthocyanin. Int. J. Food Prop. 2017, 20, 1627–1636.

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properties of soy proteins by acylation with saturated fatty acids. Food Chem. 2011, 124 (2), 596–

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25. Greenfield, N.J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2007, 1 (6), 2876-2890. 26. Nakai, S.; Kato, A. Hydrophobicity determined by a fluorescence probe method and its correlation

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27. Edwin, F.; Jagannadham, M. V. Sequential unfolding of papain in molten globule state. Biochem. Biophys. Res. Commun. 1998, 252 (3), 654-660. 28. Arroyo-Maya, I.J.; Rodiles-López, J.O.; Cornejo-Mazon, M.; Gutierrez-Lopez, G.F.; Hernández-

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Arana, A.; Toledo-Núñez, C.; Hernández-Sánchez, H. Effect of different treatments on the ability of

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α-lactalbumin to form nanoparticles. J. Dairy Sci. 2012, 95 (11), 6204-6214.

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29. Hou, F.; Ding, W.; Qu, W.; Oladejo, A. O.; Xiong, F.; Zhang, W.; He, R.; Ma, H. Alkali solution

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extraction of rice residue protein isolates: Influence of alkali concentration on protein functional,

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structural properties and lysinoalanine formation. Food Chem. 2017, 218 (1), 207–215.

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30. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of

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protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 7 (72), 248-254.

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31. Diniz, R. S.; Coimbra, J. S; dos R., Teixeira, Á. V. N. de C.; da Costa, A. R.; Santos, I. J. B.;

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Bressan, G. C., da Cruz-Rodriguez, A.M.; da Silva, L. H. M. Production, characterization and

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32. Mengual, O.; Meunier, G.; Cayre, I.; Puech, K.; Snabre, P. Characterization of instability of

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33. Miedzianka, J.; Pęksa, A. Effect of pH on phosphorylation of potato protein isolate. Food Chem. 2013, 138 (4), 2321–2326. 34. Daverey, A.; Pakshirajan, K. Sophorolipids from Candida bombicola using mixed hydrophilic

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35. Manzi, P.; Durazzo, A. Antioxidant properties of industrial heat-treated milk. J. Food Meas. Charact. 2017, 11 (4), 1690–1698. 36. Mielstien, J.B; Fife, T.H. Steric effects in the acylation of alpha-chymotrypsin. Biochem. 1969, 8 (2), 623-627. 37. Pérez-Iratxeta, C.; Andrade-Navarro, M. A. K2D2: estimation of protein secondary structure from circular dichroism spectra. BMC Struct. Biol. 2008, 8, 25. 38. Robbins, F.M.; Holmes, L. G. Circular dichroism spectra of alpha lactoalbumin. Biochim. Biophys. Acta. 1970, 221 (2), 234-240.

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39. Bi, H.; Tang, L.; Gao, X.; Jia, J.; Lv, H. Spectroscopic analysis on the binding interaction between

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tetracycline hydrochloride and bovine proteins β-casein, α-lactalbumin. J. Lumin. 2016, 178, 72–83.

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40. Schmid, F.X. Spectral methods of characterizing protein conformation and conformational

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changes. In T.E. Protein structure: A practical approach Creighton Ed., Oxford: Oxford University

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41. Shilpashree, B.G.; Arora, S.; Sharma, V.; Chawla, P.; Vakkalagadda. Changes in physicochemical

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42. Lakowicz, J. R. Time-Dependent Anisotropy Decays. In Principles of Fluorescence Spectroscopy. (3rd ed). Springer, Baltimore, Maryland, USA. 2006. Pp- 383-412. 43. Lamus, U.M.:Barrera-Arellano, D. Efecto de la lipofilización sobre las propiedades funcionales de la harina de palmiste (Elaeis guineensis). Grasas Aceites. 2005, 56, 1-8. 44. Graham, D.E.; Phillips, M.C. Proteins at liquid interfaces: I. Kinetics of adsorption and surface denaturation. J. Colloid Interface Sci. 1979, 70 (3), 403–439. 45. Mattarella, N. L.; Richardson, T. Physicochemical and functional properties of positively charged derivatives of bovine beta-lactoglobulin. J. Agric. Food Chem. 1983, 31 (5), 972-978. 46. Kitabatake, N.; Doi, E. Surface tension and foaming of protein solutions. J. Food Sci. 1982, 47, 1218-1221. 47. Lam, R. S. H.; Nickerson, M. T. The effect of pH and temperature pre-treatments on the

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2015, 60 (1), 427–434.

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48. Chobert, J. M.; Bertrand-Harb, C.; Nicolas, M. G. Solubility and emulsifying properties of caseins

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and whey proteins modified enzymically by trypsin. J. Agric. Food Chem. 1988, 36 (5), 883-892.

545

49. Pihlanto, A. Antioxidative peptides derived from milk proteins. Int. Dairy J. 2006, 16 (11), 1306–

546 547 548

1314. 50. Rival, S.G.; Boeriu, C.G.; Wichers, H. J. Caseins and casein hydrolysates. 2. Antioxidative properties and relevance to lipoxygenase inhibition. J. Agric. Food Chem. 2001, 49 (1), 295–302.

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549 550

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51. Laguerre, M.; Bayrasy, C.; Lecomte, J; Chabi, B; Decker, E.A.; Wrutniak-Cabello, C.; Cabello, G.; Villeneuve, P. How to boost antioxidants by lipophilization? Biochimie 2013, 95, 20-26.

551

52. Chen, J.; Lindmark-Mansson, H.; Gorton, L.; Akesson, B. Antioxidant capacity of bovine milk as

552

assayed by spectrophotometric and amperometric methods. Int. Dairy J. 2003, 13 (12), 927–935.

553 554

26


Page 27 of 37


FIGURE CAPTIONS Fig. 1. Zeta potential (mV) of α-LA, native and chemically lipophilized with different fatty acids, at several pH values. ▪ α-LA, • α-LA-12C,

α-LA-16C,

α-LA-18C

Fig. 2. Effect of pH on the surface (a) and interfacial (b) tensions of suspensions of α-LA, native and chemically lipophilized with different fatty acids. ▪ α-LA, • α-LA-12C,

α-LA-16C,

α-LA-18C

555

27



Page 28 of 37

Table 1. α-helix, β-strand, and random structure contents in α-LA native and modified by chemical lipophilization at different pH values α-helix (%)

β -sheet (%)

Random structure (%)

pH 3 α-LA

35

15

50

α-LA-12C

35

15

50

α-LA-16C

29

15

56

α-LA-18C

28

14

58

pH 5 α-LA

29

14

57

α-LA-12C

11

41

47

α-LA-16C

24

19

57

α-LA-18C

29

15

59

pH 7 α-LA

28

14

59

α-LA-12C

40

16

44

α-LA-16C

30

15

55

α-LA-18C

35

16

49

pH 10 α-LA

29

14

57

α-LA-12C

55

9

36

α-LA-16C

30

15

55

28


Page 29 of 37


α-LA-18C

28

15

58

α-LA= native alpha-lactalbumin; 12C to 18C represents the length of fatty acids, 12C = Lauric acid; 16C = Palmitic acid; 18C = Stearic acid.

556

29



Page 30 of 37

Table 2. Emission data for intrinsic and extrinsic fluorescence for α-LA native and modified by chemical lipophilization at different pH values Protein

pH 3

pH 5

pH 7

pH 10

λmax

NIF

λmax

NIF

λmax

NIF

λmax

NIF

IF

322

0.145

316

0.211

315

1.000

316

0.926

EF

482

1.288

483

1.016

484

1.000

483

1.092

IF

315

0.588

320

1.438

323

1.570

322

1.806

EF

485

0.319

464

0.080

458

0.020

481

0.019

IF

322

0.360

315

0.260

319

0.983

318

1.181

EF

483

0.041

430

0.031

485

0.035

484

0.024

IF

320

0.489

314

0.263

319

0.837

316

1.198

EF

482

0.034

483

0.121

528

0.211

486

0.052

α-LA

α-LA-12C α-LA-16C

α-LA-18C α-LA= native alpha-lactalbumin; 12C to 18C represents the length of fatty acids, 12C = lauric acid; 16C = palmitic acid; 18C = stearic acid. λ max = maximum emission wavelength of intrinsic (IF) or extrinsic (EF) fluorescence. NIF = normalized fluorescence intensity compared to α-LA pH 7 (value 82237 arbitrary units of fluorescence for intrinsic fluorescence; 72854 arbitrary units of fluorescence for extrinsic fluorescence).

30


Page 31 of 37


Table 3. Solubility index, emulsifying activity index, and emulsion stability for α-LA native and modified by chemical lipophilization at different pH values.

Protein

α-LA

α-LA-12C

α-LA-16C

α-LA-18C

pH 3 SI

EAI

(%)

(m2g-1)

96.47 ±

110.60 ±

0.25aA

11.65aA

54.92 ±

59.17 ±

0.17aB

3.17aB

76.17 ±

104.80 ±

2.40aC

9.79aA

76.17 ±

119.69 ±

0.79aC

14.28aA

pH 5 TSI

28.7

13.9

15.0

22.0

SI

EAI

(%)

(m2g-1)

74.78 ±

78.27 ±

0.86bA

2.13bA

3.66 ±

11.17 ±

0.69bB

1.86bB

40.91 ±

50.63 ±

0.96bC

4.24bC

46.49 ±

40.47 ±

1.71bD

4.36bD

pH 7 TSI

59.2

12.2

30.2

28.9

SI

EAI

(%)

(m2g-1)

93.29 ±

101.88 ±

8,28adA

0.39aA

64.66 ±

124.94 ±

0.93cB

11.33cB

90.63 ±

112.01 ±

3.88cC

9.90aAB

77.88 ±

96.66 ±

1.95acD

6.67cAD

pH 10 TSI

32.1

12.2

14.5

20.8

SI

EAI

(%)

(m2g-1)

98.86 ±

71.88 ±

0.54cdA

10.22bcA

66.16 ±

149.94 ±

0.59cdB

36.95cdB

85.36 ±

132.03 ±

2.58cdC

28.47aB

77.90 ±

110.78 ±

0.40cdD

7.40aB

TSI

30.6

8.0

8.5

21.6

*Results for solubility index (SI), Emulsifying Activity Index (EAI) and emulsion stability expressed as Turbiscan Stability Index (TSI) are the mean of three replicates ± SD. Values in the same row followed by different lowercase letters mean significant differences (p ≤ 0.05); values in the same column followed by different capital letters mean significant differences (p ≤ 0.05). 12C, 16C and 18C represents the length of the fatty acid; 12C = lauric acid; 16C = palmitic acid; 18C = stearic acid. 31



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Table 4. Foaming capacity and foam stability for α-LA native and modified by chemical lipophilization at different pH values.

pH 3

pH 5

pH 7

pH 10

Protein FC (%)

FS (%)

FC (%)

FS (%)

FC (%)

FS (%)

FC (%)

FS (%)

α-LA

58.33 ± 2.35

70.77 ± 4.12

62.06 ± 4.87

69.10 ± 0.19

41.66 ± 2.35

58.58 ± 0.35

45.16 ± 1.12

59.09 ± 3.01

α-LA-12C

40.16 ± 2.35

74.18 ± 5.08

21.66 ± 4.71

28.57 ± 0.11

48.27 ± 2.35

72.79 ± 3.12

65.51 ± 1.72

68.23 ± 20.3

α-LA-16C

32.75 ± 2.43

83.97 ± 0.91

25.00 ± 2.35

44.44 ± 4.05

28.33 ± 2.36

90.46 ± 0.64

31.58 ± 0.78

95.83 ± 4.81

α-LA-18C

26.66 ± 1.98

100.00 ± 0.00

31.66 ± 2.35

73.08 ± 5.44

34.48 ± 4.87

95.46 ± 6.43

44.82 ± 1.11

79.41 ± 4.16

α-LA= native alpha-lactalbumin; 12C to 18C represents the length of fatty acids, 12C = lauric acid; 16C = palmitic acid; 18C = stearic acid. FC= foaming capacity, FS= foam stability after 30 min *Results are the mean of three replicates ± SD. Values in the same row followed by different lowercase letters mean significant differences (p ≤ 0.05); values in the same column followed by different capital letters mean significant differences (p ≤ 0.05).

32


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Table 5. Antioxidant activity determined by FRAP and ABTS for α-LA native and modified by chemical lipophilization at different pH values.

pH 3

pH 5

pH 7

pH 10

Protein

FRAP

ABTS

FRAP

ABTS

FRAP

ABTS

FRAP

ABTS

α-LA

32.61 ±

68.51 ±

138.40 ±

77.77 ±

79.96 ±

80.74 ±

104.08 ±

52.96 ±

4.01aA

5.13aA

17.92bA

1.11bA

5.79cA

0.64cA

2.52dA

3.90dA

602.78 ±

1716.67 ±

772.43 ±

480 ±

1020.68 ±

847.78 ±

992.52 ±

431.11 ±

1.89aB

3.14aB

16.52bB

3.14bB

6.69bcB

31.43bcB

11.53bcdB

26.71bcdB

58.06 ±

1504.44 ±

41.33 ±

1014.44±

81.78 ±

704.44 ±

85.57 ±

1249.44 ±

0.14aC

31.43aC

5.06bC

23.57bC

1.09acA

4.71acA

0.54dC

19.64dC

47.41 ±

1346.11 ±

37.75 ±

856.11 ±

58.77 ±

980 ±

62.55 ±

209.44 ±

6.65aDC

14.93aDC

1.68bDC

5.50bDC

12.19abcC

17.28abcC

2.52cD

18.07cD

α-LA-12C

α-LA-16C

α-LA-18C

α-LA= native alpha-lactalbumin; 12C to 18C represents the length of fatty acids, 12C = lauric acid; 16C = palmitic acid; 18C = stearic acid. FRAP (Ferric Reducing Antioxidant Power) and ABTS (Antioxidant activity determined by 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) antioxidant activities were expressed in µmol of Trolox equivalents per liter of sample. Results are the mean of three replicates ± SD. Values in the same row followed by different lowercase letters mean significant differences (p ≤ 0.05); values in the same column followed by different capital letters mean significant differences (p ≤ 0.05).

33



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557

Fig. 1

34


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Fig. 2

35



558

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TABLE OF CONTENTS GRAPHIC

559 560 561 562

563

36


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37


Chemical Lipophilization of Bovine Î±-Lactalbumin with Saturated Fatty

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