Storage of micellar casein powders with and without lactose

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

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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

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Abstract

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

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

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

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

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

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

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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

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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

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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

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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

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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

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

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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

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

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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

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reinforced intermicellar interactions3,

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

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

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% 1-3.

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

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

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protein cross-links formed during storage

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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.,

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and Le at al.,

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

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

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

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

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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

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initiation of protein cross linking.

12

established that HMW complexes could be

17, 18

one could expect

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

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

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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

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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

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immediately after the asparagine residue in the polypeptide chain.

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

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

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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

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

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

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techniques (one-dimensional electrophoresis and Liquid Chromatography Mass Spectrometry

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

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

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

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

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(after powder reconstitution in water).

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2. Materials & Methods

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2.1 Dairy powder manufacture and physicochemical analysis

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Dairy powder manufacture

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

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

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

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

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

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

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

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

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

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

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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,

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

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

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

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

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

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Physicochemical analysis of dairy powders

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

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

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

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

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

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

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

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

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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.

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

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

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

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

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

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

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

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

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

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

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Bretonneux, France) respectively.

23

. The total nitrogen

and was calculated by

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2.2. Storage of MC powders

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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

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

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

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temperature has been evidenced 6.

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2.3. Characterization of changes occurring during powder storage

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One-dimensional electrophoresis and gel scanning

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

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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

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remained intact whereas they were dissociated under R conditions.

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

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

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

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

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

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

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

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

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

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

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

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carried out in triplicate.

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Mass spectrometry

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

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

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Separation/identification/quantification of peptides

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

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

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

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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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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normalized collision energy.

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

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chemical modifications

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

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abundance of peptides present in each sample.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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values were replaced by 0.

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Statistical analysis

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

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

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

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

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

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corrections.

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Color measurement of MC powders and browning index determination

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

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

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

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30

:

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BI =

[( , )] ,

(1)

276

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With

x=

(∗ ,×∗ ) (,×∗ ∗ .×∗ )

(2)

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

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al,.6.

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Determination of solubility

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

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

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

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

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

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σ=

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

× 100

(3)

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

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

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

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study the solubility evolution during ageing.

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

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Statistical analysis

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

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

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

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3. Results

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Functional properties of MC powders

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Browning index

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

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

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

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

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reached a plateau value after 60 days of storage.

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Protein solubility and pH of the protein suspension

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

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

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

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

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

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independent of the lactose content of the MC powder.

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

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

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

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

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Molecular changes

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Protein crosslinks

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

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

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

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

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

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

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

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

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

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complex formed during the technological steps involved in the powder manufacture

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

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

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

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

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

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