In vitro digestion of purified β-casein variants A1, A2, B, and I: Effects on antioxidant and...

1

J. Dairy Sci. 98 :1–12

http://dx.doi.org/ 10.3168/jds.2014-8330

© american Dairy Science association®, 2015 .

ABSTRACT

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain tech-nological properties, such as casein (CN) number and cheese yield. However, reports show that such polymor-phisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pat-tern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A1, A2, B, and I by a combination of cold stor-age, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ = 280 nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the un-digested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In par-ticular, after 60 min of pepsin followed by 5 min of pan-creatic enzyme digestion, one ≈4 kDa peptide with the N-terminal sequence 106H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the 122Ser to 122Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon diges-tion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60 min of pepsin + 120 min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also sig-nificantly increased by digestion, with the B variant reaching the highest inhibitory capacity at the end of

digestion (60 min of pepsin + 120 min of pancreatic enzymes), possibly because of the observed alternative digestion pattern. These results demonstrate that ge-netic polymorphisms affect the digestion pattern and bioactivity of milk proteins. Moreover, their capacity for radical scavenging and ACE inhibition is affected by digestion. Key words: milk protein , β-casein , genetic polymor-phism , bioactive peptide

INTRODUCTION

Bioactive peptides are defined as protein fragments that interact with, or have an effect on, bodily tissues or functions and thus may influence health positively; milk proteins are an excellent source of such peptides (Meisel, 1998; Shah, 2000; Nagpal et al., 2011). The 4 caseins (αS1, αS2, β, and κ) constitute approximately 80% of the protein in bovine milk. Casein-derived peptides have been shown to have a range of effects, such as antihypertensive, antithrombotic, antimicro-bial, opioid, immune-modulating, and mineral binding (Silva and Malcata, 2005; Phelan et al., 2009) and are therefore suitable candidates for the development of novel functional foods. Bioactive peptides are en-crypted within the primary structure of proteins and may be released through various types of enzymatic hydrolysis; that is, the targeted action of microbial or plant-derived enzymes, the action of microbial enzymes during fermentation, or the action of digestive enzymes in vitro or in the gastrointestinal tract (Korhonen and Pihlanto, 2006; Phelan et al., 2009). The set of peptides generated from any given protein depends on the speci-ficity of the proteolytic enzymes and, consequently, on the structure of the protein itself. Variations in pri-mary structure may therefore influence the bioactive potential of proteins; for example, by altering enzyme cleavage sites, modifying protein structure, or changing the behavior of the liberated peptides (Kamiński et al., 2007; Caroli et al., 2009). These and other variations can be the result of genetic polymorphisms and may suggest that some variants of proteins behave differ-ently from others with regard to certain health effects

In vitro digestion of purified β-casein variants A1, A2, B, and I: Effects on antioxidant and angiotensin-converting enzyme inhibitory capacity

B. Petrat-Melin ,* P. Andersen ,* J. T. Rasmussen ,† N. A. Poulsen ,* L. B. Larsen ,* and J. F. Young *1

* Department of Food Science, aarhus University, 8830 tjele, Denmark † Department of Molecular Biology and Genetics–Molecular nutrition, aarhus University, 8000 aarhus C, Denmark

Received May 6, 2014. Accepted October 2, 2014. 1 Corresponding author: [email protected]

2 Petrat-Melin et al.

Journal of Dairy Science Vol. 98 no. 1, 2015

(Kamiński et al., 2007). About 40% of the CN in bovine milk is β-CN (Bobe et al., 1998) and, until recently, at least 12 different variants carrying AA substitutions had been identified, designated A1, A2, A3, B, C, D, E, F, G, H1, H2, and I, with the most common being the A1, A2, and B variants (reviewed by Caroli et al., 2009). The number of described variants was increased to 15 in 2013 when the novel variants J, K, and L, described by Gallinat et al. (2013), were identified in Bos indicus breeds using DNA sequencing techniques. In a recent study involving approximately 800 Danish dairy cattle (Danish Holstein and Danish Jersey), the B variant was found to be slightly less common in Danish Holstein than the I variant (Poulsen et al., 2013).

Recently, it was demonstrated that the in vitro diges-tion of β-CN genetic variants A1, A2, and B generated different arrays of peptides, and the authors suggested that these peptide variations could have an effect on immunoglobulin-E binding activity (Lisson et al., 2013). β-Casein also exhibits antioxidative capacity (Pihlanto, 2006), and studies have shown that this ca-pacity is enhanced by digestion of the protein (Gómez-Ruiz et al., 2008; Kumar et al., 2010). Weimann et al. (2009) published the results of an in silico digestion study of κ-CN, wherein variations in the generation of angiotensin-converting enzyme (ACE) inhibitory peptides from different genetic κ-CN variants were characterized. These effects are of relevance with re-gard to ailments such as arthritis, neurodegenerative disease, cardiovascular disease, and cancer (Halliwell, 2007; Valko et al., 2007). Together, these studies indi-cate that genetic polymorphisms may indeed influence the bioactive potential of proteins upon digestion.

To investigate the digestion and bioactive potential of β-CN variants and their digestion products, the dif-ferent variants need to be available in purified form. The basis for the traditional method of separating β-CN from the other CN was described more than half a century ago by Hipp et al. (1952). That method relies on the different solubilities of the caseins in a urea solu-tion, and often involves some type of chromatography, as reviewed by Imafidon (1997). In the last 2 decades, methods have been developed, using reverse-phase HPLC (Bobe et al., 1998; Bonfatti et al., 2008), that are highly effective and able to separate variants of β- and κ-CN, as well as β-LG. However, these methods still use denaturing conditions that disrupt the native secondary structure of the proteins. For some applica-tions, such as in vitro digestion, the presence of urea can be problematic as it can cause carbamylation of Lys and Arg side chains as well as protein amino ter-minals (Stark et al., 1960; Stark, 1965; Kollipara and Zahedi, 2013). It is therefore preferable to avoid urea in the purification process, because carbamylation of

proteins may lead to changes in the pattern or extent of digestion, as well as altered chromatographic retention times. Moreover, protein modifications induced during the purification procedure could modify the behavior of the purified variants relative to the native forms (Mun and Golper, 2000). If denaturing is required in other downstream applications, an alternative chaotrope that does not cause protein modifications, such as guanidine hydrochloride, could be considered.

The aim of this work was to develop a method for purification of β-CN from milk of cows homozygous for the genetic variants A1, A2, B, and I, without the use of urea in the purification protocol. Moreover, to the best of our knowledge, the molar extinction coefficients of isolated β-CN or its variants have not been reported previously. Therefore, these were determined to facili-tate rapid protein quantification of the different vari-ants purified using the reported method. Susceptibility to digestive enzymes and the bioactivity of peptides is largely determined by AA sequence. Consequently, we investigated the in vitro digestion pattern of each of these 4 variants, as well as their bioactive potential in relation to antioxidant and ACE inhibitory capacities, upon in vitro digestion.

MATERIALS AND METHODS

Reagents and Chemicals

Angiotensin-converting enzyme (EC 3.4.15.1), pepsin (EC 3.4.23.1), pancreatin, bovine CN, fluo-rescamine, Coomassie brilliant blue G-250 (CBB G-250), o-phthaldialdehyde, AA standards, captopril, and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were all from Sigma (St. Louis, MO). 2-Aminobenzoylglycyl-p-nitrophenylalanyl-proline (ACE substrate) was purchased from Bachem (Buben-dorf, Switzerland). Polypeptide molecular weight mark-ers (3.5 to 26.6 kDa) were from BioRad Laboratories (Hercules, CA). All other reagents and chemicals were of analytical grade.

Purification of β-CN Variants

β-Casein was purified from milk samples collected by the Danish-Swedish Milk Genomics Initiative as previously described (Jensen et al., 2012a). In brief, morning milk samples from more than 800 individual cows in mid-lactation were collected, analyzed for fat and protein composition by Milkoscan (Foss Analytical, Hillerød, Denmark), and stored at −20°C until use. Ge-notyping of all cows was carried out as described previ-ously (Poulsen et al., 2013), enabling the identification of milk samples from cows that were homozygous for


DiGeStiOn anD BiOaCtiVitY OF CaSein VariantS 3

the common β-CN genetic variants A1 and A2, as well as the less frequent variants B and I. The AA substitu-tions characterizing these variants are shown in Table 1. The frozen milk samples were moved from −20°C to 4°C and then kept at 4°C for 48 h for thawing and dissociation of β-CN from the CN micelle; stirring was applied for the final 24 h. Skimming of the milk samples was then done by centrifugation at 2,600 × g and 4°C for 30 min, and discarding the top fat layer, except for the sample with the I variant, which had been skimmed before being frozen. The skim milk was then centrifuged at 150,000 × g at 4°C for 2 h, using an Optima L-80XP Ultracentrifuge (Beckman Coulter Inc., Brea, CA) and a titanium fixed-angle 70-Ti Rotor (angle 23°). This separated the soluble phase, containing the dissociated β-CN, from the colloidal phase, which contained the remaining β-CN as well as αS1-, αS2-, and κ-CN. The β-CN was then isoelectrically precipitated by addition of one-tenth final volume of 10% acetic acid, leaving for 5 min at room temperature (RT), and then add-ing one-tenth final volume of 1 M sodium acetate. The β-CN was then recovered by centrifugation at 1,000 × g at 4°C for 10 min, and washed 3 times in MilliQ H2O (Millipore, Billerica, MA). After the final washing step, the isolated β-CN was resuspended in MilliQ H2O and lyophilized before storage at −80°C until use. An overview of the study design and progression of analysis is shown in Figure 1.

Determination of Protein Concentration

by AA Analysis

Amino acid analysis was chosen for protein deter-mination for its longstanding reputation as the gold standard (Barkholt and Jensen, 1989). The purified, lyophilized β-CN variants were dissolved to 0.125 mg/mL in 0.75 M guanidine hydrochloride (GndHCl) and dried by vacuum-centrifugation (overnight at pressure = 1.2 × 10−2 mbar, 1,500 × g). The protein samples were then acid-hydrolyzed in 6 M HCl at 110°C for 20 h, vacuum-centrifuged for 2 h as above, and redissolved in 10 mM HCl. The AA composition was determined essentially as described by Barkholt and Jensen (1989), using cation exchange chromatography (CK10U col-

umn, 120 × 6 mm, Mitsubishi Chemical, Tokyo, Ja-pan), combined with postcolumn derivatization with o-phthaldialdehyde after oxidation with hypochlorite. The AA derivatives were then detected by an LC 1250 fluorescence detector (GBC Scientific Equip-ment, Melbourne, Australia), and the resulting data obtained with the Clarity software package version 2.4 (DataApex, Prague, Czech Republic). The analysis was performed in triplicate from 2 independent assays, and the protein content (%) calculated thus:

Protein % =× ×

×n

N V C

AA

AA i β

100%,

where nAA is the molar amount of a specific AA, NAA is the number of this AA in the protein sequence, Vi is the initial volume of sample, and Cβ is the concentration of β-CN under the assumption of 100% protein content in the weighed sample material. Calculations were based on the average of values obtained based on glycine and alanine residues.

Liquid Chromatography/Electrospray

Ionization–Mass Spectrometry

Liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS) was used to confirm the identity and assess the purity of the isolated β-CN variants. In summary, lyophilized β-CN preparations were solubilized in 50 mM GndHCl to a concentration of 3 µg/µL, and fresh dithioerythritol was added to a final concentration of 15 mM. The samples were filtered through a 0.2-µm polytetrafluoroethylene filter (Mini-Uniprep, Whatman, GE Healthcare Life Sciences, Piscataway, NJ). Otherwise, the analysis was carried out as previously described (Jensen et al., 2012b). All systems were controlled by ChemStation software (Agilent Technologies, Santa Clara, CA), which was also used to obtain individual peak areas from the chromatograms by integration. The areas were used to calculate the relative protein composition.

Determination of Molecular Extinction Coefficients

of the Different β-CN Variants

Lyophilized β-CN was dissolved to 1 mg/mL in 6 M GndHCl, followed by absorption measurement at 280 nm in a Cary 60 UV/Vis spectrophotometer (Agilent Technologies) using solvent as blank. Their measured molar extinction coefficients were then calculated using the concentrations obtained from the AA analysis, to-gether with the protein composition obtained from the LC/ESI-MS. The calculation of predicted absorbances

Table 1. Position of AA substitutions within the mature protein of variants of β-casein

Position

Variant1

A1 A2 B I

67 His Pro Pro93 Met Leu122 Ser Arg

1A1 is the reference sequence (Caroli et al., 2009).



(Abs) was described earlier (Edelhoch, 1967; Gill and von Hippel, 1989) and here used to create the following equation:

Predicted Abs

# , # , #

280

5 500 1 490 125

=

× + × + ××

Trp Tyr Cys

MWFraci i i

i

ii∑ ,

where #Trpi is the number of Trp residues in the ith protein, #Tyri is the number of Tyr residues in the ith protein, #Cysi is the number of Cys residues in the ith protein, MWi is the molecular weight of the ith protein, and Fraci is the relative amount of the ith protein in the sample, where i = αS1, αS2, β, κ, or whey proteins. The numeric factors for Trp, Tyr, and Cys are the extinction coefficients for these specific AA, as determined by Edelhoch (1967). The equation can be used to extract the molar extinction coefficient deriving

from individual milk proteins by dividing the predicted extinction coefficient of protein i by the total predicted extinction coefficient, and multiplying the obtained fraction by the measured extinction coefficient.

Digestive Enzyme Activities

To add equal and physiological (Kalantzi et al., 2006) proteolytic activities in the gastric and intestinal steps of the digestion, the proteolytic activities of both enzyme preparations were determined. Pepsin from porcine gastric mucosa was dissolved to 10 mg/mL in 0.1 M phosphate buffer (PB) that had been adjusted to pH 2.0 with 2 M HCl. The porcine pancreatic ex-tract pancreatin was suspended to 10 mg/mL in 0.1 M PB (pH 6.5), and vortexed periodically at RT for 30 min, followed by centrifugation at 10,000 × g and 4°C for 20 min to precipitate undissolved material and obtain a water-soluble proteolytic fraction. The super-

Figure 1. Overview of the experimental design. LC-MS = liquid chromatography-mass spectrometry.



natant was used for determination of enzyme activity in the preparation. Proteolytic activity was determined analogously for pepsin and pancreatin extract using 2% acidified bovine hemoglobin (Anson and Mirsky, 1932) and 1% bovine CN, respectively, as substrates. Enzyme solutions were incubated at 37°C with substrate in a 1:9 ratio for 10 min. Then, an equal volume of 10% TCA was added to all reactions to stop the enzymatic activity. Solutions were precipitated for 20 min at RT and centrifuged at 8,000 × g and RT for 5 min. One unit of proteolytic activity was defined as the volume of enzyme solution producing 1 mg of TCA-soluble BSA equivalents per minute, as detected by the bicin-choninic acid assay kit (Thermo Scientific, Waltham, MA) according to the manufacturer’s manual under the defined assay conditions.

In Vitro Gastrointestinal Digestion

The following procedure, based on previous studies (Calbet and Holst, 2004; Dupont et al., 2010; Ulleberg et al., 2011), was developed for the present study.

β-Casein Solutions. β-Casein was dissolved with agitation and gentle heating (50°C) as 8 mg/mL in simulated gastric fluid (30 mM NaCl) with 0.1 mM NaOH. Following complete dissolution, the pH was ad-justed to 2.0 with 2 M HCl. Casein solutions were made fresh for each digest.

Digestion. Casein solutions were equilibrated to 37°C before the addition of pepsin at 25 U/g of β-CN. The reaction was allowed to run for 60 min before add-ing 54 mM NaHCO3, which brought the pH to 6.5 and inactivated pepsin (Piper and Fenton, 1965). Then, pancreatic enzyme solution was added at 25 U/g of β-CN and the reaction was continued for an additional 120 min. All samples were heat-inactivated at 90°C for 10 min and kept at −20°C until use.

SDS-PAGE

All samples were diluted 5 times in SDS sample buf-fer (1 M Tris, 1% SDS, 2 mM dithioerythritol, 20% glycerol, 0.05% CBB G-250), and 32 µg of protein per lane was loaded to precast criterion 10 to 20% Tris-Tri-cine gels (BioRad Laboratories). Tris-Tricine gels were used because they are more suitable for separation of low-molecular-weight peptides. The gels were run in a Tris-Tricine running buffer (100 mM Tris, pH 8.3, 100 mM Tricine, 0.1% SDS) and then kept in fixing buffer (50% ethanol, 8% phosphoric acid) overnight followed by staining with CBB [0.02% CBB G-250, 2% phos-phoric acid (85%), 5% aluminum sulfate, 10% ethanol] for at least 2 h (Kang et al., 2002). Gel images were captured using a UVP Multispectral Imaging System (BioSpectrum, Cambridge, UK).

Peptide Identification by Electroblotting

and Edman Sequencing

Peptide fragments were subjected to N-terminal AA sequencing by cutting bands of interest from a polyvinylidine difluoride membrane electroblot of an unstained SDS-PAGE gel (Matsudaira, 1987). Bands were visualized by staining the membrane for 1 min in 0.1% CBB in 40% methanol and 10% acetic acid, and then destaining in 40% methanol. The N-terminal AA sequences were established by automated Edman deg-radation using a Procise Protein Sequencer (Applied Biosystems, Foster City, CA).

Degree of Hydrolysis by Quantification

of N-Terminal Amines with Fluorescamine

The degree of hydrolysis (DH) was determined by the method described by Larsen et al. (2004), which is fast and convenient. In brief, hydrolysates were mixed 1:1 with 24% TCA and precipitated on ice for at least 30 min followed by centrifugation at 14,000 × g at 4°C for 20 min. The supernatants were then used for the fluorescamine primary amine analysis (Udenfriend et al., 1972). Standard (-leucine) or sample (30 µL) with TCA was mixed with 900 µL of 0.1 M sodium tet-raborate buffer (pH 8.0), followed by addition of 300 µL of 0.2 mg/mL fluorescamine in dry acetone. Fluo-rescence was measured using excitation and emission wavelengths of 390 and 480 nm, respectively. The DH can then be calculated as follows:

DHh=−

−

×

∞

[ ] [ ]

[ ] [ ],

-NH -NH

-NH -NH

2 2

2 2

0

0

100

where [-NH2] is equal to the concentration of primary amines in the hydrolysed (h) or unhydrolysed (0) sam-ples, and [-NH2]∞ is equal to the theoretical maximal primary amine concentration assuming total digestion to free AA. [-NH2]∞ was calculated as

[ ]( )

,-NH2 ∞=

+ ×1 f C

MW

lys CN

AA

where flys is the fraction of lysine residues in the CN, CCN is the CN concentration, and MWAA is the mean molecular weight of AA in the CN.

ABTS Decolorization Assay

The ABTS decolorization assay is widely used to de-termine antioxidant capacity of food constituents (Moon and Shibamoto, 2009) and was used here to determine



the antioxidant capacity of the β-CN variants and their hydrolysates, essentially as described elsewhere (Re et al., 1999). Excess ABTS (18.7 mM) was mixed with ammonium persulfate (8.8 mM) and allowed to react overnight at RT for the formation of the ABTS•+ radi-cal. The stock solution was then diluted with 75 mM PB (pH 7.4) to give an assay absorbance at 734 nm of ≈0.7. β-Casein samples were diluted to 0.1 mg/mL with 75 mM PB (pH 7.4). A volume of 50 µL of sample or the water-soluble vitamin E derivative Trolox in 75 mM PB (pH 7.4) was mixed with 200 µL of ABTS•+ working solution, and incubated for 60 min at RT in the dark before measuring absorbance at 734 nm on a Synergy 2 Microplate reader (BioTek Instruments Inc., Winooski, VT). The extent of decolorization enables the calculation of the Trolox equivalent antioxidant capacity (TEAC).

ACE Inhibition

The internally quenched fluorescent tripeptide 2-ami-nobenzoylglycyl-p-nitrophenylalanyl-proline (ACE sub-strate) was used to assess the capacity of the β-CN hydrolysates for ACE inhibition. The method was modified from Sentandreu and Toldra (2006). Briefly, 50 µL of ACE stock solution [0.5 U/mL, 0.05 M sodium tetraborate, 1 M NaCl, 2 mg/mL BSA, 50% (vol/vol) glycerol, pH 8.2] was diluted 50 times in 0.05 M sodium tetraborate buffer with 1 M NaCl (BB, pH 8.2), and 50 µL was mixed with 50 µL of sample or control in a microtiter plate and incubated for 10 min at 37°C. Then, 200 µL of ACE substrate solution (187.5 µM in BB) was added to each well and fluorescence read-ings were initiated immediately, using excitation and emission wavelengths of 350 and 420 nm, respectively, in a Synergy 2 Microplate reader (BioTek Instruments Inc.). Readings were taken every minute for 25 min.

Data Analysis

The β-CN variants were purified from one individual sample each based on genotypic information (see Meth-ods) and subsequently validated by LC/ESI-MS in trip-licate. All other assays were carried out in 3 indepen-dent experiments, unless otherwise stated. The statisti-cal software package R (version 3.0.2; www.r-project.org) was used to analyze differences between different variants and treatments by 2-way ANOVA. Variant and digestion time were used as factors in the ANOVA. Individual comparisons were done using Tukey’s hon-est significant difference post hoc test. Differences were deemed statistically significant if P ≤ 0.05.

RESULTS

Purification of β-CN Variants

The 4 bovine β-CN variants A1, A2, B, and I were successfully purified using a combination of cold stor-age, ultracentrifugation, and acid precipitation of the obtained supernatant. The LC/ESI-MS analysis of the precipitated β-CN variants confirmed their homozy-gosity and genotypes by having only a single major β-CN peak with the expected masses of 24,018, 23,977, 24,087, and 23,959 Da for variants A1, A2, B, and I, respectively (all representing forms with 5 phosphoryla-tions; Figure 2). Using the milk composition data by Milkoscan combined with protein quantification by AA analysis and, assuming 40% of the CN is β-CN, the yield of β-CN from the purification was calculated. The yields of the 4 isolated β-CN variants A1, A2, B, and I were estimated to be between 5 and 20% of total β-CN originally present in the milk samples, and the purity relative to total protein determined by LC/ESI-MS varied from 89.2 to 93.2% (Table 2).

Molar Extinction Coefficients of β-CN Variants

The absorption at λ = 280 was measured for the purified fractions of β-CN variants dissolved in 6 M GndHCl (Table 3). The relative protein composition was determined by LC/ESI-MS using the protein de-tection wavelength of 214 nm. This was used together with the protein concentration determined by AA analysis, and molecular weight, to calculate the mo-lar extinction coefficients (ε). For the whole fractions, ε-values of 12,142 to 14,191 M−1cm−1 were obtained, deviating less than 11% from the predicted values Us-ing the relative composition of milk proteins in each of the purified preparations (Table 2), the contribution to ε from β-CN was estimated to be in the range from 9,659 to 11,448 M−1cm−1. These values deviated from the predicted values for these variants by 0 to 16%. We found no significant differences between variants in the data presented here.

In Vitro Gastrointestinal Digestion

A 2-step in vitro gastrointestinal digestion using pep-sin and pancreatic enzymes was used to estimate the digestibility and digestion pattern of the β-CN variants. The effect of digestion on antioxidant capacity and ACE inhibitory capacity of the hydrolysates was subse-quently assessed (see below). Based on the determined specific proteolytic activities of the enzyme solutions, the amount of enzyme used in the in vitro digestion corresponded approximately to an enzyme:substrate



ratio of 1:200 in the reaction. The DH for the β-CN variants after 60 min of pepsin digestion (Table 4) was around 3%. After 5 min of digestion with pancreatic enzymes, DH increased to around 20%, and after 120 min of pancreatic enzyme digestion, DH was close to 50% for all β-CN variants, with the order of DH being A1 > A2 > I > B. Degree of hydrolysis can be converted to a mean length of peptides by 100%/DH (Table 4), which provides a more intuitive measure of hydrolysis. Following digestion, the hydrolysates were analyzed by SDS-PAGE (a representative gel image is shown in Figure 3). The lanes with the intact β-CN variants showed approximately the same pattern, with one clear large band around 25 kDa corresponding roughly to the molecular weight of β-CN. In addition, for all 4 vari-ants a fainter band (approximately 50 kDa) was visible, likely a result of dimerized β-CN, as well as several more or less faint bands and smears. After 60 min of pepsin digestion, little undigested β-CN remained and several bands of lower molecular weight had appeared for all variants. The addition of pancreatic enzymes changed the digestion pattern between the β-CN vari-ants, as the B variant was missing a low-molecular-weight band around 4 kDa, compared with the A1, A2, and I variants after 5 min. After 60 min of pepsin + 120 min of pancreatic enzyme digestion, no bands were detectable, indicating that all peptides had eluted from the gel under the conditions used here. The band cor-responding to the missing band in the B variant was

electroblotted from the A1 variant onto a polyvinylidine difluoride membrane, and the peptide fragments were subsequently sequenced by Edman degradation. The strongest N-terminal sequence tag deduced from the

Figure 2. Bovine β-casein variants A1, A2, B, and I were puri-fied from milk samples obtained from cows that were homozygous at the β-casein locus, and analyzed by liquid chromatography combined with mass spectrometry. The molecular weight (Da) of each variant is indicated for each peak. Horizontal brackets indicate retention times of different milk proteins. Data are normalized to the maximum value within each individual chromatogram. All variants were analyzed in triplicate. Abs = absorbance.

Table 2. Relative content of milk proteins in the isolated β-casein variant preparations1

β-casein variant β-casein κ-casein α-casein

Whey proteins

A1 89.7 ± 1.4 2.3 ± 0.4 4.6 ± 1.7 3.5 ± 2.5A2 93.2 ± 1.7 0.5 ± 0.3 1.0 ± 0.5 3.2 ± 2.6B 89.0 ± 0.1 5.6 ± 1.4 3.4 ± 0.3 1.4 ± 1.1I 90.3 ± 1.1 2.9 ± 0.7 4.8 ± 0.8 1.7 ± 0.2

1Values were calculated as relative peak areas within each chromato-gram by liquid chromatography/electrospray ionization-mass spec-trometry. All values are expressed as mean percentage of total peak area ± SEM (n = 3).

Table 3. Molar extinction coefficients (ε; M−1cm−1) of β-casein variants at λ = 280 nm, measured in 6 M guanidine hydrochloride1

β-casein variant

ε (fraction)2 measured % Dev3

ε (β-CN)4 measured % Dev3

A1 13,398 ± 483 2.66 10,547 ± 380 −7.94A2 12,642 ± 1,033 5.38 11,238 ± 918 −1.94B 12,142 ± 948 −5.55 9,659 ± 754 −15.75I 14,191 ± 1,154 10.93 11,448 ± 931 −0.04

1Values are given as means ± SEM (n = 3).2Total purified fractions.3Deviation from predicted values.4Molar extinction coefficients deriving from β-CN alone.



resulting chromatograms from 8 degradation cycles was 106H-K-E-M-P-F-P-K-.

Antioxidant Capacity

The ABTS decolorization assay was used to deter-mine the TEAC values for the β-CN variants (Figure 4). This method has been used frequently for assessing the antioxidant capacity of food constituents in vitro (Moon and Shibamoto, 2009). By incubating the stable ABTS•+ radical with the synthetic vitamin E analog Trolox or with the β-CN variants and their hydrolysates, TEAC values for the β-CN variants were obtained. We observed significant overall effects of digestion time (P

< 0.001) and variant (P < 0.01). All 4 β-CN variants had TEAC values slightly below 0.8 µmol of Trolox equivalents/mg of protein when undigested, and we observed a small nonsignificant increase upon 60 min of pepsin digestion. After 5 min of digestion with pan-creatic enzymes, the overall TEAC values increased further compared with the undigested proteins (P < 0.001). At the end of digestion, an additional increase was seen (P < 0.001). Following digestion, we detected accumulated increases in TEAC for variants A1, A2, B, and I of 67, 59, 59, and 75%, respectively. However, these differences were not reflected in a significant interaction effect between variant and digestion time. Throughout the course of digestion, the A1 and I vari-ants showed progressively higher TEAC values than the A2 variant (P < 0.05) or the B variant (P < 0.1).

ACE Inhibition

We determined ACE inhibition using a synthetic in-ternally quenched fluorescent substrate, and the results are expressed as 100% minus the residual ACE activity after incubation with the different samples (Figure 5). The synthetic specific ACE inhibitor captopril at a con-centration of 20 nM inhibited almost all enzyme activity (98.9% inhibition). For β-CN at an assay concentration of 0.1 mg/mL, we detected some inhibition from the undigested protein for all 4 variants (9.3 to 14.3%), with the A2 and I variants having the highest inhibition. Af-ter 60 min of digestion with pepsin, inhibition increased significantly to between 44 and 54% (P < 0.001), with the A1 and B variants being the most inhibitory. After further digestion for 5 min with pancreatic enzymes, ACE inhibition increased to between 58 and 64% (P < 0.001), and the inhibition levels converged somewhat. At the end of pancreatic enzyme digestion, we observed another smaller increase in inhibition, which appeared to be the most pronounced for the B variant. However, we detected no overall significant effect of variant or its interaction with time.

DISCUSSION

The research field of milk genomics attempts to link variation at the genomic level to variation in nutrition-al, technological, and health-related properties of milk. So far, the focus has been mainly on productive, com-positional, and technological phenotypes, such as milk yield, casein number, and coagulation behavior (Martin et al., 2002; Poulsen et al., 2013) and less on nutritional and health-related properties, although the latter have been gaining interest in recent years (Kamiński et al., 2007; Caroli et al., 2009; Lisson and Erhardt, 2011). In the work presented here, the A1, A2, B, and I variants of

Table 4. In vitro gastrointestinal digestion of β-casein variants using pepsin and pancreatic enzymes1

β-CN variant

tpep (min)2

tpan (min)3

DH (%)4 MLP5

A1 60 0 3.6 ± 0.42a 27.6 ± 3.01A2 60 0 3.2 ± 0.20a 31.5 ± 2.20B 60 0 3.6 ± 0.22a 28.0 ± 1.95I 60 0 2.6 ± 0.17a 37.6 ± 2.46A1 60 5 20.4 ± 1.71b 4.9 ± 0.42A2 60 5 21.5 ± 2.47b 4.6 ± 0.55B 60 5 20.2 ± 0.89b 5.0 ± 0.20I 60 5 19.4 ± 1.38b 5.2 ± 0.33A1 60 120 55.0 ± 5.99c 1.8 ± 0.21A2 60 120 52.4 ± 5.54c 1.9 ± 0.19B 60 120 46.2 ± 3.98c 2.2 ± 0.16I 60 120 49.9 ± 4.24c 2.0 ± 0.17

a–cDifferent letters denotes significant difference (P < 0.001).1Results are shown as the mean ± SEM (n = 3; n = 2 for A1).2tpep = reaction time with pepsin.3tpan = reaction time with pancreatic enzymes.4DH = degree of hydrolysis. 5MLP (100%/DH) = mean length of peptides.

Figure 3. Representative SDS-PAGE gel of β-casein variants sub-jected to in vitro gastrointestinal digestion with pepsin and pancreatic enzymes. The A1, A2, B, and I variants are indicated at the top; for each variant, the 4 lanes are as follows: 0 = undigested, 60 = 60 min of pepsin, 65 = 60 min of pepsin + 5 min of pancreatic enzymes, 180 = 60 min of pepsin + 120 min of pancreatic enzymes. Molecular weight (kDa) markers are indicated to the left.



β-CN from milk samples of cows that were homozygous at the β-CN locus have been purified using a relatively simple, nondenaturing method. Thawing and storing the milk samples at 4°C favors the monomeric state of β-CN, which has a strong tendency to form poly-disperse micellar structures at higher temperatures, as shown by Payens and van Markwijk (1963) and, more recently, by O’Connell and colleagues (2003). The hydrophobic stretch in the primary structure of β-CN facilitates its association with the other CN through hydrophobic interactions, which are disrupted by low-ering the temperature to 4°C. Stirring was applied in an attempt to affect the calcium balance toward the dissociated state and thereby further enhance the dis-sociation of β-CN into the soluble phase, as discussed by Dalgleish and Law (1989). The analysis by LC/ESI-MS showed that the β-CN samples were around 90% pure using the reported method, albeit with a more modest yield (≈12%) than reported by Garnier et al. (1964) and Cayot et al. (1992), who reported yields of approximately 30 and 88%, respectively, using urea-based procedures. These results indicate that the present method can be used for semipreparative-scale purification of β-CN for in vitro digestion or bioactivity studies, and that it limits the risk of modifications that could influence further analysis. In addition, we found no apparent consequences of skimming the milk before

rather than after freezing, as judged by protein integ-rity from LC/ESI-MS, SDS-PAGE, and DH analyses.

As expected, the ε-values for the 4 variants did not differ significantly, because molar extinction coefficients are largely determined by the number of Tyr, Trp, and Cys residues in a protein (Edelhoch, 1967; Pace et al., 1995), and all 4 variants have the same number of these residues: 4 Tyr, 1 Trp, and 0 Cys. In the study by Pace et al. (1995), the molar extinction coefficients at 280 nm of 80 different proteins were measured and compared with their respective predicted values. In general, de-viations of less than ±5% were seen, with a small num-ber deviating ±10 to 16%, which is comparable to the deviations presented here. For the β-CN fractions, the I variant deviated the most; however, when considering only the contribution to the absorption from β-CN, the B variant deviated most. This may be ascribed, in part, to the estimation of milk protein composition of the fractions analyzed by LC using absorbance at 214 nm, which, although quite reliable, can exhibit some de-viation (Kuipers and Gruppen, 2007). Predicted molar extinction coefficients based on LC/ESI-MS analysis of protein composition can thus be used to determine the total protein content of purified β-CN samples, thereby avoiding the use of more time-consuming assessments, such as AA analysis. In addition, for compositional analysis of raw materials at the dairy, it is relevant to

Figure 4. Effect of in vitro gastrointestinal digestion with pep-sin and pancreatic enzymes on Trolox equivalent antioxidant capacity (TEAC, µmol of Trolox equivalents/mg of protein) of β-casein vari-ants. β-Casein was incubated with the ABTS radical for 60 min at room temperature and the reduction in abs(734 nm) was measured and related to that obtained with Trolox. Bars represent mean values, error bars are SEM (n = 3 (n = 2 for A1 variant)). Asterisks denote significant difference from the undigested sample (***P < 0.001).

Figure 5. Effect of in vitro gastrointestinal digestion with pep-sin and pancreatic enzymes on angiotensin-converting enzyme (ACE) inhibitory capacity of β-casein variants (see legend) at 0.1 mg/mL. Captopril concentration was 20 nM. Bars represent mean values, error bars are SEM (n = 3). Asterisks denote significant difference from the undigested sample (***P < 0.001). We observed no overall effect of variant on ACE inhibition.



know that the response factor for β-CN absorbance at 280 nm does not differ between variants.

A wide range of in vitro digestion models exists, varying greatly in complexity and equipment require-ments, and the choice of model depends largely on the end-points of interest as well as the compound or food components under investigation (Wickham et al., 2009; Hur et al., 2011). In the present study, a single isolated protein was under investigation, and a low-complexity method was developed for this relatively simple sub-strate. Moreover, the enzymes used were gastric and intestinal proteases; that is, pepsin and pancreatic enzymes, respectively. It has previously been shown that CN, whey, and their hydrolysates all have similar rates of gastric emptying, with half times between 18 and 21.5 min (Calbet and Holst, 2004). Based on this, a digestion time of 60 min for pepsin was chosen for the static system used here. Furthermore, preliminary experiments (data not shown) indicated that pancre-atic enzyme digestion reached a plateau after 90 to 120 min, and therefore we used 120 min for this step. In the present study, we observed a low DH after pepsin digestion, but SDS-PAGE revealed that little intact β-CN was present at this point. This is in good accor-dance with Dupont et al. (2010), who observed almost complete disappearance of intact β-CN after 60 min of pepsin digestion. The pattern of low-molecular-weight bands from our study is similar to that of Dupont and coworkers, even though their pepsin to β-CN ratio was approximately 10-fold higher than that presented here. Dupont et al. (2010) also observed a comparable DH when using a pepsin concentration closer to that pre-sented here, which was done in an attempt to mimic infant digestion more closely, although their observed pattern of peptides differed slightly from what was seen in the present study. Herein, after 5 min of pancreatic enzyme digestion, a rapid increase in DH occurred, and by 120 min, all detectable protein bands disappeared. Pancreatin contains several different proteolytic activities—trypsin, chymotrypsin, elastase, carboxy-peptidase A, and dipeptidase (Mullally et al., 1994); thus, even though the added level of enzyme activity was comparable to that of pepsin, a protein would be hydrolyzed at multiple sites simultaneously, which is likely the underlying reason for the rapid progression of DH. Under their specific assay conditions, Dupont et al. (2010) did not observe the complete disappearance of protein bands after duodenal digestion. Perhaps this was a consequence of the shorter digestion time used (60 min), but more likely because only 2 proteases were used—trypsin and chymotrypsin. In another study on in vitro digestion of CN, the DH after 24 h of digestion with a pancreatin:CN ratio of 1:100 reached only 28.5% (Su et al., 2012), whereas our results showed consider-

ably higher DH after just 2 h of pancreatic enzyme digestion. Su et al. (2012) did not use pepsin, used complete pancreatin (not an extract), and digested the total CN fraction. Taken together, these factors might explain the differences in DH reached. In addition, β-CN variant B exhibited an altered pattern of diges-tion evident from gel electrophoresis, which revealed that an approximately 4 kDa peptide, corresponding to about 35 AA, was missing compared with the A1, A2, and I variants. Edman sequencing of this peptide from β-CN variant A1 identified the 8 N-terminal AA as 106H-K-E-M-P-F-P-K-. The sequence of β-CN between position 106 and 35 AA toward the C-terminal covers position 122, where a Ser is replaced by an Arg in the B variant, introducing a novel trypsin cleavage site. This is the probable cause of the missing peptide, as the products of trypsin cleavage at this site would produce 2 peptides of sufficiently small size to elute from the gel completely.

Many proteins, derived from a variety of plant foods as well as animal-based foods such as milk, are known to harbor peptides with antioxidant capacity in their sequence (see Pihlanto, 2006, and Sarmadi and Ismail, 2010). The results presented here show that the intact as well as digested β-CN variants possess similar anti-oxidant capacity toward the ABTS radical but with a significantly higher radical-quenching capacity follow-ing in vitro digestion. This has previously been shown for both combined and individual CN types (Gómez-Ruiz et al., 2008; Kumar et al., 2010). The effects of digesting β-CN on antioxidant capacity in our obser-vations (≈1.7 fold) were moderately larger than those presented by Gómez-Ruiz et al. (2008), who analyzed ovine β-CN. However, Kumar et al. (2010) observed a notably greater increase in TEAC (≈4.0 fold). These differences may be a result of different digestion pro-cedures. It was previously noted that His residues and their position relative to the N-terminal might be im-portant in the antioxidative capacity of peptides (Chen et al., 1998). Furthermore, a study investigating the re-actions of individual AA with the ABTS radical showed that His, together with Cys, Trp, and Tyr, possesses quenching capacity (Aliaga and Lissi, 2000). Nine of these AA are present in the A1 and B variants, whereas only 8 are present in the A2 and I variants (the His at position 67 is replaced by Pro). As shown in the present study, subtle differences exist in the digestion pattern of the variants that may be caused, in part, by this substitution. Jinsmaa and Yoshikawa (1999) indicated that β-CN variants had very different susceptibilities to hydrolysis at position 67 with gastrointestinal enzymes. Therefore, we expect that, with respect to TEAC val-ues, the A1 and B variants would be similar, and hence, also the A2 and I variants. This could not, however, be



confirmed here, perhaps because antioxidant capacity is determined not only by the total number of ABTS-scavenging AA and DH but also by the position of these AA within individual peptides. Further studies are needed to elucidate this question.

Angiotensin-converting enzyme inhibitory capacity has been widely reported for β-CN (FitzGerald and Meisel, 2000), and it depends on the specific AA se-quence of candidate peptides. Studies have revealed that the C-terminal AA most strongly associated with ACE inhibitory capacity are hydrophobic and aromatic residues, such as Trp, Phe, and the branched-chain AA (Wu et al., 2006), in agreement with Cheung et al. (1980). However, Cheung et al. (1980) also found that Pro was favorable at a C-terminal position, and FitzGer-ald and Meisel (2000) noted that Arg and Lys at the C-terminal position appear favorable for ACE inhibition. The AA substitutions in β-CN variants A2, B, and I are all potentially activity-altering substitutions, according to findings in the mentioned studies. As expected, in the current study, a marked increase in ACE inhibition was seen upon initial digestion, which affected the A1 (5.8-fold) and B (5.4-fold) variants to a greater degree than the A2 (3.0-fold) and the I (2.6-fold) variants. This could be a consequence of the 67His residue being more permissive than the 67Pro toward pepsin cleavage after the 66Ile residue. Cleavage here would create a pep-tide with Ile in the C-terminal position. The effect of continued digestion was much less dramatic, and after 60 min of pepsin followed by 120 min (compared with only 5 min) with pancreatic enzyme digestion, none of the variants showed a significant increase in inhibi-tion. Nevertheless, the B variant did appear to increase slightly more than the other variants, perhaps because of the 122Ser to 122Arg substitution, as a peptide with a C-terminal Arg may have ACE inhibitory capacity, as discussed above. Clearly, more work is needed to fully understand the development of ACE inhibitory capac-ity during gastrointestinal digestion of β-CN variants.

In summary, the results presented here demonstrate that AA substitutions caused by genetic polymorphisms at the β-CN locus affect the manner in which these proteins are processed by the gastrointestinal system. Variations in the array of peptides and bioactive capaci-ties of the β-CN studied here suggest potential health-related implications of underlying genetic variation, which should be studied further, but show perspective for targeting raw materials for specific food products.

ACKNOWLEDGMENTS

The authors extend their gratitude to Hanne S. Møller (Department of Food Science, Aarhus Univer-sity, Denmark) for operating the LC/ESI-MS. The

authors are also grateful to Margit S. Rasmussen for invaluable assistance with the amino acid analysis and Anni Boisen (both at the Protein Chemistry Labora-tory, Department of Molecular Biology and Genetics, Aarhus University, Denmark) for carrying out the Ed-man sequencing.

REFERENCES

Aliaga, C., and E. A. Lissi. 2000. Reactions of the radical cation de-rived from 2,2-azinobis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) with amino acids. Kinetics and mechanism. Can. J. Chem. 78:1052–1059.

Anson, M. L., and A. E. Mirsky. 1932. The estimation of pepsin with hemoglobin. J. Gen. Physiol. 16:59–63.

Barkholt, V., and A. L. Jensen. 1989. Amino-acid analysis—Determi-nation of cysteine plus half-cystine in proteins after hydrochloric-acid hydrolysis with a disulfide compound as additive. Anal. Bio-chem. 177:318–322.

Bobe, G., D. C. Beitz, A. E. Freeman, and G. L. Lindberg. 1998. Separation and quantification of bovine milk proteins by reversed-phase high-performance liquid chromatography. J. Agric. Food Chem. 46:458–463.

Bonfatti, V., L. Grigoletto, A. Cecchinato, L. Gallo, and P. Carnier. 2009. Validation of a new reversed-phase high-performance liq-uid chromatography method for separation and quantification of bovine milk protein genetic variants (vol 1195, pg 101, 2008). J. Chromatogr. A 1216:1528.

Calbet, J. A., and J. J. Holst. 2004. Gastric emptying, gastric se-cretion and enterogastrone response after administration of milk proteins or their peptide hydrolysates in humans. Eur. J. Nutr. 43:127–139.

Caroli, A. M., S. Chessa, and G. J. Erhardt. 2009. Invited review: Milk protein polymorphisms in cattle: Effect on animal breeding and human nutrition. J. Dairy Sci. 92:5335–5352.

Cayot, P., J. L. Courthaudon, and D. Lorient. 1992. Purification of αs-, β- and κ-caseins by batchwise ion-exchange separation. J. Dairy Res. 59:551–556.

Chen, H. M., K. Muramoto, F. Yamauchi, K. Fujimoto, and K. Noki-hara. 1998. Antioxidative properties of histidine-containing pep-tides designed from peptide fragments found in the digests of a soybean protein. J. Agric. Food Chem. 46:49–53.

Cheung, H. S., F. L. Wang, M. A. Ondetti, E. F. Sabo, and D. W. Cushman. 1980. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-termi-nal dipeptide sequence. J. Biol. Chem. 255:401–407.

Dalgleish, D. G., and A. J. R. Law. 1989. pH-induced dissociation of bovine casein micelles. 2. Mineral solubilization and its relation to casein release. J. Dairy Res. 56:727–735.

Dupont, D., G. Mandalari, D. Molle, J. Jardin, J. Leonil, R. M. Faulks, M. S. J. Wickham, E. N. C. Mills, and A. R. Mackie. 2010. Com-parative resistance of food proteins to adult and infant in vitro digestion models. Mol. Nutr. Food Res. 54:767–780.

Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954.

FitzGerald, R. J., and H. Meisel. 2000. Milk protein-derived pep-tide inhibitors of angiotensin-I-converting enzyme. Br. J. Nutr. 84(Suppl. 1):S33–37.

Gallinat, J. L., S. Qanbari, C. Drögemüller, E. C. G. Pimentel, G. Thaller, and J. Tetens. 2013. DNA-based identification of novel bovine casein gene variants. J. Dairy Sci. 96:699–709.

Garnier, J., B. Ribadeau-Dumas, and G. Mocquot. 1964. A new method for the preparation of an immunologically homogeneous β-casein. J. Dairy Res. 31:131–136.

Gill, S. C., and P. H. von Hippel. 1989. Calculation of protein extinc-tion coefficients from amino-acid sequence data. Anal. Biochem. 182:319–326.

Gómez-Ruiz, J. A., I. Lopez-Exposito, A. Pihlanto, M. Ramos, and I. Recio. 2008. Antioxidant activity of ovine casein hydrolysates:



Identification of active peptides by HPLC-MS/MS. Eur. Food Res. Technol. 227:1061–1067.

Halliwell, B. 2007. Oxidative stress and cancer: Have we moved for-ward? Biochem. J. 401:1–11.

Hipp, N. J., M. L. Groves, J. H. Custer, and T. L. McMeekin. 1952. Separation of α-, β- and γ-casein. J. Dairy Sci. 35:272–281.

Hur, S. J., B. O. Lim, E. A. Decker, and D. J. McClements. 2011. In vitro human digestion models for food applications. Food Chem. 125:1–12.

Imafidon, G. I., N. Y. Farkye, and A. M. Spanier. 1997. Isolation, pu-rification, and alteration of some functional groups of major milk proteins: A review. Crit. Rev. Food Sci. Nutr. 37:663–689.

Jensen, H. B., J. W. Holland, N. A. Poulsen, and L. B. Larsen. 2012a. Milk protein genetic variants and isoforms identified in bovine milk representing extremes in coagulation properties. J. Dairy Sci. 95:2891–2903.

Jensen, H. B., N. A. Poulsen, K. K. Andersen, M. Hammershoj, H. D. Poulsen, and L. B. Larsen. 2012b. Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good, poor, or noncoagulation properties as reflected in protein genetic variants and isoforms. J. Dairy Sci. 95:6905–6917.

Jinsmaa, Y., and M. Yoshikawa. 1999. Enzymatic release of neocaso-morphin and beta-casomorphin from bovine beta-casein. Peptides 20:957–962.

Kalantzi, L., K. Goumas, V. Kalioras, B. Abrahamsson, J. B. Dress-man, and C. Reppas. 2006. Characterization of the human upper gastrointestinal contents under conditions simulating bioavailabil-ity/bioequivalence studies. Pharm. Res. 23:165–176.

Kamiński, S., A. Cieslinska, and E. Kostyra. 2007. Polymorphism of bovine beta-casein and its potential effect on human health. J. Appl. Genet. 48:189–198.

Kang, D. H., Y. S. Gho, M. K. Suh, and C. H. Kang. 2002. Highly sen-sitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bull. Korean Chem. Soc. 23:1511–1512.

Korhonen, H., and A. Pihlanto. 2006. Bioactive peptides: Production and functionality. Int. Dairy J. 16:945–960.

Kollipara, L., and R. P. Zahedi. 2013. Protein carbamylation: In vivo modification or in vitro artefact? Proteomics 13:941–944.

Kuipers, B. J. H., and H. Gruppen. 2007. Prediction of molar ex-tinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spec-trometry analysis. J. Agric. Food Chem. 55:5445–5451.

Kumar, R., V. Singhal, R. B. Sangwan, and B. Mann. 2010. Compara-tive studies on antioxidant activity of buffalo and cow milk casein and their hydrolysates. Milchwissenschaft 65:287–290.

Larsen, L. B., M. D. Rasmussen, M. Bjerring, and J. H. Nielsen. 2004. Proteases and protein degradation in milk from cows infected with Streptococcus uberis. Int. Dairy J. 14:899–907.

Lisson, M., and G. Erhardt. 2011. Milk protein variants and human nutrition. Ernahrungs Umschau 58:602–607.

Lisson, M., G. Lochnit, and G. Erhardt. 2013. Genetic variants of bo-vine beta- and kappa-casein result in different immunoglobulin E-binding epitopes after in vitro gastrointestinal digestion. J. Dairy Sci. 96:5532–5543.

Martin, P., M. Szymanowska, L. Zwierzchowski, and C. Leroux. 2002. The impact of genetic polymorphisms on the protein composition of ruminant milks. Reprod. Nutr. Dev. 42:433–459.

Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035–10038.

Meisel, H. 1998. Overview on milk protein-derived peptides. Int. Dairy J. 8:363–373.

Moon, J. K., and T. Shibamoto. 2009. Antioxidant assays for plant and food components. J. Agric. Food Chem. 57:1655–1666.

Mullally, M. M., D. M. O’Callaghan, R. J. FitzGerald, W. J. Donnelly, and J. P. Dalton. 1994. Proteolytic and peptidolytic activities in commercial pancreatic protease preparations and their relation-ship to some whey protein hydrolyzate characteristics. J. Agric. Food Chem. 42:2973–2981.

Mun, K. C., and T. A. Golper. 2000. Impaired biological activity of erythropoietin by cyanate carbamylation. Blood Purif. 18:13–17.

Nagpal, R., P. Behare, R. Rana, A. Kumar, M. Kumar, S. Arora, F. Morotta, S. Jain, and H. Yadav. 2011. Bioactive peptides derived from milk proteins and their health beneficial potentials: An up-date. Food Funct. 2:18–27.

O’Connell, J. E., V. Y. Grinberg, and C. G. de Kruif. 2003. Associa-tion behavior of β-casein. J. Colloid Interface Sci. 258:33–39.

Pace, C. N., F. Vajdos, L. Fee, G. Grimsley, and T. Gray. 1995. How to measure and predict the molar absorption-coefficient of a protein. Protein Sci. 4:2411–2423.

Payens, T. A., and B. van Markwijk. 1963. Some features of associa-tion of beta-casein. Biochim. Biophys. Acta 71:517–530.

Phelan, M., A. Aherne, R. J. FitzGerald, and N. M. O’Brien. 2009. Ca-sein-derived bioactive peptides: Biological effects, industrial uses, safety aspects and regulatory status. Int. Dairy J. 19:643–654.

Pihlanto, A. 2006. Antioxidative peptides derived from milk proteins. Int. Dairy J. 16:1306–1314.

Piper, D. W., and B. H. Fenton. 1965. pH stability and activity curves of pepsin with special reference to their clinical importance. Gut 6:506–508.

Poulsen, N. A., H. P. Bertelsen, H. B. Jensen, F. Gustavsson, M. Glantz, H. L. Mansson, A. Andren, M. Paulsson, C. Bendixen, A. J. Buitenhuis, and L. B. Larsen. 2013. The occurrence of non-coagulating milk and the association of bovine milk coagulation properties with genetic variants of the caseins in 3 Scandinavian dairy breeds. J. Dairy Sci. 96:4830–4842.

Re, R., N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, and C. Rice-Evans. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26:1231–1237.

Sarmadi, B. H., and A. Ismail. 2010. Antioxidative peptides from food proteins: A review. Peptides 31:1949–1956.

Sentandreu, M. A., and F. Toldra. 2006. A rapid, simple and sensi-tive fluorescence method for the assay of angiotensin-I converting enzyme. Food Chem. 97:546–554.

Shah, N. P. 2000. Effects of milk-derived bioactives: An overview. Br. J. Nutr. 84(Suppl. 1):S3–10.

Silva, S. V., and F. X. Malcata. 2005. Caseins as source of bioactive peptides. Int. Dairy J. 15:1–15.

Stark, G. R. 1965. Reactions of cyanate with functional groups of pro-teins. 3. Reactions with amino and carboxyl groups. Biochemistry 4:1030–1036.

Stark, G. R., W. H. Stein, and S. Moore. 1960. Reactions of cyanate present in aqueous urea with amino acids and proteins. J. Biol. Chem. 235:3177–3181.

Su, R., M. Liang, W. Qi, R. Liu, S. Yuan, and Z. He. 2012. Pancreatic hydrolysis of bovine casein: Peptide release and time-dependent reaction behavior. Food Chem. 133:851–858.

Udenfriend, S., S. Stein, P. Bohlen, and W. Dairman. 1972. Fluores-camine—Reagent for assay of amino acids, peptides, proteins, and primary amines in picomole range. Science 178:871–872.

Ulleberg, E. K., I. Comi, H. Holm, E. B. Herud, M. Jacobsen, and G. E. Vegarud. 2011. Human gastrointestinal juices intended for use in in vitro digestion models. Food Dig. 2:52–61.

Valko, M., D. Leibfritz, J. Moncol, M. T. D. Cronin, M. Mazur, and J. Telser. 2007. Free radicals and antioxidants in normal physi-ological functions and human disease. Int. J. Biochem. Cell Biol. 39:44–84.

Weimann, C., H. Meisel, and G. Erhardt. 2009. Short communica-tion: Bovine kappa-casein variants result in different angiotensin I converting enzyme (ACE) inhibitory peptides. J. Dairy Sci. 92:1885–1888.

Wickham, M., R. Faulks, and C. Mills. 2009. In vitro digestion meth-ods for assessing the effect of food structure on allergen break-down. Mol. Nutr. Food Res. 53:952–958.

Wu, J., R. E. Aluko, and S. Nakai. 2006. Structural requirements of angiotensin I–converting enzyme inhibitory peptides: Quantitative structure-activity relationship study of di- and tripeptides. J. Ag-ric. Food Chem. 54:732–738.

In vitro digestion of purified β-casein variants A1, A2, B, and I: Effects on antioxidant and...

Documents

Transcript of In vitro digestion of purified β-casein variants A1, A2, B, and I: Effects on antioxidant and...