Amélie Deglaire

Samira Cassia De Oliveira

Julien Jardin

Valérie Briard-Bion

Florian Kroell

Mathieu Emily

Olivia Ménard

Claire Bourlieu-Lacanal

Didier Dupont

Samira De Oliveira

Claire Bourlieu

Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic digestion at the preterm newborn stage

Keywords: GRAVY, Grand Average of Hydropathy Value, MS, Mass spectrometry, PHM, pasteurized human milk, RHM, raw human milk digestion, human milk, pasteurization, peptidomics, preterm

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Human milk is considered as the ideal food for neonates, supporting not only their nutrition but also their optimal growth and development [START_REF] Victora | Breastfeeding in the 21st century: Epidemiology, mechanisms, and lifelong effect[END_REF]. When vulnerable hospitalized neonates such as preterm infants receive donor human milk, it has to be first pasteurized (62.5 °C, 30 min) in the milk bank for sanitary reasons [START_REF] Wight | Donor human milk for preterm infants[END_REF]. Together with the associated freeze/thaw cycles, pasteurization induces some protein denaturation, which reduces the content of some native proteins and thus affect their bioactivity, such as for immunoglobulins (loss of 20 to 100%), lysozyme (loss of 20 to 69%), lactoferrin (loss of 35 to 90%), and bile salt-stimulated lipase (complete loss) [START_REF] O'connor | Human milk pasteurization: Benefits and risks[END_REF]Peila, Moro, Bertino, Cavallarin, Giribaldi, Giuliani, et al., 2016). These physical treatments also impact the human milk microstructure, as previously observed (De Oliveira, Bourlieu, Ménard, Bellanger, Henry, Rousseau, et al., 2016a;[START_REF] De Oliveira | Holder pasteurization impacts the proteolysis, lipolysis and disintegration of human milk under in vitro dynamic term newborn digestion[END_REF]. In the latter studies, we have demonstrated that human milk pasteurization affected its proteolysis kinetics, at different extent, during in vitro gastro-intestinal digestion at the preterm and term stages (De Oliveira, et al., 2016a;[START_REF] De Oliveira | Holder pasteurization impacts the proteolysis, lipolysis and disintegration of human milk under in vitro dynamic term newborn digestion[END_REF], and during in vivo gastric digestion in preterm neonates [START_REF] De Oliveira | Impact of human milk pasteurization on gastric digestion in preterm infants: A randomized controlled trial[END_REF]. More precisely, at the preterm stage, pasteurization did not significantly impact the kinetics of disappearance of the major proteins in the gastric phase, but during the intestinal phase, amino acid bioaccessibility was selectively modulated (De Oliveira, et al., 2016a). Knowledge on the kinetics of peptide release during digestion is crucial for a better understanding of the potential peptide bioactivity released in the gastrointestinal tract and on the amino acid absorption rate, the latter of which has been demonstrated to be an independent factor of modulation of the postprandial regional metabolism, at least in adult [START_REF] Deglaire | Hydrolyzed dietary casein as compared with the intact protein reduces postprandial peripheral, but not whole-body, uptake of nitrogen in humans[END_REF]. To date, only our study has investigated and showed that there was an impact of milk pasteurization on the kinetics of peptide release during in vitro dynamic digestion of term milk at the term newborn stage [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. We assume that such an impact should also be found for preterm milk digested in the preterm newborn condition but certainly at a different extent given the digestive immaturity of the preterm newborn combined to a higher protein content in the preterm human milk. The objective of the present paper was to verify this hypothesis. The kinetics of peptide release was followed during gastro-intestinal digestion of a pool of raw or pasteurized preterm human milk at the preterm newborn stage. Digestion was simulated using an in vitro dynamic model, where digestive enzymes, pH and digestive compartment emptying were continuously controlled.

Nature and abundance of peptides were determined by mass spectrometry and data analyzed by multivariate statistics.

Material and methods

All chemicals and enzymes were from commercial origin (Sigma-Aldrich, Saint Quentin Fallavier, France), except for the Rabbit Gastric Extract, obtained from GERME SA (Marseille, France).

Human milk collection and digestion

Collection of Human milk has been previously detailed by De Oliveira et al. (2016a). Briefly, mature human milk samples were obtained frozen from five women (26-34 years old) on average 6.6 ± 2.4 weeks after preterm delivery via a donor milk bank (University Hospital Center, Rennes, France). The gestation length was in average 29.8 ± 3.0 weeks of amenorrhea, ranging from 26 up to 34 weeks of gestation. After milk thawing (at 4°C, over 16 h) and pooling, half of the pool was pasteurized (62.5 °C, 30 min). Raw (RHM) and pasteurized (PHM) human milk were then stored at -20°C. Milk was analyzed by a Human Milk Analyzer (Miris, AB, Uppsala, Sweden) and consisted of 35.7 ± 0.7 g/L of fat, 13.2 ± 0.8 g/L of proteins and 78.3 ± 2.3 g/L of carbohydrates (De Oliveira, et al., 2016a).

Gastrointestinal digestion was performed on 100 mL of whole milk, as RHM or PHM, in a bicompartmental in vitro dynamic system (DIDGI®, INRA, France) simulating the preterm newborn digestive conditions, such as reviewed in [START_REF] Bourlieu | Specificity of infant digestive conditions: Some clues for developing relevant in vitro models[END_REF]. The rationale of the parameters used in the present model has been detailed in De Oliveira et al. (2016a). Briefly, fluid secretions, pH and emptying were continuously monitored. The transit time of the chyme followed the Elashoff equation with t 1/2 = 36 min and β = 1.15 for the gastric emptying and t 1/2 = 200 and β = 2.2 for the intestinal emptying (De Oliveira, et al., 2016a). The gastric pH followed the equation: pH = -0.0155 x time (min) + milk pH. The intestinal pH was maintained at 6.2. Gastric enzymes were added as rabbit gastric extract (pepsin and lipase: 120 and 8.6 U/mL of gastric content, respectively) and intestinal enzymes as porcine pancreatin (trypsin, chymotrypsin and lipase: 16, 2 and 59 U/mL of intestinal content) in addition to bovine bile salts. Enzyme units were determined based as a function of the mean body weight, estimated at 1.9 kg for a preterm newborn at a gestational age of 28 weeks and a postnatal age of four weeks (De Oliveira, et al., 2016a).

Digestions were conducted in triplicate for each matrix over 3 hours with digesta sampling every 30 or 60 min. Samples were stored at -20°C after addition of the antiprotease phenylmethylsulfonyl fluoride (PMSF) to avoid further proteolysis.

Ethical approval was granted by the Hospital Ethics Committee (n° 13-12) and donors gave their informed written consent.

Mass spectrometry

Peptide identification

Mass spectrometry (MS) analysis was conducted as described previously [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. Briefly, a nano-RSLC Dionex U3000 system fitted to a Q-Exactive mass spectrometer (Thermo Scientific, San Jose, USA) equipped with a nanoelectrospray ion source was used. Digesta samples were diluted 100 times in the injection buffer and filtered (0.45 µm cut-off) before concentration on a µ-precolumn pepMap100 (C18 column, 300 µm i.d. × 5 mm length, 5 µm particle size, 100 Å pore size; Dionex, Amsterdam, The Netherlands) and separation on a PepMap RSLC column (C18 column, 75 µm i.d. x 150 mm length, 3µm particle size, 100 Å pore size; Dionex).

Peptide separation was performed at a flow rate of 0.3 µL.min -1 using solvents A [2% (v/v) acetonitrile, 0.08% (v/v) formic acid and 0.01% (v/v) TFA in HPLC gradient grade water] and B [95% (v/v) acetonitrile, 0.08% (v/v) formic acid and 0.01% (v/v) TFA in HPLC gradient grade water]. The elution gradient first rose from 5 to 35% solvent B over 87 min, then up to 85% solvent B over 3 min before column reequilibration. The mass spectra were recorded in positive mode using the m/z range 250-2000. The resolution of the mass analyzer for m/z of 200 amu (atomic mass unit) was set in the acquisition method to 70 000 for MS and 17 500 for MS/MS. For each MS scan, the ten most intense ions were selected for MS/MS fragmentation and excluded from fragmentation for 15 s.

Peptides were identified from the MS/MS spectra using the X!TandemPipeline software [START_REF] Langella | X!Tandempipeline: A tool to manage sequence redundancy for protein inference and phosphosite identification[END_REF] against a human milk protein database [START_REF] Molinari | Proteome mapping of human skim milk proteins in term and preterm milk[END_REF] to which was added the common Repository of Adventitious Protein (http://thegpm.org/crap). The possible post-translational modifications were serine or threonine phosphorylation, methionine oxidation, lysine or arginine lactosylation, cyclisation of glutamine or glutamic acid into pyroglutamic acid and acetylation of cysteine, serine, lysine or any amino acid in Nter position. Peptides identified with an e-value < 0.01 were automatically validated. The peptide false discovery rate was less than 0.3%.

Quantification of peptides

Each identified peptide was quantified by label-free MS using the MassChroQ software [START_REF] Valot | Masschroq: A versatile tool for mass spectrometry quantification[END_REF]. A m/z width of 10 ppm was used to perform extracted ion chromatograms (XIC) of peptides in time-aligned chromatograms and the area under the curve was then quantified. When a peptide was measured with several charge states, all ion intensities were summed.

Biochemical characteristics of the peptides

Several biochemical characteristics were determined for each peptide, particularly the amino acids at cleavage sites in positions P1 (C-ter end) and P1' (N-ter end), the number of essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine), the isoelectric point, the molecular weight and the Grand Average of Hydropathy Value (GRAVY) as defined by the ExPASy "ProtParam tool" [START_REF] Gasteiger | Protein identification and analysis tools on the expasy server[END_REF]. A positive GRAVY corresponds to a hydrophobic peptide and a negative one to a hydrophilic peptide [START_REF] Kyte | A simple method for displaying the hydropathic character of a protein[END_REF].

Bioactive peptides were searched within the databases of BIOPEP [START_REF] Minkiewicz | Biopep database and other programs for processing bioactive peptide sequences[END_REF] and MBPDB [START_REF] Nielsen | Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization[END_REF] both accessed on January 2018. Only exact matching between sequences was considered.

Prediction of antimicrobial activities was examined using CAMPR3 based on the algorithms of Random Forest (RF) and Support Vector Machines (SVM), previously shown to have a good prediction performance [START_REF] Gabere | Empirical comparison of web-based antimicrobial peptide prediction tools[END_REF]. A peptide sequence was determined to have a greater likelihood of antimicrobial functionality with scores approaching 1. All peptides with scores > 0.5 on both prediction methods were considered significant for antimicrobial functionality.

NH 2 quantification

As described earlier [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF], the method was a fluorescent microplate analysis based on the reaction of ortho-phthaldialdehyde (OPA) and dithiothreitol (DTT) with primary amines resulting in 1-alkylthio-2-alkylisondole detected at 340 nm.

Enzymes predicted to be active in milk before gastrointestinal digestion

The peptide sequences as identified in milk before digestion were submitted to the online software Enzyme Predictor (http://bioware.ucd.ie), which examined the cleavage patterns and predicted the enzymes responsible for this profile based on the cleavage specificities of 35 enzymes [START_REF] Vijayakumar | Enzymepredictor: A tool for predicting and visualizing enzymatic cleavages of digested proteins[END_REF]. Enzymes were then ranked according to their likelihood of having being used to generate the hydrolysate thanks to the calculation of an odds ratio (OR). The same human enzymes as those previously considered [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF] were included. Enzymes presenting only one potential cleavage were not considered. Cleavage sites of cytosol aminopeptidase and carboxypeptidase B2 were examined manually over β-casein, such as performed previously [START_REF] Dallas | Endogenous human milk peptide release is greater after preterm birth than term birth[END_REF].

Statistical analyses

Data analyses were performed using the R software, version 3.3.1 (R Core Team, 2014). All graphical representations were based on the log10-transformed abundances [log10(abundance+1)]. An in-house program allowed the peptide mapping and their average cumulative abundances onto the parent protein. For each digestion and each phase (milk prior to digestion, gastric phase or intestinal phase), abundances of the peptides were averaged over the different digestion times of the corresponding phase, summed amino acid by amino acid and log10-transformed [log10(abundance+1)]. Missing abundances were set at 0. Log10transformed abundances were averaged by type of milk in order to be plotted along the amino acid sequence of the protein; they were compared between RHM and PHM for each amino acid using a t-test.

For each peptide identified at least on two digestions of RHM or PHM (n = 1134), a two-way analysis of variance was fitted (lm function; pasteurization, time and their interaction as factors) on the transformed abundances of the 9 time points (before digestion, 3 time points in the gastric phase and 5 in the intestinal phase) over the three digestions of RHM and PHM, such as performed previously [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. Abundance transformation consisted in abundance scaling after log-transformation [log(abundance + 1)] followed by setting the maximum abundance to 1. From the two-way analysis of variance, the "Pasteurization x Digestion time" interaction coefficients, for which the sum equalized zero over time within each milk and at each time for RHM and PHM, were used for subsequent analyses. In particular, the eight independent interaction coefficients of PHM were used for the ascending hierarchical clustering (hclus function, stats package) of the 1134 peptides based on the minimum within-cluster variance Ward's agglomeration. The number of clusters was determined thanks to the bar heights at one of the most marked jump. The function heatmap.2 was used to display the heatmap and its dendrogram. Cluster characterization was undertaken (catdes function, FactoMiner package) using the biochemical characteristics. A Chi-square test was undertaken for qualitative variables (protein origin and cleavage sites), and a one-way analysis of variance for quantitative variables (isoelectric point, molecular weight, number of essential amino acids, GRAVY). V-test indicated whether the modality category was significantly overrepresented (v > 2) or underrepresented (v < -2).

A two-way analysis of variance (with pasteurization, time and their interaction as factors) was fitted to the abundances of each bioactive peptide. When the interaction was statistically significant, a post-hoc test was performed on the model residuals (testinteractions function, phia package) using the p-value adjustment method of [START_REF] Benjamini | Controlling the false discovery rate: A practical and powerful approach to multiple testing[END_REF].

Statistical significance level was considered for p-value <0.05.

Results

Identified proteins and peptides

Twenty-seven human milk proteins (Supplementary Material 1) were at the origin of 1531 peptides (as detailed in Supplementary Material 2) identified in RHM and PHM prior to and during gastro-intestinal digestion. Identified peptides contained 6 to 48 amino acids. Some peptides (n = 162) presented modifications that were serine phosphorylation (n = 84), methionine oxidation (n = 43), lactosylation of lysine (n = 8) or arginine (n = 2), glutamine/glutamic acid cyclisation (n = 4), or acetylation (n = 21). A large proportion of the modified peptides (73%) had a sequence that was also identified either without modification or with a different modification.

Proteolysis of human milk prior to gastrointestinal digestion

Before digestion, 204 peptides were identified and derived at 82.3% from β-casein; the remaining peptides derived from α s1 -casein (7.4%), osteopontin (4.9%) and polymeric immunoglobulin receptor (4.4%). PHM and RHM presented 43 and 33 specific peptides, respectively (at 84 and 70% from β-casein) and shared 128 common peptides. The average abundances of all the peptides identified prior to digestion were plotted onto their parent protein sequences, as shown in figure 1A. These peptides covered 95% of the β-casein sequence in both RHM and PHM, with 28% of this hydrolysed sequence having a nontransformed abundance significantly higher for PHM than for RHM (4 times; P < 0.05). The greatest difference was observed between the residues 136-138 residues with a PHM/RHM ratio of 12.

For α s1 -casein (figure 1A), peptides were identified over 53% and 71% of the sequence for PHM and RHM, respectively. Non-transformed abundances were significantly lower for PHM than for RHM only between residues 84 and 109 (2 times; P < 0.05). For osteopontin and polymeric immunoglobulin receptor, peptides were identified on a limited portion of the sequence (figure 1A) and abundances were not significantly different between RHM and PHM.

Based on their high odds ratio and total number of cleavages, plasmin (which shares the same cleavage sites as trypsin) was predicted to be the most active enzyme in human milk proteolysis prior to gastrointestinal digestion (Table 1), with a similar number of cleavages between RHM and PHM (87 and 85, respectively). Cathepsin D was also assumed to be responsible of a high number of cleavages (61 and 71 for RHM and PHM), but due to its large specificity, the odds ratio was below 1. Cytosol aminopeptidase was assumed to have mainly cleaved sites located on the C-ter side of β-casein for both RHM and PHM (Supplemental material 3), while carboxypeptidase B2 mainly cleaved at the residue 96.

NH 2 release after digestion of human milk

The concentration of NH 2 released after gastric and intestinal digestion was not significantly different (P > 0.05) between RHM and PHM, with average values of 47.5 ± 15.3 and 611.2 ± 14.7 mg of NH 2 /L of milk at 90 min of gastric digestion and 180 min of intestinal digestion, respectively.

Peptide release during gastro-intestinal digestion of RHM and PHM

Peptidomic profiles of proteins

The first eight proteins being at the origin of the identified peptides were included in Figure 1, showing the average cumulative peptide abundances onto the protein sequence during digestion. The proportion of the sequence covered by peptides was at 100% during all the digestion only for β-casein (figure 1A), while it decreased from the gastric to the intestinal phase for polymeric immunoglobulin receptor and bile-salt stimulated lipase (-12 and -21%, respectively) and increased for the other proteins (+13% for α s1 -casein up to +117% for αlactalbumin). During the gastric phase, only abundances from bile-salt stimulated lipase significantly differed between RHM and PHM with greater abundances for RHM on 16% of the hydrolysed sequence (Figure 1B). During the intestinal phase, this relationship was opposite for bile-salt stimulated lipase with abundances being significantly higher for PHM (6 times higher for non-transformed abundances) on 59% of the hydrolysed sequence. A similar relationship was observed for lactoferrin during the intestinal digestion (10 times significantly higher for non-transformed abundances of PHM; 32% of the hydrolysed sequence), while for α s1 -casein cumulative abundance was higher for RHM (6 times) but on a limited proportion of the hydrolysed sequence (15%).

Kinetics of peptide release during gastrointestinal digestion

Among the 1531 peptides, 26 % of them were not included in the clustering analyses, because they were detected exclusively in RHM (n = 172) or in PHM (n= 73), or sporadically in both RHM and PHM (i.e. only detected on one out of the three digestions, n = 152). This did not greatly affect the parent protein distribution (Figure 2A).

Eight clusters resumed the kinetics of release of the 1134 peptides (Figure 2B) and the qualitative variable being the most significantly associated to the clustering was the parent protein (p-value = 6.1 E-21). One cluster (n° 4; 31% of the peptides) included peptides exerting similar kinetics along the entire gastro-intestinal digestion. These peptides were originating mostly from β-casein and to a lesser extent from κ-casein, while there was a very limited number of peptides from bile-salt stimulated lipase and lactoferrin (Table 2).

Clusters 6 and 8 (36% of the peptides) included peptides mainly produced during the gastric phase and showed a lower abundance for PHM than for RHM (Figure 2C). In cluster 6, leucine (cleavage specificity for pepsin) in position P1 was significantly more represented unlike lysine and arginine (cleavage specificity for plasmin/trypsin) significantly less represented than in the overall dataset; peptides had in average a higher molecular weight than in overall (Table 2). Cluster 8 was significantly associated to bile salt-stimulated lipase.

Clusters 1 to 3 (20% of the peptides) included peptides mostly produced in the intestinal phase, where the abundance was lower for PHM than for RHM at some digestion time (Figure 2C). In cluster 1, the proportion of α s1 -casein peptides and of arginine in position P1 was greater than in overall (Table 2). In cluster 3, there was significantly less peptides from βcasein than in overall and no peptides from bile salt-stimulated lipase (Table 2). Cluster 5 (5% of the peptides) showed peptides produced only during the intestinal phase at a constant level for RHM while decreasing for PHM (Figure 2C). These peptides were more originating from lactoferrin (46% of the cluster peptides) and less from β-casein and α s1 -casein (24% of the cluster peptides); they contained more lysine and arginine in position P1 than in overall (Table 2). Cluster 7 (9% of the peptides) was the only cluster where PHM abundance was higher during intestinal digestion (at 90 and 120 min). It included more peptides from bile salt-stimulated lipase but less from β-casein and α-lactalbumin; their molecular weight was lower than in overall (Table 2).

Bioactive peptides

The exact matching sequence search over the 1531 peptides resulted in 36 peptides having a predicted antimicrobial activity; they were originating mostly from bile-salt stimulated lipase (39% of them) and lactoferrin (19%) (data not shown). Eight peptides were found to have a proved bioactivity according to the literature, such as indexed by BIOPEP and MBPDB databases. They were all from β-casein, except for one peptide from lactoferrin (Table 3). The antimicrobial peptide β-casein(185-211) had an abundance six times lower (P < 0.05) for PHM prior to digestion but its abundance was then similar (P > 0.05) between RHM and PHM during the first hour of gastric digestion before being null for both types of milk. Two angiotensin-converting enzyme inhibitor peptides [β-casein(154-160) and β-casein(161-166)]

were present only in the intestinal phase and had abundances significantly higher (P < 0.05) for PHM than for RHM. Non-transformed abundances were two times higher for β-casein(154-160) in PHM during the intestinal digestion (except at 60 min) and four times higher for β-casein(161-166) in PHM only at 180 min of digestion. An additional bioactive peptide, not reported within the bioactive peptide databases, was the β-casein(1-18), a caseinophosphopeptide (CPP) homologue [START_REF] Wada | Bioactive peptides released from in vitro digestion of human milk with or without pasteurization[END_REF], which was present in the human milk prior to digestion in a lower proportion in PHM than in RHM (3 times lower).

This difference was maintained along the gastric digestion, while the peptide was hydrolysed rapidly in the intestinal phase.

Discussion

The present paper demonstrates that pasteurization impacted selectively the peptide release from preterm human milk prior to digestion and during gastro-intestinal digestion at the preterm newborn stage, mainly depending on the protein origin. This is the first time that a complete view of the peptides identified and released during digestion is provided, from semiquantitative peptidomic maps up to peptide clustering based on their kinetics of release.

Human milk pre-proteolysis

Before digestion, endogenous peptides were detected in human milk and originated essentially from β-casein, and to a lesser extent from α s1 -casein, osteopontin and polymeric immunoglobulin receptor, such as observed previously [START_REF] Dallas | Extensive in vivo human milk peptidomics reveals specific proteolysis yielding protective antimicrobial peptides[END_REF][START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF][START_REF] Dingess | Human milk peptides differentiate between the preterm and term infant and across varying lactational stages[END_REF][START_REF] Ferranti | Casein proteolysis in human milk: Tracing the pattern of casein breakdown and the formation of potential bioactive peptides[END_REF][START_REF] Khaldi | Predicting the important enzymes in human breast milk digestion[END_REF]Nielsen, Beverly, & Dallas, 2017 ). This confirms that indigenous milk proteases (e.g. plasmin, carboxypeptidase, cytosol aminopeptidase, kallikrein) can hydrolyse human milk proteins, particularly β-casein, within the mammary gland (Demers-Mathieu, Nielsen, Underwood, Borghese, & Dallas, 2017;Nielsen, Beverly, & Dallas, 2017) but also during milk processing (freeze-thaw cycle, pasteurization), as qualitative and quantitative differences occurred on the same pool of milk differing only by their processing (raw or pasteurized).

In the present study, plasmin was predicted, to be the most active protease in human milk, such as reported previously [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]Nielsen, Beverly, & Dallas, 2017). The number of cleavages from plasmin between PHM and RHM was not different, unlike in our previous study [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF], however PHM abundance was greater at 6 out of the 11 Lys residues (plasmin cleavage specificity) of the β-casein sequence. As discussed previously [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF], pasteurization may have activated the milk plasmin system through inactivation of plasminogen activator inhibitors and plasmin inhibitors [START_REF] Ismail | Invited review: Plasmin protease in milk: Current knowledge and relevance to dairy industry[END_REF]. Besides, denaturation of lactoferrin by pasteurization, as confirmed by reverse HPLC (signal decreased by 3 after pasteurization; unpublished data), may have contributed to this activation, as it acts as a natural inhibitor of plasminogen activation [START_REF] Zwirzitz | Lactoferrin is a natural inhibitor of plasminogen activation[END_REF]. It should be noted that the arginine and lysine residues are also cleavage specificities of kallikrein and thrombin, which have been shown to be active in preterm human milk (Demers-Mathieu, Nielsen, Underwood, Borghese, & Dallas, 2017). In addition, the arginine and lysine residues are the cleavage sites for trypsin that has been shown to be potentially present in human milk but not active (Demers-Mathieu, Nielsen, Underwood, Borghese, & Dallas, 2017).

A greater abundance for PHM was observed between the β-casein residues 130-145, although this was statistically significant only between residues 136-138. This area includes the hydrophobic amino acids that are leucine, valine, isoleucine, which are cleavage sites for both cathepsin D and elastase [START_REF] Vijayakumar | Enzymepredictor: A tool for predicting and visualizing enzymatic cleavages of digested proteins[END_REF]. However, cathepsin D, which is an acid protease, is mostly present in human milk as procathepsin D, i.e. the inactive zymogen [START_REF] Vijayakumar | Enzymepredictor: A tool for predicting and visualizing enzymatic cleavages of digested proteins[END_REF]. On the contrary, elastase has been shown to be active in human milk recently (Demers-Mathieu, Nielsen, Underwood, Borghese, & Dallas, 2017) and may have been more active in PHM in this area due to the heat-induced structural changes of β-casein (Guyomarc'h, Law, & Dalgleish, 2003) unmasking resistant area and possibly compensating for the loss of activity likely induced by heat treatment. For the same reason, cystosol aminopeptidase may also have been more active in PHM particularly at the residues 136 and 137 (Supplemental Material 3), where the abundance was 12 times greater for PHM. Because cystosol aminopeptidase is a minor protease in milk, particularly in bovine milk, few information are available on the impact of heat treatment on its activity (O'Mahony, Fox, & Kelly, 2016). Some pepsin and chymotrypsin activities have been predicted in the present paper, likely due to their overlapping specificity with elastase (Vijayakumar, Guerrero, Davey, Lebrilla, Shields, & Khaldi, 2012) as they have not been shown to be present in human milk (Demers-Mathieu, Nielsen, Underwood, Borghese, & Dallas, 2018).

Overall, as reported earlier (Demers-Mathieu, Nielsen, Underwood, Borghese, & Dallas, 2017), the present work suggests that indigenous enzymes, such as plasmin, elastase and probably cytosol aminopeptidase, were more active in pasteurized milk. As a result, some peptides were more actively produced (i.e. more abundant) in PHM or, on the contrary, more extensively degraded, such as observed for the antimicrobial peptide, β-casein(185-211). The latter was less abundant in PHM prior to digestion, maybe due to a greater activity of cytosol amino peptidase in this area (Supplemental Material 3). Nevertheless, this does not indicate whether the proteolysis degree prior to digestion was different between RHM and PHM.

Pre-proteolysis of β-casein may have contributed to its further digestion, in particular on the first 20 amino acids where the cumulative abundance remained similar before and after 30 min of gastric digestion. Indigenous enzymes, in particular the acid enzyme cathepsin D, could have contributed to gastric digestion although a minimal contribution has been previously assumed [START_REF] Demers-Mathieu | Changes in proteases, antiproteases, and bioactive proteins from mother's breast milk to the premature infant stomach[END_REF].

Human milk digestion

The distribution of the protein origin of the common peptides was similar to that observed in our previous study at the term stage [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF], with the major protein of human milk, β-casein, generating 42% of the peptides, whilst α-lactalbumin, the other major protein in human milk, generating only 3% of the peptides. This can reflect its proteolysis resistance as observed by SDS-PAGE (De Oliveira, et al., 2016a) but also probably the nonidentification of the peptides engaged in a disulphide bond (four out of 123 amino acids of the protein sequence) with our non-reducing analysis. Such as observed previously (Deglaire, et al., 2016), despite being minor proteins in terms of concentrations, bile-salt stimulated lipase and osteopontin generated almost the same number of peptides as lactoferrin and α s1 -casein. Nevertheless, this is not indicative of the proteolysis degree of these proteins.

In the present study, the proportion of peptides predominantly released in the gastric phase (36% of the peptides in 2 clusters) was lower than in the intestinal phase, while the opposite was true in the term study [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. This is certainly resulting from the decreased level of pepsin (2 times lower) combined to a greater protein content (1.2 times higher) and a faster gastric emptying (t 1/2 of 36 min) for the preterm model compared to the term one (t 1/2 of 47 min) [START_REF] Bourlieu | Specificity of infant digestive conditions: Some clues for developing relevant in vitro models[END_REF].

In the gastric phase, peptide abundances were generally lower for PHM than for RHM, particularly for peptides from bile salt-stimulated lipase, such as observed on both peptidomic mapping and clustering. On the contrary, intestinal peptide abundances from bile-salt stimulated lipase became in average higher for PHM than for RHM, as observed on the protein sequence and in clusters 7 and 8 (gathering 50% of its peptides). These gastrointestinal kinetics of peptide release are in line with that previously at the term stage [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. This indicates a delay in the proteolysis of bile salt-stimulated lipase from PHM unlike that for RHM, maybe due to some increased resistance to gastric proteolysis due to some heat-induced structural changes, probably masking pepsin cleavage sites. This heat-denaturation is already known to drastically inactivate this indigenous lipase [START_REF] Henderson | Effect of pasteurization on long chain polyunsaturated fatty acid levels and enzyme activities of human milk[END_REF]. Besides, it can be noted that no peptide was found from the residue 526 until the C-ter end of bile-salt stimulated lipase. This region includes a high number of proline and has been described, from residue 538, as a mucin-like flexible Cterminal tail with a high level of glycosylation [START_REF] Downs | Proline-rich domain and glycosylation are not essential for the enzymatic activity of bile salt-activated lipase. Kinetic studies of T-BAL, a truncated form of the enzyme, expressed in Escherichia coli[END_REF], probably preventing it from proteolysis but also from MS/MS peptide identification.

Regarding lactoferrin, data suggested a greater and faster release of its peptides in PHM, resulting in the intestinal phase in peptides being less abundant in PHM than in RHM (cluster 5 and protein sequence in supplemental Material 4). This can indicate a lower resistance to intestinal proteolysis for the heat-denaturated lactoferrin, unlike that for the native lactoferrin, a globular protein, known to be resistant to proteolysis such as discussed earlier (Deglaire et al., 2016). The concentration of native lactoferrin reaching the intestinal epithelium is thus likely to differ between PHM and RHM, which can modulate its bioactivity. As reported by [START_REF] Buccigrossi | Lactoferrin induces concentration-dependent functional modulation of intestinal proliferation and differentiation[END_REF], high lactoferrin concentrations induced epithelial cell proliferation while low lactoferrin concentrations stimulated intestinal differentiation. In addition, the greater persistence of intact peptides in the gastro-intestinal tract may have physiological consequences, as it is known to be important for gut-associated-lymphoid tissue (GALT) education and microbiota shaping [START_REF] Chatterton | Anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns[END_REF][START_REF] Newburg | Protection of the neonate by the innate immune system of developing gut and of human milk[END_REF]. It should be noted that the end of our intestinal digestion may not be fully representative of the in vivo condition, as brush border peptidases, not included in our model, can further hydrolyse the present peptides. Thus, pasteurization may impact the regulation of the early postnatal intestinal development, through modulation of lactoferrin proteolysis, but further investigations are needed.

Regarding β-casein, data suggested that its peptide abundances were in average not different between RHM and PHM and its kinetics of peptide release were opposite to those for lactoferrin or bile salt-stimulated lipase. This is well in agreement with our previous observations in the term study [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. Nevertheless, there was no emerging cluster significantly associated to β-casein with an increased gastric abundance for PHM in the present study, while this was true in the term study for a limited proportion of the peptides (11% of the total peptides, 17% of the β-casein peptides) [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. The reasons for this remain unknown.

Overall, the different kinetics of peptide release between PHM and RHM may have been somewhat compensating, together with smaller and larger peptides not taken into account here, as the overall degree of gastro-intestinal proteolysis (based on the primary amine content) was not significantly different but the intestinal bioaccessibility of some amino acids (lysine, phenylalanine, tyrosine) was increased by pasteurization (De Oliveira, et al., 2016a).

Bioactive peptides

The present bioactive peptides were identified only after exact matching sequence, although other bioactive peptides can be encrypted within larger peptides and further released in vivo by brush border enzymes not included in the present model. Most potential bioactive peptides were predicted, but not experimentally demonstrated, as having an antimicrobial activity.

Only a limited number of bioactive peptides, mostly from β-casein, was based on a demonstrated activity in the literature. Prior to digestion, two bioactive peptides, the caseinophosphopeptide (CPP) homologue β-casein(1-18) [START_REF] Wada | Bioactive peptides released from in vitro digestion of human milk with or without pasteurization[END_REF] and the antimicrobial peptide β-casein(185-211), were present in human milk as observed previously [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF][START_REF] Dingess | Human milk peptides differentiate between the preterm and term infant and across varying lactational stages[END_REF][START_REF] Wada | Bioactive peptides released from in vitro digestion of human milk with or without pasteurization[END_REF]. Their abundance were lower in PHM than in RHM prior digestion, unlike that observed in the term study [START_REF] Deglaire | Impact of human milk pasteurization on the kinetics of peptide release during in vitro dynamic term newborn digestion[END_REF]. Whether this is due to the term vs. preterm condition remains unknown. Both peptides were hydrolysed rapidly in the intestinal phase in both types of milk, unlike that observed by [START_REF] Wada | Bioactive peptides released from in vitro digestion of human milk with or without pasteurization[END_REF]. This can result from milk variability but also from the simpler and shorter model of digestion (static conditions) used by [START_REF] Wada | Bioactive peptides released from in vitro digestion of human milk with or without pasteurization[END_REF].

Regarding lactoferrin, only one opioid antagonist peptide was released during the gastric phase in a similar manner between RHM and PHM. No peptide was detected between the residues 1-49 that includes the antimicrobial lactoferricin peptide [START_REF] Zwirzitz | Lactoferrin is a natural inhibitor of plasminogen activation[END_REF], probably due to the presence of disulphide bonds between Cys 19 and Cys 36 and between Cys 9 and Cys 45 [START_REF] Farnaud | Lactoferrin-a multifunctional protein with antimicrobial properties[END_REF][START_REF] Wada | Bioactive peptides released from in vitro digestion of human milk with or without pasteurization[END_REF]. However, looking at individual bioactive peptides and at their abundances should be considered with care; further confirmation would be required.

Conclusion

To conclude, the present study demonstrated that pasteurization impacted the human milk peptidome prior to digestion mainly originating from β-casein and induced different kinetics of peptide release during gastro-intestinal digestion mainly for the heat-denaturated proteins (bile-salt stimulated lipase and lactoferrin), such as observed in the term stage. When considering the overall proteolysis, there was no impact of pasteurization; however, it may induce different concentrations of intact peptides within the gastro-intestinal, which may have further physiological consequences. Table 3. Bioactive peptides identified within the entire peptide dataset (n=1531) using an exact matching search the databases of BIOPEP and MBPDB. Results obtained from the online software Enzyme Predictor. Cleavages from cytosol aminopeptidase and carboxypeptidase B2 were examined manually on β-casein and appear only in supplementatl figure 3.

List of tables and figures

2 Odds ratio (OR) were calculated on the present enzymes as OR= (a * d) / (b *c), where a is the total number of sites cleaved by enzyme "A" at the peptide termini ("total number of cleavages"), b is the total number of all sites cleaved by all enzymes except "A"; c is the total number of sites enzyme "A" could have cleaved within the current peptides ("number of expected cleavages within peptides"), and d is the number of sites all enzymes, aside from A, could have cleaved within the current peptides.

Figure 1 .

 1 Figure 1. Mapping of the average cumulative peptide abundances of RHM and PHM onto

3

  Standard errors (SE) were calculated as : SE = exp (1/a +1/b +1/c +1/d) 1/2[START_REF] Vijayakumar | Enzymepredictor: A tool for predicting and visualizing enzymatic cleavages of digested proteins[END_REF].

2

  Effects were determined using a two-way analysis of variance with Pasteurization, Time and their interaction as factors.

  Figure 1 Deglaire

Table 1 :

 1 Predicted enzymes and cleavage counts for proteolysis of human milk as raw or pasteurized prior to digestion.

Table 2 .

 2 Peptide biochemical characteristics significantly associated (p < 0.05) with clusters.

Table 1 :

 1 Predicted enzymes and counts for proteolysis of human milk as raw or pasteurized before gastrointestinal digestion 1 1

Table 2 .

 2 Peptide biochemical characteristics significantly associated (p < 0.05) with clusters1,2 .

			Qualitative modalities				Quantitative modalities
		Modality	Cla/Mod 3	Mod/Cla 4	Overall 5 (n= 1134)	v.test	p.value	Modality	Mean ± SD in category	Mean ± SD in overall (n= 1134)	v.test p.value
		protein = CASA1	14.43	19.44	8.55	3.01	0.0027
		P1' = M	19.23	6.94	2.29	2.24	0.0251
		P1' = S	15.22	9.72	4.06	2.18	0.0290
	Cluster 1	P1 = R protein = FAS	11.02 40.00	19.44 2.78	11.20 0.44	2.13 2.08	0.0336 0.0373
		protein = TRFL	11.11	16.67	9.52	1.97	0.0483
		P1 = T	0.00	0.00	4.14	-2.02	0.0429
		P1' = K	0.00	0.00	4.59	-2.16	0.0304
		P1' = D	0.00	0.00	4.59	-2.16	0.0304
		protein = AMY2B	42.86	2.86	0.62	2.28	0.0224	aliphatic index	94.41 ± 42.47 87.58 ± 37.07 1.98 0.0476
	Cluster 2	P1' = L	13.82	20.00	13.40	1.98	0.0472	MW 6	1.75 ± 0.67	1.92 ± 0.8	-2.26 0.0235
		P1' = P	1.67	0.95	5.29	-2.31	0.0212	isoelectric point	5.4 ± 1.55	5.85 ± 1.99	-2.45 0.0143
		protein = BT1A1	22.22	12.50	2.38	3.39	0.0007
		P1' = G	14.29	12.50	3.70	2.64	0.0082
		protein = FAS	40.00	4.17	0.44	2.39	0.0168
		P1' = K	11.54	12.50	4.59	2.25	0.0241
		P1' = D	11.54	12.50	4.59	2.25	0.0241
	Cluster 3	protein = AMY2B	28.57	4.17	0.62	2.12	0.0344
		P1 = F	9.21	14.58	6.70	1.98	0.0482
		P1 = L	1.79	8.33	19.66	-2.12	0.0338	aliphatic index	76.84 ± 42.38 87.58 ± 37.07 -2.05 0.0403
		protein = CASB	2.74	27.08	41.89	-2.14	0.0324	GRAVY	-0.7 ± 0.87	-0.42 ± 0.74	-2.62 0.0088
		protein = CEL	0.00	0.00	7.50	-2.29	0.0219	isoelectric point	4.9 ± 1.43	5.85 ± 1.99	-3.37 0.0007
		P1' = L	0.66	2.08	13.40	-2.64	0.0084
		protein = CASB	38.53	51.55	41.89	4.43	<0.0001
		protein = CASK	55.81	6.76	3.79	3.36	0.0008
		P1' = N	53.85	5.92	3.44	2.94	0.0033
	Cluster 4	P1' = H P1 = M	54.84 18.87	4.79 2.82	2.73 4.67	2.72 -2.04	0.0066 0.0412	GRAVY	-0.52 ± 0.76	-0.42 ± 0.74	-3.04 0.0024
		P1 = F	17.11	3.66	6.70	-2.86	0.0042
		protein = CEL	8.24	1.97	7.50	-5.23	<0.0001
		protein = TRFL	6.48	1.97	9.52	-6.51	<0.0001
		protein = TRFL	25.00	45.76	9.52	7.53	<0.0001
		P1 = K	13.37	38.98	15.17	4.58	<0.0001
	Cluster 5	P1' = T P1 = R	14.29 9.45	10.17 20.34	3.70 11.20	2.27 2.10	0.0232 0.0355
		protein = CASA1	1.03	1.69	8.55	-2.11	0.0347	essential AA	5.07 ± 1.57	7.02 ± 3.65	-4.23 <0.0001
		protein = CASB	2.95	23.73	41.89	-2.96	0.0031	MW	1.42 ± 0.44	1.92 ± 0.8	-4.97 <0.0001
		P1 = D	65.00	5.70	1.76	4.34	<0.0001
		P1 = L	29.15	28.51	19.66	3.63	0.0003	essential AA	8.62 ± 3.98	7.02 ± 3.65	7.39 <0.0001
		P1' = P	40.00	10.53	5.29	3.62	0.0003	MW	2.24 ± 0.85	1.92 ± 0.8	6.63 <0.0001
		P1 = M	33.96	7.89	4.67	2.42	0.0157	isoelectric point	6.46 ± 2.21	5.85 ± 1.99	5.20 <0.0001
		protein = ALBU	50.00	2.63	1.06	2.27	0.0232	GRAVY	-0.29 ± 0.63	-0.42 ± 0.74	2.98 0.0030
	Cluster 6	P1' = K P1' = N	9.62 7.69	2.19 1.32	4.59 3.44	-2.01 -2.07	0.0440 0.0383
		protein = OSTP	10.87	4.39	8.11	-2.41	0.0160
		P1 = Y	9.68	3.95	8.20	-2.77	0.0056
		protein = CASK	4.65	0.88	3.79	-2.84	0.0046
		P1 = R	11.02	6.14	11.20	-2.84	0.0045
		P1 = K	11.05	8.33	15.17	-3.37	0.0007
		protein = CEL	20.00	18.68	7.50	3.66	0.0002
		P1' = K	19.23	10.99	4.59	2.65	0.0082
	Cluster 7	P1' = G	19.05	8.79	3.70	2.33	0.0198	isoelectric point	5.4 ± 1.61	5.85 ± 1.99	-2.24 0.0254
		protein = LALBA	19.35	6.59	2.73	2.04	0.0414	essential AA	5.51 ± 2.58	7.02 ± 3.65	-4.14 <0.0001
		protein = CASB	6.11	31.87	41.89	-2.03	0.0425	MW	1.57 ± 0.65	1.92 ± 0.8	-4.39 <0.0001
		protein = CEL	32.94	15.91	7.50	4.17	<0.0001
	Cluster 8	P1' = T P1' = P	28.57 25.00	6.82 8.52	3.70 5.29	2.19 1.96	0.0286 0.0498
		P1' = G	2.38	0.57	3.70	-2.68	0.0073

Table 3 .

 3 Bioactive peptides identified within the entire dataset (n = 1531) using an exact matching search within the databases of BIOPEP and MBPDB Peptide position on the parent protein without taking into account the signal peptide.

	Parent protein	Peptide position 1	Peptide sequence	Activity	Pasteurization effect 2	Time effect 2	Pasteurization x Time effect 2
		51-58	YPFVEPIP	opioid agonist	NS	***	NS
		54-59	VEPIPY	immunomodulating	NS	***	NS
		105-117	SPTIPFFDPQIPK	stimulating proliferation	NS	***	NS
	β-casein	125-130	HLPLPL	anti-amnestic	NS	***	NS
		154-160	WSVPQPK	ACE inhibitor/ antioxidant	***	***	***
		161-166	VLPIPQ	ACE inhibitor	NS	***	*
		185-211 QELLLNPTHQIYPVTQPLAPVHNPISV	antimicrobial	NS	***	**
	Lactoferrin 319-324	YLGSGY	opioid antagonist	NS	***	NS
	1

Acknowledgments

The authors thank Frederic Carrière and GERME SA for providing rabbit gastric extract. The authors also thank the volunteer mothers for their donations of breast milk samples as well as Emelyne Dirson and her colleagues from the bank milk of the Rennes University Hospital Center for their involvement in the project. This work was supported by INRA (French National Institute for Agricultural Research) as well as by CNPq (Brazilian National Council for Scientific and Technological Development) by funding a Ph.D. scholarship for the author S. D. O. Most authors were participants of the Food and Agriculture COST (European Cooperation in Science and Technology) Action FA1005 "Improving health properties of food by sharing our knowledge on the digestive process (INFOGEST)." The authors have declared no conflict of interest.

Supporting information

Supplemental material 1. Proteins (n = 27) from which derived the identified peptides before and during digestion of raw and pasteurized milk. Supplemental material 2. Dataset of all the unique peptides (n = 1531) identified before and during digestion of raw and pasteurized milk. Supplemental material 3. Mapping of β-casein hydrolysis prior to digestion with cleavage sites of plasmin, cytosol amino peptidase and carboxypeptidase B2.

Qualitative modalities

Quantitative modalities

Modality

Cla/Mod