Storage of micellar casein powders with and without lactose

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Functional Structure/Activity Relationships

Storage of micellar casein powders with and without lactose: Consequences on color, solubility and chemical modifications Sarah Nasser, Paulo De Sa Peixoto, Anne Moreau, Thomas Croguennec, Fabrice Bray, Christian Rolando, Frederic J. Tessier, Alain Hedoux, and Guillaume Delaplace J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06147 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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Journal of Agricultural and Food Chemistry

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Storage of micellar casein powders with and without lactose: Consequences on color, solubility and chemical modifications

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Sarah Nasser a, b, c, d, Paulo De Sa Peixoto, b, c Anne Moreau b, c, Thomas Croguennec g, Fabrice Bray e, Christian Rolando e, Frédéric J Tessier f, Alain Hédoux b, d, Guillaume Delaplace b, c

26

Abstract

27

During storage, a series of changes occurs for dairy powders such as protein lactosylation,

28

formation of Maillard reaction products (MRP) leading to powder browning and increase of

29

insoluble matter. The kinetics of protein lactosylation and MRP formation are influenced by

30

lactose content of the dairy powder. However, influence of lactose in the formation of

31

insoluble matter and its role in the underlying mechanisms is still a subject of speculation.

32

In this study, we aim to investigate the role of lactose in the formation of insoluble matter in a

33

more comprehensive way than the existing literature. For that, two casein powders with

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radically different lactose content, standard micellar casein (MC) powder (MC1) and a

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lactose-free (less than 10 ppm) MC powder (MC2), were prepared and stored under controlled

36

conditions for different periods of time.

a-Centre National Interprofessionnel de l’Economie Laitière, F-75009 Paris, France b-Univ.Lille, CNRS, INRA, ENSCL, UMR 8207, UMET, Unité Matériaux et Transformations, F59 000, Lille, France c-INRA, UR 638, Processus aux Interfaces et Hygiène des Matériaux, F-59651 Villeneuve d’Ascq, France d-UMET, UMR CNRS 8207, F-59655 Villeneuve d’Ascq, France e-Miniaturisation pour la Synthèse, l’Analyse & la Protéomique (MSAP), USR CNRS 3290, Université de Lille 1 Sciences et Technologies, 59655 Villeneuve d’Ascq Cedex, France f-Univ. Lille, U995 - LIRIC - Lille Inflammation Research International Center, F-59000 Lille, France g-STLO, Agrocampus Ouest, INRA, 35000 Rennes, France

Corresponding author: Guillaume Delaplace [email protected] Inra CERTIA, Bâtiment PIHM, Unité Matériaux et Transformations Institut National de la Recherche Agronomique, 369 rue Jules Guesde, BP 20039, 59651, Villeneuve d'Ascq, France

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Powder browning index measurements and solubility tests on reconstituted powders were

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performed to study the evolution of the functional properties of MC powders during aging.

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Proteomic approaches (one-dimensional electrophoresis and Liquid Chromatography Mass

40

Spectrometry (LCMS) and innovative Label-Free Quantification methods were used to track

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and quantify the chemical modifications occurring during the storage of the powders.

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Reducing the amount of lactose limited the browning of MC powders but had no effect on the

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loss of solubility of proteins after storage suggesting that the action of lactose, leading to the

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production of MRC, does not promotes the formation of insoluble matter. Electrophoresis

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analysis did not reveal any links between the formation of covalent bonds between caseins

46

and loss in solubility, regardless of the lactose content. However, LCMS analyses have shown

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that different levels of chemical modifications occur during the MC powder storage,

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depending on the presence of lactose. An increase of protein lactosylation and acetylation was

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observed for the powder with higher lactose content while an increase of protein deamidation

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and dephosphorylation was observed for that containing lower lactose. The decrease of pH in

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the presence of lactose, due to Maillard reaction (MR), may explain the difference in the

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chemical modifications of the two powders. In view of the present results, it is clear that

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lactose is not a key factor promoting insolubility and for formation of cross-links between

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caseins during storage. This suggests that lactosylation is not the core reaction given rise to

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loss in solubility.

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Key-words : Micellar casein; storage; lactose, functionality, chemical modifications

58 59

1. Introduction

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During storage, milk powder behaves as a complex reaction system. A large number of

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chemical, physical and biochemical reactions can occur, especially under harsh conditions

63

(e.g. long storage time, high temperature) and with increasing powder water activity 1-5. These

64

reactions have negative impacts on the functional properties 6 and nutritional quality 7 of the

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powder. Some changes, such as color change, can be detected can be detected visually and

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browning index can be used as markers of the powder quality

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modifications of the functional properties, occur during storage and are only detectable after

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reconstitution. This is for instance the case for dispersion and solubilisation abilities of the

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milk protein powder 9. A fast and complete dispersion and solubilisation of the milk proteins

70

is however a prerequisite for the effective expression of other functionalities (gelling,

71

emulsifying, etc.).

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Some studies focus on identifying the cause of protein solubility loss by investigating powder

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particle changes occurring at grain scale. Sadek et al.,

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on the particle surface in the early stage of drying. Mimouni et al.,

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thin layer played a role in resisting the immediate erosion of the particle surface and retarding

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the release of individual micelles during milk protein powder dispersion and solubilisation.

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This thin surface layer became more and more compact and uniform during storage. The

78

reinforced intermicellar interactions3,

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dissolution of stored milk powders. These changes were shown to be particularly marked

80

during the storage of micellar casein (MC) powder with a content of micellar caseins up to 90

81

% 1-3.

82

Other studies were conducted to identify the underlying mechanisms at the molecular level for

83

the loss of protein solubility of dairy powders. In particular attempts for i) characterizing the

84

protein cross-links formed during storage

85

destabilize protein structure

8,12-16

11

10

6, 8

. Other changes, such as

showed that a thin layer was formed 3

also observed that this

were assumed to be responsible for the slow

9,12

ii) identifying the chemical modifications that

were carried out. Anema et al.,


9

and Le at al.,

12


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demonstrated the formation of high-molecular-weight (HMW) complexes during the storage

87

of high-protein dairy powders using two-dimensional gel electrophoresis (2-DE). Proteins

88

aggregate through non-covalent and covalent interactions, the latter involve either disulfide

89

bonds (sensitive to reducing agents) or covalent non-disulfide cross-links (resistant to

90

reducing agents). Anema et al., 9 and Le at al.,

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mainly stabilized by non-covalent cross-links and covalent non-disulfide cross-links. Their

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presence is positively correlated to the loss of protein solubility. Many of these studies

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investigating loss of solubility establish a correlation between the Maillard Reaction Products

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(MRP) and aggregation 15,16. Since MPR are formed by a series of chemical reactions between

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lactose and a lysine residue of the protein (protein lactosylation)

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lactosylation to be a key factor promoting aggregation leading-to-insolubility in a sample.

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Indeed, an increase of the protein lactosylation during the storage of milk products has been

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demonstrated by Anema et al., 9 and Le et al.,8,12,15,16. Although these authors suggested that

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MR was at the source of protein cross-linking and an alteration of the solubility of high-

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protein dairy powders stored at elevated temperature (up to 50 °C) and/or at elevated relative

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humidity (44-86 %), it has not been formally demonstrated that the loss of protein solubility

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during storage could not occur in the absence of lactose. This ambiguity was also recently

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pointed out by Gazi and Huppertz 13 who mentioned that since lactose content in high-protein

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dairy product is only between 1 and 3 %, it could be speculative that it was responsible for the

105

initiation of protein cross linking.

12

established that HMW complexes could be

17, 18

one could expect

106 107

Aside from the Maillard reaction route, other chemical modifications occur during milk

108

powder ageing. Although some of them could be responsible for the formation of HMW

109

complexes and/or the loss of protein solubility, they have seldom been tracked in depth during

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storage. Figure S1 shows the most common products of the reactions studied in this work.


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Protein dephosphorylation consists in the β-elimination of phosphoserine, forming

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dehydroalanine (DHA) that can react with nucleophilic amino acids (e.g., lysine, cysteine,

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histidine) in the same or adjacent proteins, producing lysinoalanine (LAL), lanthionine or

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histidinoalanine cross-links19. Protein acetylation was rarely studied during the storage of

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dairy products, although it is responsible for the neutralization of the positive charges and the

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modification of the size of lysine side chain, resulting in changes of conformation and

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interactions between proteins 20. Protein deamidation during powder storage has been reported

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12,14

, but further studies are required to associate this chemical modification to the formation

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of HMW complexes and the loss of protein solubility. The deamidation consists in the

120

cyclisation of an asparagine residue into a succinimide group in a flexible zone of the protein

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structure. This reaction is favoured at pH > 6 and requires preferentially a glycine residue

122

immediately after the asparagine residue in the polypeptide chain.

123 124

To clarify the role of the Maillard reaction route and the other chemical modifications in the

125

formation of HMW complexes and loss of protein solubility during casein powder storage,

126

two casein powders with different lactose contents (standard micellar Casein (MC) powder,

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MC1, and lactose-free MC powder, MC2) were prepared and stored under controlled

128

conditions at 60 °C for different periods. The protein chemical modifications (lactosylation,

129

dephosphorylation, deamidation and acetylation) were tracked with the aid of proteomic

130

techniques (one-dimensional electrophoresis and Liquid Chromatography Mass Spectrometry

131

(LCMS)) coupled to Label-Free Quantification method and the formation of HMW

132

complexes were studied by a combination of reducing (R) / non- reducing (NR) one

133

dimension-electrophoresis. The techno-functional properties of MC powders with or without

134

lactose during aging were characterized by measuring browning index and protein solubility

135

(after powder reconstitution in water).



136 137

2. Materials & Methods

138

2.1 Dairy powder manufacture and physicochemical analysis

139

Dairy powder manufacture

140

A MC concentrate obtained through microfiltration (pore size = 0.1 µm) of skimmed milk was

141

purchased from Ingredia (Arras, France). The MF concentrate was separated into two batches

142

of equivalent volume. One batch was diafiltered with reverse osmotic water in order to reduce

143

the amount of residual lactose. A pilot installation (Carbosep TECH-SEP, Rhône-Poulenc,

144

France) equipped with dual ceramic membranes (Tami Industries, Nyons, France) with a total

145

membrane surface area of 12.6 m2 and a molecular weight cut-off of 8 kDa was used. The

146

flow rate of the retentate was set at 200 L.h-1. The reverse osmotic water was added in

147

continuously and the rate of diafiltration was equal to 3 diavolumes.

148

The retentates have been obtained by membrane microfiltration according to the protocol

149

described by Pierre et al. 21 . The powders have been obtained by spray-dried using a protocol

150

close to the one described by Schuck et al., 22 with some changes as described below. A pilot

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plant spray dryer (GEA Niro A/S, Søeborg, Denmark) type Mobile Minor (UMR STLO,

152

Rennes, France) was used. Inlet and outlet temperatures were set at 180 °C ± 2 °C and 80 °C

153

±2 °C, respectively. The retentates were injected in the spray drier at 30 mL.min-1 using a

154

peristatic pump (Watson Marlow 520 S). The retentate batch spray dried immediately after

155

ultrafiltration gave the standard MC powder (MC1). The lactose-free retentate batch by

156

diafiltration before spray drying gave a lactose-free MC powder (MC2).

157 158

Physicochemical analysis of dairy powders

159

The powders were analysed for their composition, water activity, and powder particle size.

160

The nitrogen contents (total nitrogen, non-casein nitrogen, non-protein nitrogen) of initial


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23

161

state-fresh powder were determined as described by Schuck et al.,

162

content, the non-casein nitrogen content corresponding to the soluble fraction at pH 4.6, and

163

the non-protein nitrogen content corresponding to the soluble fraction after protein

164

precipitation with TCA 12%, were determined by the Kjeldhal method. Nitrogen contents

165

were converted into protein contents using 6.38 as multiplying factor. These analyses are

166

reported in Table 1. The ash content were determined according to standard 27/1964 IDF.

167

The total dry matter was determined as described by Schuck et al.,

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weight loss after drying 1 g of sample mixed with sand in a forced air oven at 105 °C for 5 h.

169

Noteworthy, one can observe that the sample where a diafiltration step has been added has a

170

higher content in total protein, non-casein protein and ash. This indicates that this additional

171

step induces some changes in the ionic composition of the sample. Lactose content was

172

determined using a High Performance Liquid Chromatograph (HPLC) chain (Dionex,

173

Germering, Germany) linked to a 6.5 mm diameter and 300 mm length column “house-

174

packed” with an ion exchange resin Aminex A-6 (Biorad, St Louis Mo., USA). The elution

175

was conducted at 60 °C with 5 mM H2SO4 buffer at a flow rate of 0.4 mL.min-1. Free lactose

176

content was detected using a differential refractometer (model RI 2031 plus, Jasco). The

177

cations and the anions were quantified by atomic absorption spectrometry (Varian 220FS

178

spectrometer, Les Ulis, France) and ion chromatography (Dionex DX 500; Dionex, Voisin-le-

179

Bretonneux, France) respectively.

23

. The total nitrogen

and was calculated by

180 181

2.2. Storage of MC powders

182

After manufacture, MC powders were packaged in individual cans of 50g and stored either at

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a controlled temperature of 4 °C (further named as “reference powder”) or at 60 °C for

184

different periods up to 3 months. Although 60 °C may seem extreme storage temperatures, a



185

relation between long-term storage at ambient temperature and short-time storage and high

186

temperature has been evidenced 6.

187 188

2.3. Characterization of changes occurring during powder storage

189

One-dimensional electrophoresis and gel scanning

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The formation of covalent interactions (disulfide bonds and non-disulfide bonds) between

191

proteins during the storage of MC powders were monitored by Tricine-sodium dodecyl

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sulphate polyacrylamide gel electrophoresis (Tricine-SDS–PAGE) under non-reducing (NR)

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and reducing (R) conditions. Under NR conditions, the disulfide-linked protein complex

194

remained intact whereas they were dissociated under R conditions.

195

Tricine-SDS-PAGE was performed at room temperature in a SE 600 Series vertical Slab Gel

196

Unit (Hoefer Scientific instrument, San Francisco, US). Sample and gel preparation were

197

based on the procedure of Pardo and Natalucci,25. SDS PAGE was performed at 30 V until the

198

samples had completely left the stacking gel and then the voltage was increased to 90 V until

199

the tracking dye reached 4/5 of the gel. Immediately after the protein migration, the gel was

200

fixed, stained and destained, also according to Pardo and Natalucci,25.

201

The image of the gel was captured with a PC scanner and saved in JPEG format. The image

202

was processed using gel analysis module of ImageJ software to obtain densitograms.

203

Integrated intensities of the protein bands were determined using ImageJ. The relative

204

quantities of the proteins were estimated by measuring the intensity of each protein band and

205

expressed as a percentage of the total band intensity. All Tricine-SDS-PAGE analysis was

206

carried out in triplicate.

207 208

Mass spectrometry


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Nano HPLC coupled to Q Exactive plus Mass Spectrometry was used to identify and to get

210

relative quantification of all the four chemical modifications studied in this work.

211 212

Separation/identification/quantification of peptides

213

The quantity of proteins was measured by BCAssay method. 100 µg of proteins of the MC

214

powders were processed in the 10 kDa filtration units (Millipore) using eFASP protocol

215

(enhanced Filter Aided Samples Preparation) 26.Trypsin was used for the digestion of proteins

216

with a ratio of 1/50 (trypsin/protein). Peptides were extracted and the solvent was evaporated.

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Samples were diluted in buffer A of nanoHPLC and analysed in triplicate.

218

The peptides mixtures were analysed using a nanoflow HPLC instrument (U3000 RSLC

219

Thermo Fisher Scientific) coupled on-line to a quadrupole-Orbitrap mass spectrometer (Q

220

ExactiveTM Plus, Thermo Scientific) with a nanoelectrospray ion source. The principle of this

221

analytical method is that the charged peptides will “fly” around an inner attractive electrode in

222

an analogous way of a satellite orbiting around a planet. The peptides cycle around the inner

223

electrode on elliptical trajectories which depend of their mass. 1 µL of peptide mixture

224

(corresponding to 500 ng of proteins) was loaded onto the pre-concentration trap (Thermo

225

Scientific, Acclaim PepMap100 C18, 5 µm, 300 µm i.d × 5 mm) using partial loop injection,

226

for 5 min at a flow rate of 10 µL/min with buffer A (5% acetonitrile and 0.1% formic acid).

227

Peptides were separated on analytical column (Acclaim PepMap100 C18, 3 µm, 75 mm i.d. ×

228

500 mm) with a linear gradient of 5–50% buffer B (75% acetonitrile and 0.1% formic acid) at

229

a 250 nL/min flow rate and a 45 °C controlled temperature. The total time for a LC MS/MS

230

run was about 240 min long. MS data were acquired using a top-20 data-dependent method

231

dynamically choosing the most abundant precursor ions from the survey scan for HCD

232

fragmentation27. Dynamic exclusion duration was 60 s. Isolation of precursors was performed

233

with a 1.6 m/z window and MS/MS scans were acquired with a starting m/z of 80. Survey



234

scans were acquired in the Orbitrap analyzer with m/z range of 350–1600 with 70,000

235

resolutions at m/z 200. Resolution for HCD spectra was set to 35,500 at m/z 200 and 28 eV

236

normalized collision energy.

237

Label Free Quantification (LFQ) intensities treatment: Identification and quantification of

238

chemical modifications

239

LFQ is based on the comparison of the different MS signal intensity to determine the relative

240

abundance of peptides present in each sample.

241

The acquired raw files were analyzed with MaxQuant software (version 1.5.3.30) using the

242

Andromeda search engine28. Proteins were identified by searching MS and MS/MS data of

243

peptides in the decoy UniProt-Bovine database (Version June 2016, 59,345 entries)

244

supplemented with 262 frequently observed contaminants and forward/reverse sequences. The

245

precursor mass and fragment mass were identified with an initial mass tolerance of 10 ppm

246

and 20 ppm, respectively. The search included variable modifications, i.e. the oxidation of

247

methionine and proline, deamidation of asparagine and glutamine, lactosylation of arginine

248

and lysine, level of phosphorylation of serine residue. Minimal peptide length was set to six

249

amino acids and a maximum of three mis-cleavages was allowed. The false discovery rate

250

(FDR) was set to 0.05 for peptide and protein identifications. MS runs from casein powder

251

were analyzed with the “match between runs” option and a 20 min retention time window. In

252

the case of identified peptides that are all shared between two proteins, these were combined

253

and reported as one protein group. Proteins matching to the reverse database were filtered out.

254

For the proteins that were identified with single peptide, detailed information about the

255

MS/MS spectrum, peptide sequence, precursor m/z is provided (Table 2). LFQ intensities for

256

respective protein groups were uploaded in Perseus and analyzed29. Briefly, contaminant

257

proteins, resolved by post traductional modification-sites only and identified by reverse decoy

258

were removed from the dataset. Raw LFQ intensities were logarithmized by Log2. At least


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259

three LFQ values per protein group needed to be present for the analysis. Non-quantified

260

values were replaced by 0.

261

Statistical analysis

262

The relative frequency of modifications is calculated using the label-free statistical probability

263

identification method of the software Perseus (Max Planck Institute, Martinsried, Germany).

264

The software output is the relative number of chemical modification found in all peptides

265

analysed for a given sample. Significant evolutions were determined using ANalyse Of

266

VAriance (ANOVA) test with Permutation-based FDR < 0.05 method to perform testing

267

corrections.

268 269

Color measurement of MC powders and browning index determination

270

The principle is that there is a correlation between the amount of chemical reactions,

271

subsequent to lactosylation, and the brown colour of the sample. Browning of powders was

272

monitored with browning index (BI), calculated from a formula combining the L*a*b* values

273

30

:

274

275

BI =

[( , )] ,

(1)

276

277

With

x=

(∗ ,×∗ ) (,×∗ ∗ .×∗ )

(2)

278 279

More details for browning index determination of stored powder can be found in Nasser et

280

al,.6.

281 282

Determination of solubility



283

To measure solubility, solutions of 5% (w/w) MC powder have been prepared in distilled

284

water for 1 h under stirring under room temperature. Then, 50 ml of the MC solution has been

285

transferred into 50 ml centrifugation tubes and centrifuged (Sigma, Labozentrifugen GmbH,

286

Osterode am Harz, Germany) at 700 × g at 20◦C for 20 min. The amount of soluble material

287

(σ) in the MC powders was calculated using the following equation 9 :

288

289

σ=

!" #$ %&' ()*&+"+" , !" #$ -#.)"#+

× 100

(3)

290 291

It represents the solids in the ultracentrifugal supernatant, expressed as a percentage of total

292

soluble solids in the whole solution. The σ values of aged powder samples were compared to

293

that of the reference powder, and this σ(aged)/σ(reference) ratio in percentage was used to

294

study the solubility evolution during ageing.

295 296

More details for solubility determination of stored powder can be found in Nasser et al., 6.

297 298

Statistical analysis

299

Student's t-tests with a 0.05 level of significance were used to measure the significance of the

300

differences between aged samples and reference powder. As is widely known, the differences

301

are statistically non significant when p > 0.05 and statistically significant when p < 0.05

302 303

3. Results

304

Functional properties of MC powders


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305


Browning index

306

BI evolution of the MC powders stored at 60 °C as a function of time is illustrated in Figure

307

1. The colour of MC2 (lactose-free powder) did not evolve, even after 3 months of storage. In

308

contrast, the browning index of MC1 increased as soon as the first hours of storage and it still

309

increased rapidly during the first 15 days of storage. Then the BI increase levelled off and

310

reached a plateau value after 60 days of storage.

311 312

Protein solubility and pH of the protein suspension

313

The protein solubility of MC powders stored at 60 °C is plotted as a function of storage time

314

in Figure 2. The fresh powders exhibited an excellent solubility (100%). The protein

315

solubility exhibited a sharp decrease in the first 8 hours of storage, then, the decrease slowed

316

down until reaching a plateau at 25% after 15 days of storage. The decrease of protein

317

solubility with storage time was nearly identical for the two powders, and consequently was

318

independent of the lactose content of the MC powder.

319

The pH of the MC powder reconstituted in water solution were determined after various

320

periods of storage. The pH of MC2 suspensions was constant all along the storage of the

321

powder (pH 6.97). In contrast, the pH of MC1 suspensions decreased during the storage

322

period. It reached a value of 6.53 after 60 days’ storage at 60 °C.

323 324

Molecular changes

325 326

Protein crosslinks

327

Figure S2 and S3 shows the electrophoresis separation under NR and R conditions of the

328

major proteins (αS1-casein (αS1-csn); αS2-casein (αS2-csn); β-casein (β-csn); and κ-casein (κ-

329

csn); β-lactoglobulin (β-lg); α-lactalbumin (α-lb)) in MC1 and MC2 powders stored at 60 °C.



330

On the SDS-PAGE under reducing conditions, the evolution of band intensities of HMW

331

complexes and individual caseins for MC1 and MC2 powders during 2, 5 and 15 days storage

332

at 60 °C are compared to reference (Figure 3). These storage times were selected as the

333

solubility of MC1 and MC2 powders were largely depreciated after only 5 days of storage.

334

Prior to storage, the MC powders already contained a significant amount of HMW complexes

335

that must correspond to natural high molecular weight proteins present in milk and, possibly,

336

complex formed during the technological steps involved in the powder manufacture

337

Storage conditions induced only a very slight evolution in the amount of HMW complexes

338

regardless of the presence of lactose. The amount of HMW complexes increased from 28.32%

339

to 36.11% after 5 days of storage .Similar evolution was noted for MC2, with an increase of

340

HMW complexes from 24.9% to 31.71%. Impact of lactose in formation of HMW complexes

341

during storage seems minimal. Finally, the amounts of individual caseins and whey proteins

342

were not significatively different in the powders stored up to 5 days (p

Storage of micellar casein powders with and without lactose

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