Effects of Industrial Heating Processes of Milk-Based Enteral Formulas

Article pubs.acs.org/JAFC

Effects of Industrial Heating Processes of Milk-Based Enteral Formulas on Site-Specific Protein Modifications and Their Relationship to in Vitro and in Vivo Protein 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, Kanagawa Pref. 252-8583, Japan

‡

ABSTRACT: Heat treatments are applied to milk and dairy products to ensure their microbiological safety and shelf lives. Types of heating processes may have different effects on protein modifications, leading to different protein digestibility. In this study, milk-based liquid nutritional formulas (simulating enteral formulas) were subjected to steam injection ultra-high-temperature treatment or in-can sterilization, and the formulas were investigated by proteomic methods and in vitro and in vivo digestion assays. Proteomic analyses revealed that in-can sterilization resulted in higher signals for Nε-carboxymethyllysine and dephosphorylation of Ser residues in major milk proteins than in steam-injected formula, reflecting the more severe thermal process of in-can sterilization. In vitro and in vivo digestion assays indicated that steam injection improved protein digestibility, supposedly by denaturation, while the improvement seemed to be overwhelmed by formation of aggregates that showed resistance to digestion in in-can sterilized formula. Adverse effects of heat treatment on protein digestibility are more likely to be manifested in milk-based formulas than in cow’s milk. Although the differences might be of limited significance in terms of amino acid bioavailability, these results emphasize the importance of protein quality of raw materials and selection of heating processes. KEYWORDS: enteral formula, heat treatment, milk proteins, protein digestibility, protein modification, mass spectrometry

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lysinoalanine and lanthionine (Figure 1e,f).5,6 These crosslinked products also inhibit enzymatic proteolysis, thereby decreasing protein digestibility. Furthermore, disulfide bond interchanges occur between proteins,7 and some of the nonnative complexes resist digestion.8 Characterization of protein modifications has therefore been of nutritional interest in terms of evaluation of protein quality.9−11 We previously investigated proteins in cow’s milk treated by various industrial heating processes, using a proteomic technique and established methods for in vitro and in vivo digestion,12 and found that industrial heating may improve protein digestibility by denaturation but the improvement is likely offset by heat-derived modifications resulting in decreased protein digestibility. However, adverse effects of heat treatment on protein digestibility seem more pronounced in milk-based infant/enteral formula than in cow’s milk.13−16 We assumed that this discrepancy could be explained by the previous observations that protein modifications were more extensive in formulas than in cow’s milk,9,10,17 but direct comparisons of the relationship between protein modifications and protein digestibility have rarely been made for infant/enteral formulas. In this study, milk-based enteral formulas, with milk proteins derived from skim milk powder and sodium caseinate, were manufactured using two types of industrial heat processes: steam injection UHT treatment and in-can sterilization. These formulas as well as unheated formula were subjected to proteomic

INTRODUCTION Milk protein has high nutritional value and is therefore often used in infant/enteral formulas. Raw materials other than milk protein generally include carbohydrates such as lactose, maltodextrin, and oligosaccharides, vegetable fat or the combination of vegetable fat and fish oil, vitamins, and minerals; they are blended into an emulsified formula, with heat treatment to ensure biological safety and to prolong shelf life. Some formula products are sold in liquid (ready-to-use) form, commonly exposed to ultra-high-temperature (UHT) treatment (135−150 °C for 2−6 s) or in-can sterilization (>110 °C for 10−30 min), and the combination with aseptic packaging makes them distributable at ambient temperature for months. Effects of heat treatment on protein digestibility/amino acid bioavailability have been investigated extensively.1 Heat treatment principally unfolds tertiary and secondary structures of protein and increases the digestibility.2 However, nonenzymatic post-translational modifications occur concomitantly in proteins. The Maillard reactions modify the side chains of proteins; ε-amino groups of lysine (Lys) residues primarily react with reducing sugars to form the Amadori products such as lactulosyllysine and fructosyllysine (Figure 1a,b), which subsequently undergo oxidization to form Nε-carboxymethyllysine (CML; Figure 1c).3 Blocking of the ε-amino groups of Lys residues hinders tryptic digestion as well as the action of other digestive enzymes, thereby impairing protein digestibility.4 Further, the blocked Lys is no longer bioavailable, reducing the biological value of protein.5 In addition, β-elimination reactions proceed in phosphoserine and cystine/cysteine (Cys) residues to yield dehydroalanine (Figure 1d), which subsequently reacts with Lys and Cys residues to form cross-linked products such as © 2015 American Chemical Society

Received: Revised: Accepted: Published: 6787

May 2, 2015 July 8, 2015 July 10, 2015 July 10, 2015 DOI: 10.1021/acs.jafc.5b02189 J. Agric. Food Chem. 2015, 63, 6787−6798

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of heat-derived protein modifications. Lactulosyllysine (a) and fructosyllysine (b) are early Maillard reaction products, which are subsequently oxidized to Nε-carboxymethyllysine (CML; c) as an advanced Maillard reaction product. Dehydroalanine (d) is the precursor of cross-linked products, lysinoalanine (e), and lanthionine (f).

analysis and in vitro/in vivo digestion as done previously,12 and the relationship between protein modifications and protein digestibility is discussed.

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layer was removed. The tiny pellet, formed after centrifugation, was thoroughly mixed with the defatted supernatant. We confirmed, using a Dumas method (Sumigraph NC-220F, Sumica Chemical Analytic Center, Tokyo, Japan) that no notable loss in protein content (as measured by nitrogen (N) amount) was accompanied by removal of fat layer. Protein concentrations were determined by the Bradford method,18 and were adjusted to 7.5 g/L with Milli-Q water. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing/nonreducing conditions to separate the proteins (Bio-Rad, Hercules, CA). Fifty micrograms of protein was loaded for each sample onto Mini-PROTEAN TGX Any kD gel (Bio-Rad). Electrophoresis was run at 120 V for 50 min, and gels were stained with Bio-Safe Coommassie Stain (Bio-Rad). Proteomic Analysis. Proteomic analysis was conducted at the Proteomic Core Facility at the University of California, Davis, as described previously,12 in order to site-specifically semiquantify lactulosyllysine, fructosyllysine, CML, dehydroalanine formed from phosphoserine and Cys, and phosphorylation of serine (Ser) residues in major milk proteins such as α-lactalbumin (α-LA), β-lactoglobulin (β-LG), and CNs. In-Gel Digestion. In-gel reduction, alkylation, and tryptic digestion were done using similar protocols as published previously.19 Briefly, lyophilized formulas were reconstituted with Fisher Optima water (Fisher Scientific, Pittsburgh, PA), loaded onto a Novex 10% Trisglycine gel (Life Technologies, Grand Island, NY), and applied to electrophoresis at 80 V for 20 min. The fraction containing the entire protein band was excised, and the gel piece was washed twice with 100 mM ammonium bicarbonate (AmBic) and dehydrated twice with 100% acetonitrile (ACN). The gel piece was reduced using 10 mM dithiothreitol (in 100 mM AmBic) at 56 °C for 30 min. The gel piece was then alkylated using 55 mM iodoacetamide (in 100 mM AmBic) at room temperature (RT) for 20 min in the dark. The gel piece was washed twice with 100 mM AmBic, dehydrated twice with 100% ACN, and then mechanically dried using vacuum centrifugation. Finally, sequencing grade modified trypsin (Promega, Madison, WI; in 50 mM AmBic) was added in a 1:30 ratio (trypsin:protein) to rehydrate the gel. After replacing the excess solution with 50 mM Ambic, the gel piece 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.

MATERIALS AND METHODS

Milk Samples. Milk-based liquid formulas, simulating enteral formulas, were manufactured at the pilot plant of Morinaga Milk Industry (Zama, Japan) with ingredients such as high-heat skim milk powder, sodium caseinate, vegetable fat, maltodextrin, vitamins, minerals, and emulsifiers. The ratio of casein (CN) to whey protein was approximately 7:1. The heating conditions were set to mimic those for the manufacturer’s enteral formula products (Table 1).

Table 1. Heat Treatment of Milk-Based Liquid Formulasa treatment

holding temp/time

F0 value

steam injection in-can sterilizationb

151 °C for 4 s 123 °C for 8 min

65 20

a

Heating conditions were set by reference to those of the manufacturer’s commercial products. bThis heat treatment involves slow rates of temperature rise/fall, which also contributed to the F0 value. The heated formulas were homogenized at 30 MPa, followed by lyophilization. The lyophilized formula powders were stored at 4 °C until use. Nutrient composition of the products was determined by the Analytical Research Center of Morinaga Milk Industry (Table 2).

Table 2. Nutrient Composition of Lyophilized Enteral Formulas component

g/100 g

protein fat carbohydrate ash water

18.1 12.3 65.5 3.2 0.9

Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Milk Samples. Six grams of lyophilized formulas were reconstituted with 24 mL of Milli-Q water (Millipore, Billerica, MA), subjected to centrifugation at 8500g for 30 min twice, and the upper fat 6788

DOI: 10.1021/acs.jafc.5b02189 J. Agric. Food Chem. 2015, 63, 6787−6798

Article


Figure 2. Electrophoresis of proteins in milk-based enteral formulas. Formula samples were applied to SDS-PAGE analysis under (a) nonreducing and (b) reducing conditions. Fifty micrograms of protein was loaded on a gel, and the gel was stained with Bio-Safe Coommassie Stain. the digestion times,24 and pH of the gastric digestion,24−28 had been reviewed carefully and selected accordingly. 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 (Sigma-Aldrich; 2% in 1 mM HCl) was added to the sample in a 1:12.5 ratio (pepsin:protein). Samples were placed in a water bath equipped with an incubation shaker (Yamato Scientific, Tokyo, Japan) in the dark at 140 rpm at 37 °C for 30 min. After incubation, 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 a water bath equipped with an incubation shaker in the dark at 140 rpm at 37 °C for 60 and 240 min. After 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 digestion assays was determined using a Dumas method (Sumigraph NC-220F) and with a procedure as described previously.29 Briefly, total N and nonprotein N [NPN; soluble fraction after trichloroacetic acid (TCA; 12% final concentration30) 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 (i.e., pepsin digestion at pH 2.0/4.0 for 30 min, and pepsin digestion at 2.0/4.0 for 30 min + pancreatin digestion at pH 7.0 for 60 min). In Vivo Digestion. A suckling rat pup model was used to investigate in vivo digestion of milk samples as described previously.12,31 The study was approved by the Animal Research Committee of Morinaga Milk Industry (protocol no. 14-006). Sprague−Dawley rats with litters of 12 pups (6 males + 6 females) were obtained commercially (Charles River Japan, Yokohama, Japan), maintained in polycarbonate cages with wood shavings in a temperature-, humidity-, and light-controlled facility (24 °C, humidity 55%, and 12 h light/dark circle), and allowed to consume water and standard rat chow diet (F-2, Funabashi Farm, Funabashi, Japan) ad libitum. Pups were randomly assigned as 3 males + 3 females/formula 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 unheated, steam-injected, or in-can sterilized formula (reconstituted and defatted as above; protein concentration was adjusted to 30 g/L), in a 1:36 v/w ratio (formula (mL)/body weight (g)), as done in our previous cow’s milk study.12 Pups were euthanized with CO2 asphyxiation at 0.5, 1, or 2 h after intubation. As experiment control, 3 male pups + 2 female pups

Liquid Chromatography coupled with tandem mass spectrometry (LC-MS/MS). 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). 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 buffer A (0.1% formic acid) and B (100% ACN), with 2% B to 35% B over 70 min, 35% B to 80% B for 5 min, 80% B for 2 min, and 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 normalized collision energy of 27% was used for fragmentation. A 5 s duration was used for the dynamic exclusion. Data Analysis. 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 digestion enzyme trypsin. Iodoacetamide derivative of Cys was specified in X! Tandem as a fixed modification. One of the targeted modifications [lactulosyllysine (+324.105647), fructosyllysine (+162.052824), CML (+58.005479), phosphorylation on Ser, threonine and tyrosine (+79.966331), dehydration of Ser (−18.010565), or dehydroalanine on Cys (−33.987721)], as well as acetylation of the N-terminus, ammonialoss of the N-terminus, pyro-glutamination of the N-terminus, and oxidation of methionine and tryptophan were specified in X! Tandem as variable modifications. Scaffold (version Scaffold_4.1.1, Proteome Software Inc., Portland, OR) and Skyline (version 1.4.0.4421, MacCoss Lab, Department of Genome Science, University of Washington) were used to validate MS/MS based peptide and protein identifications,20−22 and to determine retention time and peak areas of peptides with targeted modifications, respectively. 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), and they were summed up to obtain higher confidence. For normalization between samples, the summed peak area was normalized by dividing it with the exclusive spectrum counts of all modified and nonmodified peptides derived from the parental protein, in Scaffold. In Vitro Digestion. Powdered formulas were reconstituted with Milli-Q water and defatted, and protein concentration was adjusted to 7.5 g/L as above. Six mL of samples were aliquoted into 15 mL centrifuge tubes. Samples were subjected to in vitro digestion as described previously.12 Conditions for the in vitro digestion in general,23 6789


Article

Journal of Agricultural and Food Chemistry Table 3. Lactulosyllysine in Milk Proteinsa protein

residue

amino acid

α-LA

59−79

Lys62

109−122

Lys114

β-LG

αs1-CN

1−14

Lys8

41−60 76−83 76−91

Lys47 Lys77 Lys83

78−91 84−101

Lys83 Lys91

125−138

Lys135

139−148

Lys141

3−22 35−42

Lys7 Lys36

Table 4. Fructosyllysine in Milk Proteinsa

charge

steam injectedb

in-can sterilizedb

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

0.9 1.0 1.1 0.8

1.3 1.6 0.8 0.7

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

2.2 6.5 9.4 1.1 1.0 0.7 0.9 2.3 1.0 1.1 1.4 1.1 3.6 3.7

1.0 2.9 6.1 0.9 0.3 0.4 0.5 1.8 0.4 0.6 0.8 0.7 1.8 1.9

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

6.0 1.2 1.8 1.8 2.2 4.2 2.1 1.6

30.3 1.5 1.7 1.1 0.8 14.8 8.1 1.7

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

2.4 1.9 1.5 2.5 6.1 2.3 1.4 1.2

1.7 0.9 1.3 2.4 5.7 0.8 1.3 1.2

80−90

Lys82

104−119

Lys105

120−132

Lys124

22−32 25−41 151−160 171−181

Lys24 Lys33 Lys152 Lys173

172−181 198−205

Lys173 Lys199

β-CN

170−183

Lys176

[2+] [3+]

1.5 1.6

2.1 2.2

κ-CN

22−34

Lys24

61−68 69−97

Lys63 Lys86

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

1.0c 1.0c 1.8 5.9

1.1c 1.0c 2.0 2.2

αs2-CN

charge

steam injectedb

in-can sterilizedb

Lys8 Lys47 Lys47

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

2.1 4.4 2.7

0.5 3.8 2.7

Lys3 and 7 Lys7 Lys7 Lys83

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

6.2 3.3 3.3 1.4 1.7 1.0c 1.0c 2.4 1.4

29.8 3.0 1.6 2.1 2.1 5.4c 0.9c 1.6 1.0

protein

residue

amino acid

β-LG

1−14 40−60 41−60

αs1-CN

3−22 4−22 6−22 80−90 104−119

Lys105

120−132

Lys124

αs2-CN

79−91 113−125 151−160 198−205

Lys80 Lys113 Lys152 Lys199

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

6.0 23.5 1.6 5.8

2.4 14.5 1.2 1.2

β-CN

28−40

Lys30

κ-CN

170−183 23−34

Lys176 Lys24

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

2.3 3.0 20.4 3.1

7.6 15.0 22.6 1.9

a

Formula samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying tryptic peptides containing fructosyllysine in major milk proteins. bPeak areas of the specific peptides are normalized to unheated formula. cPeak areas are normalized to steam-injected formula because those of unheated formula were below the detection limit. (5 μg of protein) were applied to reducing SDS-PAGE, and proteins were transferred to a Trans-Blot Turbo PVDF membrane (Bio-Rad). The membrane was blocked with 2% bovine serum albumin (in PBS containing 0.05% Tween-20 (PBST)) for 45 min at RT. After the membrane was washed three times with PBST, it was probed with rabbit anti-bovine β-LG antibody (Bethyl Laboratories Inc., Montogomery, TX) in a 1:20000 dilution (antibody:blocking buffer) at RT for 45 min. After washing three times with PBST, the membrane was probed with donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase (GE Healthcare, Pewaukee, WI) in a 1:20000 dilution (antibody:blocking buffer) at RT for 45 min. After washing three times with PBST, β-LG was detected by Pierce ECL Western Blotting Substrate (Thermo Scientific), with the chemiluminescence signal imaged by a ChemiDoc XRS+ (Bio-Rad). Blood samples were taken by cardiac puncture using syringes treated with ethylenediaminetetraacetic acid disodium (Sigma-Aldrich). They were subjected to centrifugation at 1700g for 10 min, and upper plasma layers were collected and stored at −80 °C until analysis. Amino acid patterns were analyzed for the plasma samples. The samples were subjected to TCA (5% final concentration) precipitation, and soluble fractions were filtrated through 0.2 μm PVDF filters (Thomson Instrument Company, Oceanside, CA). The filtrates were applied to an amino acid analyzer (L-8900, Hitachi High-Technologies, Tokyo, Japan). Statistical Analysis. Data on in vitro protein digestibility and coagulates in stomach (weight and pH) and plasma amino acid concentrations obtained from in vivo digestion assays were analyzed by one-way ANOVA and post-tested by Tukey test using JMP software (version 5.1.1, SAS Institute, Cary, NC), and significance was demonstrated at p < 0.05.

a

Formula samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying trypic peptides containing lactullosyllysine in major milk proteins. bPeak areas of the specific peptides are normalized to unheated formula. cPeak areas are normalized to steam-injected formula because those of unheated formula were below the detection limit. were separated from their dams for 6 h and euthanized with CO2 asphyxiation without formula samples administered. Coagulates in the stomach were collected and weighed. 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. β-LG was set as an index of in vivo protein digestibility, as done previously.12 The perfusates

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RESULTS Electrophoresis of Milk Proteins. Formula samples were applied to SDS-PAGE, and the band patterns were compared 6790


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in-can sterilized formulas had higher signals for lactulosyllysine and fructosyllysine than unheated formula at many modified sites in major milk proteins, but no clear trend was observed between steam injected formula and in-can sterilized formula. Similarly, steam injected and in-can sterilized formulas had higher signals for CML at most of the modified sites in CNs in comparison with unheated formula, but the signals were principally higher in in-can sterilized formula than in steam-injected formula. As for targeting cross-linked structures, lysinoalanine and lanthionine are not compatible with the analysis of Maillard reaction products, and therefore their precursor, dehydroalanine on Ser and Cys residues, was analyzed instead (Table 6).

(Figure 2). Proteins smeared in a wide range of molecular weight for all three formulas under reducing/nonreducing conditions. While band patterns of unheated and steam injected formulas were similar, in-can sterilized formula showed a notably different band pattern; bands of intact major milk proteins such as α-LA, β-LG, and CNs decreased and a smear band above 37 kDa increased. All three formulas exhibited strong smear bands around 250 kDa under nonreducing conditions, during which disulfide bonds are kept intact. In contrast, under reducing conditions, a smear band at the high molecular weight fraction was observed only for in-can sterilized formula. These results suggest that milk proteins are associated prior to heat treatment, and that in-can sterilization caused newly formed protein modifications that are considered noncovalent interactions other than disulfide bonds. Proteomic Analysis. Formula samples were applied to LC-MS/MS analysis following in-gel tryptic digestion for identifying and semiquantifying modifications involved in protein digestibility. It should be noted that some peptides containing the specified modifications were identified but could not be quantified probably due to low quantity. Formation of lactulosyllysine and fructosylysine was targeted as early Maillard reaction products (Tables 3 and 4) because raw materials include skim milk powder and maltodextrin, which contain reducing sugars such as lactose and glucose. Formation of CML was also targeted as an advanced Maillard reaction product (Table 5). Maillard reaction products could be semiquantified in all the major milk proteins. Steam injected and

Table 6. Dehydroalanine Derivatives on Ser and Cys Residues in Milk Proteinsa

charge

steam injectedb

in-can sterilizedb

protein

residue

α-LA

1−10 59−79

Lys5 Lys62

[2+] [3+]

0.7 0.9

0.8 0.5

1−14 125−138

Lys8 Lys135

[2+] [3+]

1.8 0.8

1.8 0.5

4−22

Lys7

23−36

Lys34

80−90

Lys83

120−132

Lys124

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

3.4 2.3 25.1 32.8 0.7 1.0 1.4 1.8

5.1 2.6 81.6 72.0 0.6 0.8 2.4 1.1

33−45

Lys41

171−181 198−205

Lys173 Lys199

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

2.2 2.0 2.1 1.9

3.1 2.6 4.7 3.5

β-CN

168−176 170−183

Lys169 Lys176

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

0.8 1.8 1.4

5.1 2.3 1.1

κ-CN

22−34

Lys24

[2+] [3+]

2.6 1.8

4.0 2.7

β-LG

αs1-CN

αs2-CN

residue

amino acid

charge

steam injectedb

in-can sterilizedb

α-LA

63−79 63−79

Ser69 Cys77

[2+] [3+]

1.2 2.8

0.6 5.7

β-LG

145−162

Ser150

[2+]

0.8

0.4

80−90

Ser88

106−119

Ser115

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

3.4 4.6 3.3

2.2 2.8 2.5

αs2-CN

25−41 33−45

Ser31 Cys36

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

1.7 0.6 1.4

1.1 1.2 2.4

β-CN

168−176

Ser168

[3+]

3.2

4.3

κ-CN

64−86 69−83

Ser69 Ser82

[2+] [2+]

0.7 1.2

0.4 0.6

αs1-CN

Table 5. Nε-Carboxymethyllysine in Milk Proteinsa amino acid

protein

a

Formula samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying tryptic peptides containing dehydroalanine in major milk proteins. bPeak areas of the specific peptides are normalized to unheated formula.

Formation of dehydroalanine has been reported to reflect thermal history to a considerable extent.32 It was identified at 18 Ser/Cys residues in major milk proteins and could be quantified at 11 of these sites. Steam-injected and in-can sterilized formulas had higher signals than unheated formula at about half of the identified sites. No clear trend was observed between steam injected formula and in-can sterilized formula. In addition to the above-mentioned modifications, phosphorylation of Ser was semiquantified (Table 7), as it may provide further information in the context of heat treatment of proteins.33 In-can sterilized formula had the lowest signals at all the modified sites of CNs, while steam injection did not appear to have any significant effects on phosphorylation. Thus, dephosphorylation of CNs was caused only by in-can sterilization. In Vitro Digestion of Formula Samples. SDS-PAGE analysis of formula samples after in vitro digestion is shown in Figure 3. While proteins were digested to a limited extent by pepsin at pH 4.0, peptic digestion proceeded at pH 2.0 and digestion patterns were quite similar for the three formulas except for smear bands resisting digestion in in-can sterilized formula. Protein digestion was facilitated extensively by pancreatin treatment. However, undigested smear bands remained more pronounced in in-can sterilized formula compared with the other

a

Formula samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying tryptic peptides containing Nε-carboxymethyllsine in major milk proteins. bPeak areas of the specific peptides are normalized to unheated formula. 6791


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Journal of Agricultural and Food Chemistry Table 7. Phosphorylation of Ser in Milk Proteinsa protein

residue

amino acid

charge

steam injectedb

in-can sterilizedb

αs1-CN

35−58 37−58

Ser41, 46, and 48 Ser41, 46, and 48

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

1.2 0.9 0.8 1.7 1.0 1.4 1.4 1.3 1.5

0.4 0.3 0.3 0.7 0.5 0.7 0.7 1.0 1.1

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

1.2 1.3 1.1 1.5 1.2 1.1

0.3 0.3 0.5 0.6 0.2 0.5

[2+] [3+]

0.7 1.2

0.4 0.9

αs2-CN

β-CN

43−58 103−119 104−119

Ser46 and 48 Ser115 Ser115

106−119

Ser115

25−41

Ser31

126−137

Ser129 and 131

137−150 138−149

Ser143 Ser143

30−48

Ser35

a

Formula samples were treated with trypsin, followed by LC-MS/MS analysis for identifying and semiquantifying tryptic peptides containing phosphoserine in major milk proteins. bPeak areas of the specific peptides are normalized to unheated formula.

Figure 3. Electrophoresis of proteins in milk-based enteral formulas after in vitro digestion. Formula samples were subjected to in vitro digestion using pepsin and pancreatin. Both infant (a-c) and adult (d-f) digestion conditions were simulated by adopting different pH values during the peptic digestion. Fifty micrograms of protein (before digestion) was loaded on a gel, and the gel was stained with Bio-Safe Coommassie Stain. Pep and Pan denote pepsin and pancreatin, respectively.

In Vivo Digestion of Milk Samples. White coagulates, likely acid-precipitated CNs, were observed in stomachs of the rat pups after intubation with the formula samples. Weights of the coagulates decreased over time (Figure 5a), but we observed no significant difference, at any time point, among the groups fed the three formulas. The pHs were also quite similar

two formulas, which could be recognized even after digestion for 240 min. In vitro protein digestibility was determined for some of the digestion conditions using a Dumas method in combination with TCA precipitation (Figure 4). Steam-injected formula showed the highest protein digestibility for all the digestion conditions, although the differences did not reach statistical significance. 6792


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Figure 4. Digestibility of proteins in milk-based enteral formulas after in vitro digestion. Protein digestibility was defined as the increase in NPN after in vitro digestion, which was determined by a combination of TCA precipitation and a Dumas method. Each digestion and the subsequent procedures were conducted six times. No significant difference was observed among the formulas for the same digestion conditions. Pep and Pan denote pepsin and pancreatin, respectively.

Figure 5. Coagulates in stomach of suckling rat pups. Rat pups fed different formula samples were killed at 0.5, 1, and 2 h after intubation, and coagulates in the stomach were obtained. Weight (a) and pH (b) were measured, and data are expressed as means ± SD (n = 3 males + 3 females). No significant difference was observed among the formula-fed pups at any time point. 6793


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Figure 6. Western blotting of β-LG in upper/lower intestine perfusates from suckling rat pups. Rat pups fed different formula samples were killed at 0.5, 1, and 2 h after intubation, and the upper intestines (a) and lower intestines (b) were perfused with PBS containing protease inhbitor. Five micrograms of protein was loaded and β-LG was detected by Western blotting as an index of in vivo protein digestibility.

antibody appeared to react with endogenous proteins in rats as well as partially digested CNs (data not shown), which hindered interpretation of the results and no conclusion could be drawn from the assays. To further explore protein quality, as part of evaluating amino acid bioavailability, plasma amino acid patterns were analyzed. In pups fed unheated formula, total and essential plasma amino acids increased at 0.5 h, decreased at 1.0 h, and stayed at the similar level at 2.0 h (Figure 7). This trend held true for most of the respective amino acids (Table 8). In pups fed steam-injected and in-can sterilized formulas, the increases in total and essential plasma amino acid levels were less responsive and the concentrations were also lower for most of the respective amino acids than those in unheated formula-fed pups at 0.5 h; the differences were significant for leucine in steam-injected formula-fed pups and isoleucine in in-can sterilized formulafed pups. When it comes to Lys, the amino acid of major interest in the context of proteomic analysis, the unheated formula-fed pups exhibited the highest plasma level, followed by steam-injected and in-can sterilized formula-fed pups. However, the differences did not reach statistical significance.

among the three formulas and decreased only slightly over time (Figure 5b). Western blotting of β-LG was conducted for upper intestine perfusates (Figure 6a,b) in order to assess in vivo digestibility as done previously.12 Each time point was assayed six times, using three male and three female pups per sample, and we observed no trend in terms of sex difference. While β-LG was barely observed in intact form in the perfusates obtained, fragmented forms and/or smear bands, supposedly degradation products from β-LG, were detected at fractions with molecular weights lower than that of β-LG. The smear bands generally became weaker over time. At 0.5 and 1.0 h, smear bands were weaker for the steam-injected formula-fed pups in 5 out of the 6 pups assayed. In contrast, at 2.0 h, results were inconsistent and the trend observed at 0.5 and 1.0 h was not confirmed. Western blotting of β-LG was also conducted for lower intestine perfusates. Like upper intestine perfusates, bands were observed principally in smear forms (Figure 6c,d). Bands were less intense than those in upper intestine perfusates, and they also became weaker over time. The bands for steam-injected formula were the weakest in all the six assays at 0.5 and 1.0 h, while the bands were hardly recognized and the trend was not observed at 2.0 h. Further, western blotting of CNs was conducted using a commercial anti-CN antibody that recognizes αs- and β-CNs because they dominate proteins in the formulas. However, the

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DISCUSSION Aiming to elucidate the relationship between protein modifications and protein digestibility of milk-based infant/enteral 6794


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unheated enteral formula, while it was quantifiable only at 3 Ser residues in raw milk proteins of our previous study,12 although some caution is needed for making a direct comparison between the two independent studies. Thus, proteins were associated with each other to a considerable extent in ingredients of enteral formulas, which may obscure the existence of newly formed aggregates caused by subsequent industrial heating (especially by steam injection) as shown in our electrophoretic analysis. Proteomic analysis further elucidated effects of heat treatment on proteins. Both steam injection and in-can sterilization progressed the Maillard reaction in major milk proteins, and the highest signals for CML were observed at many Lys residues of CNs in in-can sterilized formula. These results indicate that incan sterilization facilitates the reaction to a greater extent, resulting from its more severe thermal history including slow rates of temperature rise/fall and longer hold time at high temperature. The severity of the in-can sterilization process is also likely to be manifested as a progression of dephosphorylation of CNs in the formula, while this was not found in steaminjected formula. This notion may also be supported by a study of Birlouez-Argon et al.; they reported higher CML amounts in sterilized infant formula than in UHT formula.17 In contrast to CML and dephosphorylation, formation of lactulosyllysine, fructosyllysine, and dehydroalanine generally proceeded after both steam injection and in-can sterilization, and we observed only minor and inconsistent difference between the two heat treatments. Both lactulosyl/fructosyllysine and dehydroalanine are intermediates of CML and cross-linked products, respectively.3,6,36 Rufián-Henares et al. formulated an infant/enteral formula-resembling mixture, dissolving whey protein isolate and lactose/maltodextrin in phosphate buffer, and they reported a time-induced increase and subsequent decrease in furosine contents after heating the mixture at 140 °C. They concluded that formation of Amadori products would first be facilitated, which is followed by degradation into advanced Maillard reaction products.37 van Boekel reported that dehydroalanine formation paralleled heating time in β-CN solutions at 110 and 120 °C, but the amounts got saturated over time when heated at 130 and 140 °C,32 suggesting its conversion into cross-linked products. Thus, formation of these intermediate structures may not always parallel intensity of the heat treatment. Electrophoretic analyses of in vitro digestion assays demonstrated that in-can sterilized formula had smeared bands that persistently resisted pepsin/pancreatin digestion. Protein digestibility determined by a Dumas method in combination with TCA precipitation showed that steam-injected formula exhibited higher digestibility than unheated formula, while milk proteins in in-can sterilized formula were digested to an extent similar to those in unheated formula. Similarly, although it was only assessed by immuno-reactive materials for β-LG in in vivo digestion assays, β-LG in steam-injected formula showed more extensive digestion than in unheated formula, while β-LG in in-can sterilized formula was digested to an extent similar to that in unheated formula. Thus, as reported previously,13 UHT treatment appears superior to in-can sterilization in terms of protein digestibility; steam injection principally improved protein digestibility, possibly by denaturation, but the heat-induced improvement in protein digestibility may be overwhelmed by adverse effects caused by formation of proteolytic-resistant aggregates to a greater extent in in-can sterilized formula. In contrast, it seems that both steam injection treatment and in-can sterilization equally had negative impact on amino acid bioavailability, as manifested by lower levels in plasma amino acids

Figure 7. Plasma total and essential amino acid concentration in suckling rat pups. Rat pups fed different formula samples were killed at 0.5, 1, and 2 h after intubation, and blood was collected by cardiac puncture. Plasma was separated by centrifugation and applied to amino acid analysis. Total and essential amino acid levels are expressed as means ± SD (n = 3 males + 3 females) except for baseline, where it is shown as means ± SD (n = 3 male + 2 female pups). No significant difference was observed among the formula-fed pups at any time point.

formulas, enteral formulas with two types of industrial heating processes were industrially prepared, and these formulas as well as unheated formula were investigated as we did previously for cow’s milk.12 Electrophoresis of the three formulas showed that in-can sterilization greatly influenced protein associations, while steam injection did not exert any significant effects. This observation was partly in disagreement with our previous study,12 where both in-can sterilization and steam injection of raw milk affected protein band patterns (although the former had greater effects). This discrepancy may partly be attributed to protein damage potentially present in raw materials for enteral formulas; proteins are generally heat-treated and sometimes subjected to long terms of storage as raw materials. In the case of CN ingredients, industrial isolation involves isoelectric precipitation followed by neutralization at alkaline pH. These processes/conditions facilitate protein modifications such as formation of Amadori products and cross-linked products.34,35 Proteomic analysis is also likely to unravel protein damage in raw materials; dehydroalanine formation could be identified and quantifiable at 8 Ser and 2 Cys residues in proteins of 6795


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Journal of Agricultural and Food Chemistry Table 8. Mean Concentration of Plasma Amino Acids of Suckling Rat Pupsa 0.5 h (μmol/mLc) baseline (μmol/mLb) Essential Val Leu Ile Lys Met Phe Trp His Thr Nonessential Tyr Asp Asn Ser Glu Gln Pro Gly Ala Arg

1.0 h (μmol/mLc)

2.0 h (μmol/mLc)

unheated

steam injected

in-can sterilized

unheated

steam injected

in-can sterilized

unheated

steam injected

in-can sterilized

0.143 0.118 0.079 0.422 0.049 0.073 0.059 0.113 0.214

0.168 0.126 0.088 0.479 0.054 0.085 0.075 0.126 0.234

0.146 0.097d 0.074 0.425 0.054 0.072 0.067 0.117 0.239

0.139 0.104 0.069e 0.393 0.057 0.081 0.082 0.114 0.204

0.119 0.069 0.052 0.350 0.048 0.073 0.071 0.112 0.206

0.115 0.074 0.055 0.326 0.043 0.061 0.055 0.100 0.182

0.109 0.063 0.051 0.334 0.045 0.062 0.062 0.102 0.189

0.130 0.085 0.064 0.364 0.054 0.087 0.078 0.107 0.191

0.119 0.081 0.059 0.330 0.052 0.079 0.069 0.124 0.191

0.131 0.090 0.067 0.407 0.054 0.089 0.065 0.106 0.190

0.229 0.017 0.057 0.270 0.117 0.538 0.087 0.384 0.256 0.167

0.254 0.014 0.059 0.256 0.110 0.600 0.105 0.334 0.324 0.193

0.233 0.012 0.057 0.286 0.118 0.545 0.121 0.385 0.342 0.169

0.228 0.013 0.057 0.251 0.118 0.592 0.100 0.344 0.356 0.159

0.233 0.012 0.044 0.240 0.115 0.467 0.084 0.372 0.288 0.136

0.180 0.017 0.044 0.226 0.129 0.437 0.079 0.319 0.273 0.143

0.188 0.012 0.042 0.214 0.107 0.429 0.073 0.326 0.244 0.134

0.210 0.016 0.051 0.241 0.105 0.499 0.078 0.387 0.272 0.132

0.216 0.014 0.048 0.247 0.126 0.467 0.072 0.498 0.275 0.128

0.196 0.016 0.049 0.224 0.115 0.479 0.071 0.366 0.260 0.143

a

Three-letter codes are used for all the amino acids. Note that cysteine (measured as cystine) were below the detection limit in all the plasma samples. bEach amino acid concentration is expressed as mean value (n = 3 male + 2 female pups). cEach amino acid concentration is expressed as mean value (n = 3 male + 3 female pups). dP < 0.05 (vs unheated formula-fed pups at 0.5 h). eP < 0.05 (vs unheated formula-fed pups at 0.5 h).

rates for enteral formulas caused by their poorer protein digestibility. Even though experimental settings for the Western blots were somewhat different, immuno-reactive materials from β-LG could be detected in lower small intestine of formula-fed rats, but not in cow’s milk-fed rats, further supporting the inferiority in protein digestibility of enteral formulas. Differences in protein digestibility between cow’s milk and enteral formulas are further supported by some studies,13,16 and these may be explained by protein damage in raw materials as discussed above34,35 and the fact that proteins are further damaged by subsequent heating in production of formula products. In conclusion, heat treatment of milk protein-based enteral formulas facilitates protein modifications and apparently attenuates protein digestibility and amino acid bioavailability. These adverse effects are more likely to be prominent in enteral formulas in comparison with our previous study on cow’s milk. In-can sterilization may have greater impact on protein modifications and digestibility than steam injection treatment, but the differences might be of limited significance in terms of amino acid bioavailability. It should be noted that the conditions for heat treatments used here may not be representative of UHT treatment and in-can sterilization in general, and the observations reported here may not always be applicable to other similar products. Still, raw materials and types of heat treatment should be carefully selected in terms of protein quality, for achieving better nutritional potential of milk-based enteral formulas.

of rat pups fed these heated formulas at a specific time point compared with those of unheated formula-fed pups. Further, this trend was the case for most of the amino acids (but not for specific amino acids), and an involvement of decreased protein digestibility, rather than damage/modifications of specific amino acids, is therefore suggested. One possible explanation may be that digesta from steam-injected formula, as well as those from in-can sterilized formula, may contain large amounts of small proteolytic-resistant peptides that cannot be detected by SDSPAGE/Western blotting nor be precipitated by TCA (12% final concentration), which would significantly compromise amino acid absorption. However, amino acid bioavailability discussed here was evaluated only by amino acid levels at a single time point, warranting further investigation with expanded experimental conditions. In a proteomic context, the extent of CML formation and dephosphorylation of Ser residues implicates the greatest protein damage in in-can sterilized formula, followed by in steam-injected formula. Progressed formation of proteolyticresistant structures can therefore be suggested, as previously discussed by Cattaneo et al.,34 but it should be remembered that protein modifications such as lysinoalanine and lanthionine per se could not be measured due to technical limitations. When it comes to comparisons between our two studies,12 protein digestibility of milk-based enteral formulas may be inferior to that of cow’s milk, although there are some differences in milk protein composition and experimental procedures. Values for in vitro protein digestibility determined for unheated, steaminjected, and in-can sterilized formulas were principally lower than those for raw, steam-injected, and in-can sterilized cow’s milk. In our in vivo study, rats fed enteral formulas exhibited a slower decrease in weights of coagulates in stomach and insignificant changes in their pH over time in comparison with cow’s milk-fed rats, indirectly suggesting slower gastric emptying

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1-530-752-8347. Fax: +1-530-752-3564. E-mail: [email protected]. 6796


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Journal of Agricultural and Food Chemistry Notes

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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 also want to thank Dr. Brett Phinney and Darren Weber, the 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; CN, casein; N, nitrogen; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; Ser, serine; α-LA, α-lactalbumin; β-LG, β-lactoglobulin; AmBic, ammonium bicarborate; ACN, acetonitrile; RT, room temperature; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; NPN, nonprotein N; TCA, trichloroacetic acid; PBS, phosphate buffered saline; PBST, PBS containing 0.05% Tween-20

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Effects of Industrial Heating Processes of Milk-Based Enteral Formulas

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