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Effects of Different Industrial Heating Processes of Milk on Site-Specific Protein Modifications and Their Relationship to in Vitro and in Vivo Digestibility Yasuaki Wada†,‡ and Bo Lönnerdal*,† †

Department of Nutrition, University of California, Davis, One Shields Avenue, Davis, California 95616, United States Nutritional Science Institute, Morinaga Milk Industry Co., Ltd., 5-1-83 Higashihara, Zama 252-8583, Japan

‡

ABSTRACT: Heating processes are applied to milk and dairy products to ensure their microbiological safety and shelf lives. However, how differences in “industrial” thermal treatments affect protein digestibility is still equivocal. In this study, raw milk was subjected to pasteurization, three kinds of ultra-high-temperature (UHT) treatment, and in-can sterilization and was investigated by in vitro and in vivo digestion and proteomic methods. In-can sterilized milk, followed by UHT milk samples, showed a rapid decrease in protein bands during the course of digestion. However, protein digestibility determined by a Kjeldahl procedure showed insignificant differences. Proteomic analysis revealed that lactulosyllysine, which reflects a decrease in protein digestibility, in α-lactalbumin, β-lactoglobulin, and caseins was higher in in-can sterilized milk, followed by UHT milk samples. Thus, industrial heating may improve the digestibility of milk proteins by denaturation, but the improvement is likely to be offset by heat-derived modifications involved in decreased protein digestibility. KEYWORDS: heating processes, milk proteins, protein digestibility, Maillard reaction, mass spectrometry

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INTRODUCTION Milk is heat-treated in the dairy industry to ensure microbiological safety and to prolong shelf life. Pasteurization is the most common way to heat milk, typically at 72 °C for 15 s, which allows storage at 4 °C for a few weeks. Milk is also sterilized by ultra-high-temperature (UHT; 135−150 °C for 2−6 s) treatment, and the combination with aseptic packaging makes it distributable at ambient temperature for months. Similar to milk, ready-to-use (liquid) infant/enteral formulas are sterilized for the purpose of distribution at ambient temperature, and UHT treatment and in-can sterilization (>110 °C for 20−30 min) are commonly used. These types of heat treatment can affect their sensory, physicochemical, and nutritional qualities. The nutritional value of milk protein, which is generally recognized as excellent, is influenced by heating processes.1 Unfolding of tertiary and secondary structures (denaturation) is the principal change caused by heat treatment, and this generally increases protein digestibility, as is commonly known for legume proteins.2 However, nonenzymatic posttranslational modifications also occur in milk proteins, which influence their nutritional properties negatively. The so-called Maillard reaction modifies the side chain of the protein; ε-amino groups of lysine (Lys) residues represent the primary target for an attack by reducing sugars such as lactose to form the Amadori product lactulosyllysine (Figure 1a), which can subsequently undergo a series of advanced reactions leading to a variety of modified structures such as Nε-carboxymethyllysine (CML; Figure 1b).3 Blocking of the ε-amino groups of Lys residues hinders tryptic digestion as well as the action of other digestive enzymes and, as a consequence, reduces protein digestibility.4−6 The blocked Lys is no longer bioavailable, leading to a reduction in the biological value of milk protein.7 Furthermore, © 2014 American Chemical Society

Figure 1. Chemical structures of heat-derived protein modifications. Lactulosyllysine (a) and Nε-carboxymethyllysine (CML; b) are early/ advanced Maillard reaction products, respectively. Dehydroalanine (c) is the precursor of cross-linked products lysinoalanine (d) and lanthionine (e).

β-elimination reactions occur to phosphoserine and cystine/ cysteine (Cys) residues to form dehydroalanine (Figure 1c), which subsequently reacts with Lys and Cys residues to give cross-linked products such as lysinoalanine and lanthionine (Figure 1d,e)8,9 They also reduce protein digestibility by inhibiting enzymatic proteolysis.10 In addition, heating causes disulfide bond interchanges between proteins,11 and some of the non-native complexes are resistant to digestion.12 It is therefore important to identify and quantify these chemical Received: December 16, 2013 Accepted: April 10, 2014 Published: April 10, 2014 4175

dx.doi.org/10.1021/jf501617s | J. Agric. Food Chem. 2014, 62, 4175−4185

Journal of Agricultural and Food Chemistry

Article

modifications from the standpoint of nutritional quality control of milk and dairy products. The recent advent of highly sensitive mass spectrometry (MS) technology has enabled direct determination of modifications in milk proteins, and the combination with enzymatic digestion prior to MS analysis has further allowed site-specific identification and semiquantification.13−15 The aim of this study was to use proteomic techniques to compare protein modifications of milk proteins exposed to different industrial heat processes. The same reference raw milk was subjected to pasteurization, three kinds of UHT (plate heat exchange, steam infusion, and steam injection) treatment, and in-can sterilization. Protein digestibility of milk samples was characterized in both in vitro and in vivo experiments. Milk samples were also applied to in-gel tryptic digestion followed by liquid chromatography (LC)-MS/MS analysis for simultaneous identification and semiquantification of modifications such as formation of lactulosyllysine, CML, and dehydroalanine. Lactulosyllysine and CML are early/advanced Maillard reaction products, respectively, and serve as markers of the thermal history. Dehydroalanine was selected instead of lysinoalanine and lanthionine because it is compatible with the analysis of lactulosyllysine and CML, whereas lysinoalanine and lanthionine are not. Formation of dehydroalanine has also been reported to reflect the thermal history, similar to lysinoalanine and lanthionine.16

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Table 3. Nutrient Composition of Lyophilized Raw and In-Can Sterilized Milk

MATERIALS AND METHODS

Table 1. Heat Treatment of Milk Samples holding temp/time

F0

reference producta

pasteurization plate UHT steam infusion steam injection in-can sterilization

73 °C for 15 s 140 °C for 2 s 135 °C for 2 s 151 °C for 4 s 121 °C for 20 min

0 2.6 0.8 65 20

milk milk milk liquid enteral formula liquid enteral formula

raw

in-can sterilized

protein fat carbohydrate ash water

25.5 28.7 38.2 5.5 2.1

25.5 28.3 38.2 5.6 2.4

fat layer was removed. The tiny pellet, formed after centrifugation, was thoroughly mixed with the skimmed milk fraction. Protein concentrations of skimmed milk were determined by using the Bradford method,19 and they were adjusted to be the same among samples by dilution with deionized water. SDS-PAGE was performed under reducing/nonreducing conditions to separate the proteins (Bio-Rad, Hercules, CA, USA). Protein concentrations were determined by using the Bradford method, and 50 μg of protein was loaded for each sample onto 15% acrylamide resolving gel (pH 8.8) and 6% acrylamide stacking gel (pH 6.8). Electrophoresis was run at 150 V for 90 min, and gels were stained with Coomassie Brilliant Blue R-250 (SigmaAldrich, St. Louis, MO, USA). Proteomic Analysis. Proteomic analysis was conducted at the Proteomic Core Facility at the University of California, Davis, to sitespecifically semiquantify lactulosyllysine, CML, phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr), and dehydroalanine formed from phosphoserine and Cys residues. In-Gel Digestion. In-gel reduction, alkylation, and tryptic digestion were done as published previously.20,21 Briefly, milk samples were reconstituted with deionized water and applied to electrophoresis to excise the fraction containing the entire protein band. The gel piece was dehydrated using acetonitrile (ACN), reduced with dithiothreitol, and alkylated using 55 mM iodoacetamide. Following further dehydration, sequencing grade modified trypsin (Promega, Madison, WI, USA; in 50 mM ammonium bicarbonate) was added in a 1:30 ratio (trypsin/protein), and the gel was incubated overnight at 37 °C. While the supernatant was collected into a new tube, 60% ACN (in 0.1% trifluoroacetic acid) was added to the remaining gel piece, which was then sonicated and centrifuged for collecting another supernatant. The supernatants were then dried by vacuum centrifugation. Protein Identification. Digested peptides were analyzed by a Thermo Scientific Q Exactive Orbitrap mass spectrometer in conjunction with a Paradigm MG4 HPLC (Michrom Bio Resources, Auburn, CA, USA). The digested peptides were loaded onto a Michrom C18 trap and desalted before they were separated using a Michrom 200 μm × 150 mm Magic C18 AQ reverse phase column. A flow rate of 2 μL/min was used. Peptides were eluted using a 90 min gradient composed of buffers A (0.1% formic acid) and B (100% ACN), with 2−35% B over 70 min, 35−80% B for 5 min, 80% B for 2 min, then a decrease from 80 to 2% B in 1 min, and held at 2% B for 12 min. A spray voltage of 2.2 kV was used with a transfer capillary temperature of 200 °C. A MS survey scan was obtained for the m/z range of 300− 1600. MS/MS spectra were acquired using a top 15 method, where the top 15 ions in the MS spectra were subjected to high-energy collisional dissociation. An isolation mass window of 2.0 m/z was used for the precursor ion selection, and a normalized collision energy of 27% was used for fragmentation. A 5 s duration was used for the dynamic exclusion. Database Searching. Tandem mass spectra were extracted and charge states deconvoluted and deisotoped. All MS/MS samples were analyzed using X! Tandem (The GPM, thegpm.org; version TORNADO (2010.01.01.4)). X! Tandem was set up to search the

Milk Samples. Raw milk was heat-processed at the pilot plant of Morinaga Milk Industry Co., Ltd. (Zama, Japan), and the heating conditions were set by reference to those of the manufacturer’s commercial products (Table 1). The heated milk samples were

treatment

component (g/100 g)

a

Heating conditions were set by reference to those of the manufacturer’s commercial products.

homogenized at 30 MPa, followed by lyophilization. The reference raw milk was lyophilized without homogenization. The lyophilized milk powders were stored at 4 °C until use. Indices of thermal history such as whey protein nitrogen index (WPNI) and lactulose contents were determined in all of the lyophilized milk samples by the Analytic Center of Morinaga Milk Industry,17,18 and the results are shown in Table 2. On the basis of the results that in-can sterilized milk showed the highest lactulose and the lowest WPNI values, compositions such as protein, fat, carbohydrate, water, and ash were compared for lyophilized raw milk and in-can sterilized milk with standard methods in Morinaga Milk Industry, confirming that no notable change occurred after heat treatment (Table 3). Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Milk Samples. Milk powder samples were reconstituted with Milli-Q water (Millipore, Billerica, MA, USA) and subjected to centrifugation at 8500g for 30 min twice, and the upper

Table 2. Indices of Thermal History for Lyophilized Milk Samples component

raw

pasteurized

plate UHT

steam infused

steam injected

in-can sterilized

WPNI (mg/g) lactulose (mg/100 g)

7.51 20.2

6.91 19.6

1.75 204

4.29 42.0

2.22 241

1.33 1490

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digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 20 ppm. Iodoacetamide derivative of Cys was specified in X! Tandem as a fixed modification. One of the targeted modifications [lactulosyllysine on Lys (+324.105647), CML on Lys (+58.005479), phosphorylation on Ser, Thr, and Tyr (+79.966331), dehydration of Ser (−18.010565), or dehydroalanine on Cys (−33.987721)], as well as acetylation of the N-terminus, ammonia loss of the N-terminus, pyro-glutamination of the N-terminus, and oxidation of methionine and tryptophan, were specified in X! Tandem as variable modifications. Criteria for Protein Identification. Scaffold (version Scaffold_4.1.1, Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications, taking into consideration the Peptide Prophet algorithm, Protein Prophet algorithm, and false-discovery rates.22−24 Proteins that contained similar peptides and could not be differentiated on the basis of MS/MS analysis alone were grouped to satisfy the principles of parsimony. Peptide Semiquantification. Scaffold data were imported into Skyline (version 1.4.0.4421, MacCoss Lab, Department of Genome Science, University of Washington) to determine retention time and peak areas of peptides with targeted modifications. On the basis of the retention time, three different monoisotopic peak areas (M, M + 1, M + 2) were integrated for each specified peptide with Xcalibur (version 2.2 SP1.48, Thermo Scientific, Waltham, MA, USA), and they were summed to obtain higher confidence. For normalization between samples, the summed peak area was divided by the exclusive spectrum counts of all modified and nonmodified peptides derived from the parental protein, in Scaffold. In Vitro Digestion. Milk powder samples were reconstituted and defatted as above and subjected to in vitro digestion.25 Simulated gastric digestion was started with adjusting the pH to 2.0 (adult model) or 4.0 (infant model) with 1 M HCl. Porcine pepsin (SigmaAldrich; 2% in 1 mM HCl) was added to the sample in a 1:12.5 ratio (pepsin/protein). Samples were placed in an incubating shaker (New Brunswick Scientific, Edison, NJ, USA) at 140 rpm at 37 °C for 5, 10, 15, or 30 min. After this incubation, the pH of the samples was adjusted to 7.0 with 0.1 mol/L NaHCO3. Simulated intestinal digestion was performed following the gastric digestion (at pH 2.0 or 4.0). After the pH was adjusted to 7.0, pancreatin (Sigma-Aldrich; 0.4% in 0.1 mol/L NaHCO3) was added to the samples in a 1:62.5 ratio (pancreatin/protein). Samples were placed in an incubator shaker at 140 rpm at 37 °C for 5, 10, 30, or 60 min. After the incubation, the enzymes were inactivated in a water bath at 85 °C for 3 min. The digested samples were applied to reducing SDS-PAGE as described above, and each lane was loaded with the equivalent of 50 μg of protein before digestion. Protein digestibility of the samples subjected to gastric/intestinal digestion was determined using a Kjeldahl procedure as described previously.26,27 Briefly, total nitrogen (N), and nonprotein N [NPN; soluble fraction after trichloroacetic acid (TCA; 12% final concentration) precipitation] were measured in milk samples before/after the digestion. Protein digestibility was defined as the ratio of (NPNafter digestion − NPNbefore digestion)/(total Nbefore digestion − NPNbefore digestion). Protein digestibility was analyzed six times for each pH condition/time period tested. In Vivo Digestion. A suckling rat pup model was used to investigate in vivo digestion of milk samples.25,28−30 The study was approved by the International Animal Care and Use Committee of the University of California, Davis (protocol 17128), which is accredited by the American Association for the Accreditation of Laboratory Animal Care. Sprague−Dawley rats with litters of 12 pups (6 males + 6 females) were obtained commercially (Charles River, Wilmington, MA, USA), maintained in polycarbonate cages with wood shavings in a temperature-, humidity-, and light-controlled facility (22 °C, humidity 60%, and 12 h light/dark cycle), and allowed to consume deionized water and standard rat chow diet (Ralston Purina, St. Louis, MO, USA) ad libitum. Pups were randomly assigned as 3 males + 3 females/milk sample/time point. After acclimation to facility conditions, on day 14 postpartum, pups were separated from their dams for 6 h and then intubated with one of six types of milk (reconstituted, defatted, and

Figure 2. Electrophoresis of milk proteins. Milk samples were applied to SDS-PAGE analysis under (a) reducing and (b) nonreducing conditions. Fifty micrograms of protein was loaded onto a 15% gel, and the gel was stained with Coomassie Brilliant Blue R-250.

adjusted to 30 g/L of protein), in a 1:36 v/w ratio [milk (mL)/body weight (g)]. Pups were euthanized with CO2 asphyxiation at 0.5, 1, or 2 h after intubation. Stomach contents were collected and weighed. The small intestine was cut into upper and lower parts, and each part was perfused twice with 0.2 mL of phosphate-buffered saline (PBS) containing protease inhibitor cocktail (P2714, Sigma-Aldrich). Collected perfusates were stored at −80 °C until analysis. The perfusates underwent Western blotting. On the basis of the results obtained in the in vitro study, β-lactoglobulin (β-LG) was used as a marker protein of in vivo digestibility. The perfusates (5 μg of protein) were applied to reducing SDS-PAGE, and proteins were transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare, Pewaukee, WI, USA). The membrane was blocked with 2% bovine serum albumin [in PBS containing 0.05% Tween-20 (PBST)] for 45 min at room temperature. After the membrane was washed three times with PBST, it was probed with rabbit anti-bovine β-LG antibody (Bethyl Laboratories Inc., Montogomery, TX, USA) in a 1:20000 dilution (antibody/blocking buffer) at room temperature for 45 min. After three washings with PBST, the membrane was probed with donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase (GE Healthcare) in a 1:20000 dilution (antibody/blocking buffer) at room temperature for 45 min. After three washings with PBST, β-LG was detected by Pierce ECL Western Blotting Substrate (Thermo Scientific). Statistical Analysis. Data on protein digestibility (Kjeldahl procedure) and stomach contents resulting from in vivo digestion were analyzed by one-way ANOVA and post-tested by the Tukey test 4177



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Table 4. Lactulosyllysine Derivatives of Lys in Milk Proteinsa protein

amino acid

charge

rawb

pasteurized

plate UHT

steam infused

steam injected

α-LA

41−60

Lys98

[3+] [4+]

1.0 1.0

1.1 0.9

3.0 2.1

2.4 1.4

3.7 2.4

9.8 6.0

β-LG

41−60 76−83 76−101 84−101 125−138

Lys47 Lys77 Lys83 or 91 Lys91 Lys135

[3+] [2+] [4+] [3+] [2+] [3+]

1.0 1.0 1.0 1.0 1.0 1.0

1.0 1.4 1.2 1.1 1.0 1.1

1.6 4.4 4.5 1.7 3.0 2.8

2.1 2.0 1.6 1.6 2.3 2.1

2.5 5.5 2.1 2.0 4.0 4.0

3.6 16.5 10.2 2.4 6.2 4.8

αS1-CN

4−22

Lys7

35−42 80−90

Lys36 Lys83

104−119

Lys105

120−132 125−151

Lys124 Lys132

[3+] [4+] [2+] [2+] [3+] [2+] [3+] [2+] [2+] [3+]

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

0.8 1.1 1.2 1.0 1.9 1.8 2.6 3.7 1.5 2.1

2.6 3.1 2.7 1.8 4.1 26.9 5.6 11.9 3.9 5.4

2.0 1.8 2.4 1.5 1.9 5.6 2.0 2.8 1.6 2.3

3.0 3.3 3.8 4.6 4.9 46.2 7.7 9.4 4.9 5.3

6.3 6.1 13.7 12.4 9.7 505.3 62.3 101.6 19.8 24.0

25−41

Lys32

71−91

Lys76 or 80

171−181

Lys173

174−188

Lys181

198−205

Lys199

[2+] [3+] [3+] [4+] [2+] [3+] [2+] [3+] [4+] [2+] [3+]

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

0.5 0.6 1.1 1.2 1.5 1.6 1.6 1.2 1.5 1.1 1.2

3.1 2.4 4.1 3.5 2.8 2.4 3.9 2.5 2.3 1.8 1.5

1.5 1.3 1.7 1.7 1.7 1.8 2.4 1.6 1.6 0.8 0.8

3.2 3.1 4.3 3.6 3.3 3.1 3.3 2.9 2.5 2.1 2.3

17.1 21.2 10.1 8.5 6.2 5.4 8.0 4.9 4.0 4.2 4.1

β-CN

170−183

Lys176

[2+] [3+]

1.0 1.0

0.9 1.1

1.4 1.5

1.1 1.0

1.5 2.1

5.8 4.5

κ-CN

22−34

Lys24

35−68 69−97

Lys46 Lys86

[2+] [3+] [4+] [3+] [4+]

1.0 1.0 1.0 1.0 1.0

1.9 2.0 1.9 1.0 1.6

5.6 4.9 6.4 4.1 4.7

4.1 3.5 2.6 3.3 2.3

6.1 5.2 6.2 4.7 3.8

12.1 11.3 20.1 34.7 31.1

αS2-CN

residue

in-can sterilized

a

Milk samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying trypic peptides containing lactullosyllysine in milk proteins. bPeak areas of the specific peptides are expressed as mean values (n = 2); normalized to raw milk.

using Prism 4 (version 4.03 GraphPad Software Inc., San Diego, CA, USA), and significance was demonstrated at p < 0.05.

sterilization modified the proteins to a greater extent by forming new disulfide bonds and possibly other noncovalent interactions. Proteomic Analysis. Milk samples were applied to LC-MS/ MS analysis following in-gel tryptic digestion for identifying and semiquantifying modifications involved in protein digestibility, such as lactulosyllysine, CML, and dehydroalanine on Ser and Cys residues, in major milk proteins. Relative amounts of quantified peptides with the above modifications are shown in Tables 4−6. It should be noted that some of the peptides containing the specified modifications were identified but could not be quantified probably because of low abundance. In-can sterilized milk had the highest signal intensity for lactulosyllysine at all of the modified sites in α-LA, β-LG, and CNs, followed by plate UHT and steam-injected milk. A similar trend was observed for CML; the highest signals were observed at Lys77

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RESULTS Electrophoresis of Milk Proteins. Milk samples were applied to SDS-PAGE, and the band patterns were compared (Figure 2). A notable difference was observed for in-can sterilized milk; proteins smeared in a wide range of molecular weights, and the bands of intact milk proteins decreased under reducing/nonreducing conditions. This trend, although to a lesser extent, was also observed for the three UHT-treated milk samples under nonreducing condition, where disulfide bonds are kept intact. These results suggest that pasteurization essentially does not induce any protein associations; the three kinds of UHT treatments caused new disulfide bond formation between proteins, facilitating protein aggregation, and in-can 4178



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Table 5. Carboxymethyllysine in Milk Proteinsa amino acid

charge

rawb

pasteurized

plate UHT

steam infused

steam injected

in-can sterilized

41−70 76−83

Lys47, 69, or 70 Lys77

125−138

Lys135

[3+] [2+] [3+] [3+]

1.0 1.0 1.0 1.0

0.3 0.9 2.2 0.6

0.2 1.2 3.0 0.6

0.3 0.1 0.4 0.8

0.2 2.7 6.9 1.1

0.1 3.9 11.2 0.8

4−22

Lys7

23−36 80−90

Lys34 or 36 Lys83

[3+] [4+] [3+] [2+] [3+]

nd nd 1.0 1.0 1.0

1.0c 1.0c 22.1 3.4 3.4

1.7 1.5 68.7 20.9 4.6

1.0 1.0 18.9 3.7 0.9

1.7 1.6 63.8 20.1 4.9

4.9 3.8 552.0 142.5 16.2

71−80 151−160 171−181

Lys76 Lys152 Lys173

198−205

Lys199

[2+] [2+] [2+] [3+] [2+]

1.0 1.0 1.0 1.0 1.0

2.9 1.2 6.7 26.5 2.7

10.1 0.8 11.3 35.1 9.0

1.5 1.2 2.4 13.6 1.1

18.2 2.1 11.5 20.5 3.4

28.6 8.2 54.8 115.6 55.0

β-CN

98−107 170−183

Lys105 Lys176

[2+] [2+] [3+]

nd 1.0 1.0

nd 4.3 2.3

nd 10.8 3.4

nd 2.9 1.8

nd 6.6 2.9

1.0c 77.6 15.6

κ-CN

22−34

Lys24

[2+] [3+]

1.0 1.0

0.9 0.8

1.8 1.2

2.6 1.3

4.1 3.2

20.0 8.7

protein β-LG

αS1-CN

αS2-CN

residue

a

Milk samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying trypic peptides containing carboxymethyllysine in milk proteins. bPeak areas of the specific peptides are expressed as mean values (n = 2); normalized to raw milk. cPeak areas of the specific peptides are expressed as mean values (n = 2); normalized to other milk samples when the peak areas from raw milk were below the detection limit.

Table 6. Dehydroalanine Derivatives of Ser in Milk Proteinsa protein

residue

amino acid

charge

rawb

pasteurized

plate UHT

steam infused

steam injected

in-can sterilized

αS1-CN

104−119

Ser115

106−119

Ser115

[2+] [3+] [2+]

1.0 1.0 1.0

0.6 0.4 0.4

1.7 1.0 0.8

0.7 0.5 0.5

1.4 0.8 1.4

1.2 0.6 1.1

33−48

Ser35

[2+]

1.0

0.3

0.6

0.3

0.3

0.3

β-CN a

Milk samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying trypic peptides containing dehydroalanine on Ser in milk proteins. bMeasured values of peak areas are shown (n = 1); normalized to raw milk.

in β-LG and all of the modified sites in CNs for in-can sterilized milk, which was followed by plate UHT and steam-injected milk. Some of the lactulosyllysine/CML sites could not be solely assigned, such as lactulosyllysine of Lys83/91 in β-LG, lactulosyllysine of Lys76/80 in αS2-CN, CML of Lys47/69/70 in β-LG, and CML of Lys34/36 in αS1-CN. However, by taking into consideration the fact that CML should be converted from lactulosyllysine and that trypsin can never recognize lactulosyllysine as a cleavage site,3,5 it can be assumed that lactulosyllysine of Lys76/80 in αS2-CN would be Lys76, CML of Lys47/69/70 in β-LG would be Lys47, and CML of Lys34/ 36 in αS1-CN would be Lys 36. If this is true, all of the quantified CML sites would overlap the quantified lactulosyllysine sites except Lys152 in αs2-CN and Lys105 in βs2-CN. It should be noted that lactulosyllysine was also identified (but not quantifiable) at these sites (data not shown). In contrast with lactulosyllysine/CML, dehydroalanine could be quantified only at three sites in all of the major proteins. These signals were weak, and no clear trend was observed among the milk samples, indicating that dehydroalanine formation in the milk samples is very limited.

In addition to the above-mentioned modifications, phosphorylation on Ser, Thr, and Tyr was also semiquantified as it may provide further information in the context of heat treatment and proteins; milk proteins have generally been considered to be dephosphorylated after heat treatment of milk,31 whereas proteins can be phosphorylated in the presence of phosphate after dry heating.32,33 In this study, phosphorylation could be quantified at three Ser residues in αS1-CN, αS2-CN, and β-CN (Table 7). In general, they appear to be dephosphorylated after heating, but to a very limited extent, although phosphorylation of Ser46 increased in in-can sterilized milk. In Vitro Digestion of Milk Samples. SDS-PAGE analysis of milk samples after in vitro digestion is shown in Figure 3. A rapid decrease in α-LA, β-LG, and CN bands was observed during the course of digestion in in-can sterilized milk, indicating that in-can sterilization facilitated enzymatic digestion of these proteins. The bands for α-LA and β-LG, but not CN, decreased over time in the three UHT-treated milk samples, but this did not occur as rapidly as in in-can sterilized milk. The digestibility appeared to be plate UHT > steam injected > steam-infused milk, according to the remaining band 4179



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Table 7. Phosphorylation of Ser in Milk Proteinsa protein

residue

amino acid

charge

rawb

pasteurized

plate UHT

steam infused

steam injected

in-can sterilized

αS1-CN

119−134

Ser130

[2+] [3+]

1.0 1.0

0.7 0.8

1.0 0.7

0.9 0.7

0.7 0.8

0.5 0.5

αS2-CN

40−56

Ser46

[2+] [3+]

1.0 1.0

2.1 2.1

0.9 0.6

1.8 1.4

0.7 0.4

4.0 4.3

β-CN

45−63

Ser50

[2+] [3+]

1.0 1.0

0.9 0.8

0.7 0.6

1.0 1.1

0.8 0.7

0.7 0.6

a

Milk samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying trypic peptides containing phosphorylation on Ser in milk proteins. bPeak areas of the specific peptides are expressed as mean values (n = 2); normalized to raw milk.

Figure 3. Electrophoresis of milk proteins after in vitro digestion. Milk samples were subjected to in vitro digestion using pepsin and pancreatin for different time periods. Both infant (a-h) and adult (i-p) digestion conditions were simulated by adopting different pH values during the peptic digestion. Fifty micrograms of protein (before digestion) was loaded onto a 15% gel, and the gel was stained with Coomassie Brilliant Blue R-250. 4180



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Figure 4. Protein digestibility of milk proteins after in vitro digestion. Protein digestibility was defined as the increase in NPN after in vitro digestion, which was determined by the combination of TCA precipitation and a Kjeldahl procedure. Each digestion and the subsequent procedures were conducted six times, and data are expressed as means ± SD. “a”, p < 0.05 (in-can sterilized vs raw milk); “b”, p < 0.05 (in-can sterilized vs raw or steam-injected milk).

intensities for α-LA and β-LG. In contrast to in-can sterilized milk and the three UHT-treated milk samples, pasteurized milk showed the same band patterns as raw milk throughout the digestion experiments, suggesting that pasteurization does not affect the digestibility of the milk proteins to any significant extent. At the final time points of each peptic/pancreatic digestion, the digestibility of total milk protein was determined using a Kjeldahl procedure (Figure 4). In-can sterilized milk showed the highest digestibility of all the digestion conditions. However, the differences in total protein digestibility among the samples seem equivocal compared with the observations for the above SDS-PAGE. In Vivo Digestion of Milk Samples. White coagulates, which are likely acid-precipitated CNs, were observed in the stomachs of rat pups after intubation with the milk samples (Figure 5a). Pups fed in-can sterilized milk had the smallest contents at 0.5 and 1 h, which was statistically lower at 1 h compared with the plate UHT milk-fed group (p < 0.05). At 2 h, there were no notable differences in stomach contents among the milk samples. This indicates that extensive heat treatment such as in-can sterilization can affect coagulation of CN negatively as previously reported for pasteurized milk.34 Furthermore, a notable decrease in the pH of the stomach content was observed in the in-can sterilized milk-fed group at 2 h (Figure 5b), which may also corroborate damaged casein coagulation potential, manifested as impaired buffering activity. Perfusates from the upper small intestine showed complex protein band patterns by SDS-PAGE (data not shown). Because β-LG exhibited a prominent difference in in vitro digestion among the milk samples, this protein was chosen to assess in vivo digestibility and was analyzed by Western blotting (Figure 6). At 0.5 h, strong β-LG bands were observed for raw milk and pasteurized milk in its intact form as well as partially fragmented forms, whereas intact bands were barely detected in the three UHT-treated milk samples and in-can sterilized milk. At 1 h, β-LG was still visible in raw milk, but the bands were barely detectable or invisible for the remaining five milk samples. At 2 h, no β-LG band was detectable for any of the milk samples. Western blotting of β-LG was also conducted for the lower

intestine perfusates, but the bands were very faint and no clear differences were observed among the milk samples (data not shown).

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DISCUSSION MS techniques have extensively been utilized to characterize chemical modifications of milk proteins. However, the link between chemical characterization of the proteins and their nutritional consequences has rarely been discussed. In this study, heated milk samples were manufactured from the same reference raw milk and they were investigated in terms of both protein digestibility and proteomic characterization. Sites of protein modifications have been a major interest in proteomic studies of milk proteins, and lactulosyllysine sites are often identified. In this study, α-LA had only one lactulosylation site at Lys98, which is the main lactulosylation site in this protein. This modification has been identified in UHT milk and powdered infant formulas.35−37 As for β-LG, all five lactulosylation sites have previously been identified in pasteurized milk, UHT milk, and/or powdered infant formulas.5,35,37 Among them, Lys47 is the best studied site because of its high reactivity.38,39 Semiquantification has also been conducted using various kinds of dairy products.15 All of the lactulosyllysine sites in CNs, except Lys199 in αS2-CN, agree with previous studies.5,37 Thus, the general agreement of the lactulosyllysine sites substantiates the validity of this study, and the new lactulosyllysine site (Lys199 in αS2-CN) could be found probably due to methodological differences. With regard to semiquantification of protein modifications, our results look controversial at first glance in that both lactulosyllysine and CML were quantifiable in all heat-treated milk samples as well as raw milk (despite the absence of heat treatment). However, this observation is reasonable because some studies could quantify furosine, an indirectly measured product from Amadori compounds, and CML in raw milk samples.40,41 The highest signals for lactulosyllysine and CML were observed at almost all of the sites in in-can sterilized milk, reflecting the severe thermal history resulting from slow rates of temperature rise/fall and the longest holding time at high temperature. To a lesser extent, the signals for the three UHT-treated milk 4181



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Figure 5. Stomach contents of suckling rat pups. Rat pups fed different milk samples were euthanized at 0.5, 1, and 2 h after intubation, and the stomach contents were obtained. Weight (a) and pH values (b) were measured, and data are expressed as means ± SD (n = 3 males + 3 females). “a”, p < 0.05 (in-can sterilized vs plate UHT milk).

samples were also higher, probably because of the high holding temperature. However, steam-infused milk tended to show the lowest signals among the UHT-treated milk samples. The lowest heat damage of steam-infused milk among the three UHT-treated milk samples could arise not just from the lowest holding temperature but also from the dilution effect derived from the steam infusion.42 No CML was identified in α-LA, and there were no distinct differences in CML amounts among the milk samples at the sites of β-LG compared with those of CNs. This possibly implies that CNs, the dominant proteins in cow’s milk, might be competitive with whey proteins for progress of the Maillard reaction. Both of the electrophoretic analyses in the in vitro and in vivo digestion assays demonstrated that in-can sterilization, followed by the three UHT treatments, facilitated enzymatic digestion of the major milk proteins, especially of β-LG. Facilitated digestion of intact milk proteins has also been observed in a study similar to ours; raw milk, pasteurized milk,

Figure 6. Western blotting of β-LG in upper intestine perfusates from suckling rat pups. Rat pups fed different milk samples were euthanized at 0.5, 1, and 2 h after intubation, and the upper intestines were perfused with PBS containing protease inhibitor. Five micrograms of protein was loaded, and β-LG was detected by Western blotting as an index of in vivo protein digestibility. 4182



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limited after heating raw milk). In the context of ingredient manufacturing, industrial CN isolation involves isoelectric precipitation followed by neutralization at alkaline pH, and the alkaline treatment would facilitate the formation of crosslinks such as lysinoalanine and lanthionine.9 Furthermore, some of the above modifications proceed during long storage of ingredients/products at ambient temperature, possibly augmenting the modifications.52 In conclusion, industrial heating may improve the digestibility of proteins in raw milk by denaturation, but the improvement is likely to be offset by heat-derived modifications involved in the decrease in protein digestibility. However, this phenomenon may not hold true for dairy products such as infant/enteral formulas. Continuing studies are in progress to verify how protein modifications and consequential protein digestibility would differ between cow’s milk samples and infant formulas.

and sterilized milk were applied to in vitro digestion, and proteins in sterilized milk but not pasteurized milk exhibited better digestion.43 With regard to β-LG, it has been shown in several studies that heat treatment promotes its digestion.12,34,44,45 These positive effects of heat treatment on protein digestion can be attributed to protein denaturation, accelerating exposure of peptide bonds to digestive enzymes. Denaturation of milk proteins was apparent as WPNI values decreased for in-can sterilized milk followed by the three UHT-treated milk samples, and a faster gastric emptying rate/impaired gastric buffering activity of in-can sterilized milk was observed for in vivo digestion. In contrast to the facilitated protein digestion discussed above, protein digestibility of milk samples, determined by a combination of in vitro digestion and a Kjeldahl procedure, showed insignificant differences among the milk samples. Similar results have been reported previously,46,47 with some of the studies reporting positive effects accompanied by adverse consequences; some amino acids were not digestible, and small fragments resistant to digestion were formed.43,44 Plausible explanations for these negative outcomes would be the fact that heating facilitates the formation of lactulosyllysine, the aggregation of proteins via cross-linked products such as lysinoalanine and lanthionine, and some newly formed disulfide bonds; these chemical structures inhibit the action of trypsin as well as other digestive enzymes, leading to a decrease in protein digestibility. In this study, in-can sterilized milk manifested the greatest formation of lactulosyllysine at many Lys residues in major milk proteins, such as α-LA, β-LG, and CNs, to an extent followed by plate UHT and steam-injected milk. With regard to protein aggregation, whereas proteomic analysis indicated that formation of lysinoalanine in all of the milk samples seems quantitatively limited, the results of SDS-PAGE suggest that the three kinds of UHT treatment caused new disulfide bond formation and that in-can sterilization modified the proteins to a greater extent by forming new disulfide bonds and possibly other noncovalent interactions. Thus, it can be speculated that a denaturation-mediated improvement in protein digestibility might be “offset” by the concomitant lactulosyllysine formation and protein aggregation involved in reducing protein digestibility. It should be added that the surface hydrophobicity of milk proteins, determined by a SDS-binding method,48 showed only insignificant differences among milk samples (data not shown). Heating decreases surface hydrophobicity of caseins (the majority of total milk protein), whereas progress of the Maillard reaction increases surface hydrophobicity of total milk proteins. This observation may therefore reflect another “offset” chemistry in milk proteins. However, the possibility should be taken into consideration that total milk protein digestibility was practically the same because the heat treatment facilitates protein digestion but the resulting peptides might still be too large to be TCA-soluble. Whereas no adverse effects on protein digestibility of heated raw milk were observed in this study even after in-can sterilization (despite the greatest formation of Amadori product on Lys residue causing inhibition of digestive enzymes such as trypsin), it seems that a decrease in protein digestibility has mostly been reported for milk-based infant/enteral formulas.27,49−51 In general, the thermal history of milk-based formulas is more extensive than that for cow’s milk because milk proteins are heat-treated as both “ingredients” and as “products (i.e., formulas)”, which would promote the formation of Amadori products and cross-links (whereas our data of dehydroalanine suggested formation of cross-links may be

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AUTHOR INFORMATION

Corresponding Author

*(B.L.) Phone: (530) 752-8347. Fax: (530) 752-3564. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Keisuke Miyazaki, Junichi Hashimoto, Hiroyoshi Yabuki, and Yuki Hayakawa, Morinaga Milk Industry, for preparing for the milk samples. We thank Dr. Brett Phinney and Darren Weber, University of California, Davis, for performing the proteomic analysis.

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ABBREVIATIONS USED UHT, ultra-high-temperature; Lys, lysine; CML, Nε-carboxymethyllysine; Cys, cysteine; MS, mass spectrometry; LC, liquid chromatography; WPNI, whey protein nitrogen index; SDSPAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; N, nitrogen; NPN, nonprotein N; TCA, trichloroacetic acid; β-LG, β-lactoglobulin; PBS, phosphate-buffered saline; PBST, PBS containing 0.05% Tween-20; ACN, acetonitrile; Ser, serine; α-LA, α-lactalbumin; CN, casein

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REFERENCES

(1) Mauron, J. Influence of processing on protein quality. J. Nutr. Sci. Vitaminol. (Tokyo) 1990, 36 (Suppl. 1), S57−S69. (2) Kakade, M. L.; Evans, R. J. Chemical and enzymatic determinations of available lysine in raw and heated navy beans (Phaseolus vulgaris). Can. J. Biochem. 1966, 44, 648−650. (3) van Boekel, M. A. J. S. Effect of heating on Maillard reactions in milk. Food Chem. 1998, 62, 403−414. (4) Mauron, J.; Mottu, F.; Bujard, E.; Egli, R. H. The availability of lysine, methionine and tryptophan in condensed milk and milk powder. In vitro digestion studies. Arch. Biochem. Biophys. 1955, 59, 433−451. (5) Marvin, L. F.; Parisod, V.; Fay, L. B.; Guy, P. A. Characterization of lactosylated proteins of infant formula powders using twodimensional gel electrophoresis and nanoelectrospray mass spectrometry. Electrophoresis 2002, 23, 2505−2512. (6) Rerat, A.; Calmes, R.; Vaissade, P.; Finot, P. A. Nutritional and metabolic consequences of the early Maillard reaction of heat treated milk in the pig. Significance for man. Eur. J. Nutr. 2002, 41, 1−11. (7) Finot, P. A.; Magnenat, E. Metabolic transit of early and advanced Maillard products. Prog. Food Nutr. Sci. 1981, 5, 193−207. 4183



Article

(8) Swaisgood, H. E.; Catignani, G. L. Digestibility of modified milk proteins: nutritional implications. J. Dairy Sci. 1985, 68, 2782−2790. (9) Friedman, M. Chemistry, biochemistry, nutrition, and microbiology of lysinoalanine, lanthionine, and histidinoalanine in food and other proteins. J. Agric. Food Chem. 1999, 47, 1295−1319. (10) Robbins, K. R.; Baker, D. H.; Finley, J. W. Studies on the utilization of lysinoalanine and lanthionine. J. Nutr. 1980, 110, 907− 915. (11) Patel, H. A.; Singh, H.; Anema, S. G.; Creamer, L. K. Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk. J. Agric. Food Chem. 2006, 54, 3409−3420. (12) Peram, M. R.; Loveday, S. M.; Ye, A.; Singh, H. In vitro gastric digestion of heat-induced aggregates of β-lactoglobulin. J. Dairy Sci. 2013, 96, 63−74. (13) Fenaille, F.; Morgan, F.; Parisod, V.; Tabet, J. C.; Guy, P. A. Solid-state glycation of β-lactoglobulin monitored by electrospray ionisation mass spectrometry and gel electrophoresis techniques. Rapid Commun. Mass Spectrom. 2003, 17, 1483−1492. (14) Meltretter, J.; Seeber, S.; Humeny, A.; Becker, C. M.; Pischetsrieder, M. Site-specific formation of Maillard, oxidation, and condensation products from whey proteins during reaction with lactose. J. Agric. Food Chem. 2007, 55, 6096−6103. (15) Meltretter, J.; Becker, C. M.; Pischetsrieder, M. Identification and site-specific relative quantification of β-lactoglobulin modifications in heated milk and dairy products. J. Agric. Food Chem. 2008, 56, 5165−5171. (16) van Boekel, M. A. J. S. Heat-induced deamidation, dephosphorylation and breakdown of caseinate. Int. Dairy J. 1999, 9, 237−241. (17) Harland, H. A.; Ashworth, U. S. A rapid method for estimation of whey proteins as an indication of baking quality of nonfat dry-milk solids. Food Res. 1947, 12, 247−251. (18) Mikami, H.; Ishida, Y. Post-column fluorometric detection of reducing sugars in high performance liquid chromatography using arginine. Bunseki Kagaku 1983, 1, E207−E210. (19) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (20) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68, 850−858. (21) Alvarado, R.; Tran, D.; Ching, B.; Phinney, B. S. A comparative study of in-gel digestions using microwave and pressure-accelerated technologies. J. Biomol. Technol. 2010, 21, 148−155. (22) Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 2002, 74, 5383− 5392. (23) Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 2003, 75, 4646−4658. (24) Elias, J. E.; Gygi, S. P. Target-decoy search strategy for mass spectrometry-based proteomics. Methods Mol. Biol. 2010, 604, 55−71. (25) Jou, M. Y.; Du, X.; Hotz, C.; Lönnerdal, B. Biofortification of rice with zinc: assessment of the relative bioavailability of zinc in a Caco-2 cell model and suckling rat pups. J. Agric. Food Chem. 2012, 60, 3650−3657. (26) Hambraeus, L.; Forsum, E.; Abrahamsson, L.; Lönnerdal, B. Automatic total nitrogen analysis in nutritional evaluations using a block digestor. Anal. Biochem. 1976, 72, 78−85. (27) Rudloff, S.; Lönnerdal, B. Solubility and digestibility of milk proteins in infant formulas exposed to different heat treatments. J. Pediatr. Gastroenterol. Nutr. 1992, 15, 25−33. (28) Sandström, B.; Keen, C. L.; Lönnerdal, B. An experimental model for studies of zinc bioavailability from milk and infant formulas using extrinsic labeling. Am. J. Clin. Nutr. 1983, 38, 420−428.

(29) Lönnerdal, B.; Du, X.; Morris, K.; Rai, G.; Waworuntu, R. TGF β2 in human milk and infant formula resists digestion ain a suckling rat pup model. J. Pediatr. Gastroenterol. Nutr. 2010, 50, E199. (30) Lönnerdal, B.; Mendoza, C.; Brown, K. H.; Rutger, J. N.; Raboy, V. Zinc absorption from low phytic acid genotypes of maize (Zea mays L.), barley (Hordeum vulgare L.), and rice (Oryza sativa L.) assessed in a suckling rat pup model. J. Agric. Food Chem. 2011, 59, 4755−4762. (31) Singh, H. Heat stability of milk. Int. J. Dairy Technol. 2004, 57, 111−119. (32) Li, C. P.; Salvador, A. S.; Ibrahim, H. R.; Sugimoto, Y.; Aoki, T. Phosphorylation of egg white proteins by dry-heating in the presence of phosphate. J. Agric. Food Chem. 2003, 51, 6808−6815. (33) Li, C. P.; Hayashi, Y.; Enomoto, H.; Hu, F.; Sawano, Y.; Tanokura, M.; Aoki, T. Phosphorylation of proteins by dry-heating in the presence of pyrophosphate and some characteristics of introduced phosphate groups. Food Chem. 2009, 114, 1036−1041. (34) Scanff, P.; Savalle, B.; Miranda, G.; Pelissier, J. P.; Guilloteau, P.; Toullec, R. In vivo gastric digestion of milk proteins. Effect of technological treatments. J. Agric. Food Chem. 1990, 38, 1623−1629. (35) Siciliano, R.; Rega, B.; Amoresano, A.; Pucci, P. Modern mass spectrometric methodologies in monitoring milk quality. Anal. Chem. 2000, 72, 408−415. (36) Lund, M. N.; Olsen, K.; Sørensen, J.; Skibsted, L. H. Kinetics and mechanism of lactosylation of α-lactalbumin. J. Agric. Food Chem. 2005, 53, 2095−2102. (37) Arena, S.; Renzone, G.; Novi, G.; Paffetti, A.; Bernardini, G.; Santucci, A.; Scaloni, A. Modern proteomic methodologies for the characterization of lactosylation protein targets in milk. Proteomics 2010, 10, 3414−3434. (38) Fogliano, V.; Monti, S. M.; Visconti, A.; Randazzo, G.; Facchiano, A. M.; Colonna, G.; Ritieni, A. Identification of a βlactoglobulin lactosylation site. Biochim. Biophys. Acta 1998, 1388, 295−304. (39) Leonil, J.; Molle, D.; Fauquant, J.; Maubois, J. L.; Pearce, R. J.; Bouhallab, S. Characterization by ionization mass spectrometry of lactosyl β-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose-binding site. J. Dairy Sci. 1997, 80, 2270−2281. (40) Ahmed, N.; Mirshekar-Syahkal, B.; Kennish, L.; Karachalias, N.; Babaei-Jadidi, R.; Thornalley, P. J. Assay of advanced glycation endproducts in selected beverages and food by liquid chromatography with tandem mass spectrometric detection. Mol. Nutr. Food Res. 2005, 49, 691−699. (41) Pereda, J.; Ferragut, V.; Quevedo, J. M.; Guamis, B.; Trujillo, A. J. Heat damage evaluation in ultra-high pressure homogenized milk. Food Hydrocolloids 2009, 23, 1974−1979. (42) Nangpal, A.; Reuter, H. Reference diagram for furosine content in UHT milk. Kieler Milchw. Forsch. 1990, 42, 77−86. (43) Dupont, D.; Mandalari, G.; Mollé, D.; Jardin, J.; RoletRépécaud, O.; Duboz, G.; Léonil, J.; Mills, C. E.; Mackie, A. R. Food processing increases casein resistance to simulated infant digestion. Mol. Nutr. Food Res. 2010, 54, 1677−1689. (44) Desrosiers, T.; Bergeron, G.; Savoie, L. Effect of heat treatments on in vitro digestibility of delactosed whey protein as determined by the digestion cell technique. J. Food Sci. 1987, 52, 1525−1528. (45) Kitabatake, N.; Kinekawa, Y. Digestibility of bovine milk whey protein and β-lactoglobulin in vitro and in vivo. J. Agric. Food Chem. 1998, 46, 4917−4923. (46) alKanhal, H. A.; al-Othman, A. A.; Hewedi, F. M. Changes in protein nutritional quality in fresh and recombined ultra high temperature treated milk during storage. Int. J. Food Sci. Nutr. 2001, 52, 509−514. (47) Lacroix, M.; Léonil, J.; Bos, C.; Henry, G.; Airinei, G.; Fauquant, J.; Tomé, D.; Gaudichon, C. Heat markers and quality indexes of industrially heat-treated [15N] milk protein measured in rats. J. Agric. Food Chem. 2006, 54, 1508−1517. (48) Hiller, B.; Lorenzen, P. C. Surface hydrophobicity of physicochemically and enzymatically treated milk proteins in relation 4184



Article

to techno-functional properties. J. Agric. Food Chem. 2008, 56, 461− 468. (49) Sarwar, G.; Peace, R. W.; Botting, H. G. Differences in protein digestibility and quality of liquid concentrate and powder forms of milk-based infant formulas fed to rats. Am. J. Clin. Nutr. 1989, 49, 806−813. (50) Sarwar, G.; Peace, R. W. The protein quality of some enteral products is inferior to that of casein as assessed by rat growth methods and digestibility-corrected amino acid scores. J. Nutr. 1994, 124, 2223−2232. (51) Rutherfurd, S. M.; Moughan, P. J. Digestible reactive lysine in selected milk-based products. J. Dairy Sci. 2005, 88, 40−48. (52) Ford, J. E.; Hurrell, R. F.; Finot, P. A. Storage of milk powders under adverse conditions. 2. Influence on the content of water-soluble vitamins. Br. J. Nutr. 1983, 49, 355−364.

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