Bioavailability of peptides from casein hydrolysate in vitro: Amino acid compositions of peptides affect the antioxidant efficacy and resistance to intestinal peptidases

    Bioavailability of peptides from casein hydrolysate in vitro: Amino acid compositions of peptides affect the antioxidant efficacy and resistance to intestinal peptidases Chan Wang, Bo Wang, Bo Li PII: DOI: Reference:

S0963-9969(15)30284-2 doi: 10.1016/j.foodres.2015.12.013 FRIN 6115

To appear in:

Food Research International

Received date: Revised date: Accepted date:

31 August 2015 2 December 2015 18 December 2015

Please cite this article as: Wang, C., Wang, B. & Li, B., Bioavailability of peptides from casein hydrolysate in vitro: Amino acid compositions of peptides affect the antioxidant efficacy and resistance to intestinal peptidases, Food Research International (2015), doi: 10.1016/j.foodres.2015.12.013

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ACCEPTED MANUSCRIPT Bioavailability of peptides from casein hydrolysate in vitro: Amino acid compositions of peptides affect the antioxidant efficacy and

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resistance to intestinal peptidases

Running title: Amino acid compositions affect casein peptide bioavailability

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Chan Wang, * Bo Wang, * Bo Li, *, †, 1

* College of Food Science and Nutritional Engineering, China Agricultural University, Beijing

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100083, China.

† Key Laboratory of Functional Dairy, Ministry of Education, Beijing 100083, China. 1

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Corresponding author, Bo Li

Address: P.O. Box 294, Qinghua East Road 17, Haidian District, Beijing 100083, People’s Republic of China

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Email: [email protected]

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Fax: 86-10-62738988

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Phone: 86-10-62738988

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ACCEPTED MANUSCRIPT Abstract Measurements of in vitro bioactivity to support health benefits of bioactive compounds should be accomplished with estimates of their bioavailability to bolster nutritional significance to

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health claims. In vitro bioavailability of casein and casein peptides (casein hydrolysate and four peptide fractions) that measured by the amount of peptide nitrogen are discussed. Antioxidant

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activities during gastrointestinal digestion and Caco-2 cell absorption were investigated as indices of peptide degradation. The antioxidant capacities of Trolox equivalent and oxygen radicals were used for assessing antioxidant efficacy of surviving peptides. Results showed that casein

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hydrolysate improved bioavailability compared to casein. Amino acid composition of peptides affected the resistance of peptides to digestive enzymes and intestinal peptidases. The acidic

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peptide fractions had higher bioavailability and a higher residual ratio of antioxidant activity. The peptides in the digest and absorbate of acidic fraction F1 with the highest bioavailability (23.14%)

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and the residual ratio of antioxidant activity were identified, and 12 intact, absorbed peptides (IAP)

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were obtained. Eleven of twelve of the IAPs were from β-Casein, and their amino acid components were rich in acidic and hydrophobic amino acids. Identification of IAPs might

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provide insight into the mechanism of how peptide structure provides resistance against peptidases by Caco-2 cells. Keywords

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Antioxidative peptides, Casein, Bioavailability, Intact absorbed peptide, Caco-2 cell

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ACCEPTED MANUSCRIPT 1. Introduction Bioactive peptides are defined as specific protein fragments that have a positive effect on body functions and conditions that may affect health ultimately (Miguel et al., 2008). Peptides

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from various dietary sources have been shown to have a positive impact on health by functioning as opiates, immunomodulators, anticarcinogens, antimicrobials, anticariogenics, antioxidants,

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antithrombotic agents, and antihypertensive (Korhonen & Pihlanto, 2006; Ohsawa et al., 2008). Bioactive peptides are vital components of functional foods, a research area of high interest due to their therapeutic value and importance in the food industry (Segura-Campos, Chel-Guerrero,

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Betancur-Ancona & Betancur-Ancona, 2011). Among the numerous bioactive peptides, antioxidant peptides are peptides in which researchers are interested. Antioxidant peptides have

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positive effects on protecting our body against oxidative damage produced by free radicals or reactive oxygen species (Zhang et al., 2009).

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The physiological activity of active peptides thus far reported was evaluated directly after

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enzymatic preparation, separation, and purification (Li, Chen, Wang, Ji & Wu, 2007; Kim et al., 2009). However, one of the greatest challenges when developing the nutraceutical and functional

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food products is proving the in vivo efficacy of their bioactive components. The gastrointestinal (GI) tract is known to be a major barrier for the oral administration of bioactive components. Recently, simulated gastric and intestinal digestions have been applied to evaluate gastrointestinal

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stability of antioxidant peptides (You, Zhao, Regenstein, & Ren, 2010). However, the barrier of intestinal absorption, which contains numerous peptidases, deserves more attention for assessing actual bioavailability. Caco-2 cell models are recognized as a tool to simulate intestinal absorption by expressing numerous brush border membrane peptidases similar to those of human intestinal epithelium (Ohsawa et al., 2008). In vitro GI digestion and Caco-2 cell absorption models are the most rapid tools to estimate bioavailability of a bioactive substance (Hur, Lim, Decker & Mc-Clements, 2011). Implementation of the potential physiological effect of bioactive peptides depends largely on its ability to remain intact until reaching the target organ after oral intake (Miguel et al., 2008). Previous research indicated that the ability of peptides to resist enzymatic attack depends partly on their amino acid composition. Peptides with proline (P) and hydroxyproline residues generally are able to resist degradation by digestive enzymes (Segura-Campos et al., 2011). In our previous 3

ACCEPTED MANUSCRIPT research, we studied the GI digestion stability of casein-derived peptides with different charge properties, and the results showed that negatively charged fractions, which contained a high concentration of acidic amino acids, had stronger resistance to GI digestive enzymes. However,

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whether acidic peptides can resist peptidases following absorption has not been investigated. Casein is the main protein in milk, and various bioactive peptides have been identified from with opiate-like (Silva & Malcata, 2005), immunomodulatory

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casein hydrolysates

(Malinowski et al., 2014), antibacterial (Tomita et al., 1991), antihypertensive (del Mar Contreras et al., 2009), and antioxidant properties (Rival, Fornaroli, Boeriu & Wichers, 2001). In this

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paper, antioxidant peptides from casein were chosen as a model of bioactive peptides for assessing its bioavailability. The casein was hydrolyzed by proteases to obtain antioxidant hydrolysates.

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Antioxidant peptides with different charges were separated from the hydrolysates using ion exchange chromatography. A three-stage in vitro model system of simulated gastric juice -

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intestinal juice and Caco-2 cell monolayers was used to simulate the process of human

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gastrointestinal digestion and absorption of peptides. Peptide nitrogen content and antioxidant activities were used as the indices for peptide degradation and bioavailability. The intact absorbed

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peptides (IAP) were identified from acidic fractions using nano UPLC-ESI-qTOF-MS. 2. Materials and methods 2.1. Materials

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Casein (C3400), alcalase (P4860, ≥2.4 U/g), pepsin (P7000, ≥250U/mg), pancreatin (P1750, 4×USP specifications), TNBS (P2297, picrylsulfonic acid solution), ABTS (A1888, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), L-glutathione reduced, and trolox (238813, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma-Aldrich (Shanghai, China). Dulbecco modified Eagle’s minimal essential medium (DMEM), fetal bovine serum (FBS), non-essential amino acids (NEAA), penicillin, streptomycin, and trypsin–EDTA were products of Hyclone (Thermo Scientific). Other reagents were purchased from Beijing Chemical Reagent Co., Ltd. (Beijing, China). 2.2. Preparation of casein hydrolysate Casein hydrolysate was prepared with alcalase, and the hydrolysis process was carried out using the method reported by Ao and Li (2013), with some modification. The casein was mixed with distilled water as a substrate for the production of protein hydrolysate. The enzymatic 4

ACCEPTED MANUSCRIPT hydrolysis was performed at the optimal pH of 8.0. The substrate concentration of the hydrolysate was 5%, and the ratio of enzyme to substrate was 2% (w/w). The mixture was maintained at a constant pH during hydrolysis using 0.5 M NaOH and 0.5 N HCl. The hydrolysis process was

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carried out in a water bath shaker at 60℃ for 4 h. Inactivation of the enzymes was accomplished by heating for 10 min in boiling water and the pH was adjusted to 7.0 with 0.01M HCl. Then, the

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casein hydrolysate was centrifuged at 8000 g for 15 min at 4 ℃. The supernatant was collected, freeze-dried, and stored at -80 ℃.

2.3. Separation of casein hydrolysate by SP sephadex C-25

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Casein hydrolysate was further separated by cation exchange chromatography to obtain casein peptide fractions with different charges. Ion-exchange chromatography (IEC) is a versatile

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method for separating peptides based on exploiting differences in electrostatic charge (Kang & Frey, 2003). Lyophilized casein hydrolysate was dissolved in 20 mM sodium acetate buffer with

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pH 4.0 at a ratio of 1:2 (w/v) and filtered through a 0.45 μm filter. Then the filtrate (1 mL) was

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applied to a SP-Sephadex C-25 (GE Healthcare life science) cation exchange column (2.6 ×25 cm) equilibrated previously with buffer A ( 20 mM sodium acetate buffer with pH 4.0). The column

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was eluted with buffer A from 0 to 120 min, and then eluted with buffer A containing 0.5 M NaCl from 120 to 300 min at a flow rate of 2 mL/min, and the elution was monitored by UV absorbance at 220 nm. The elution profiles were produced using a HD-A chromatography data handling

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system (Shanghai Qingpu Huxi Instruments Factory, Shanghai, China). Fractions (F1-F4) were collected separately and concentrated using a rotary evaporator, and then the concentrated solutions were dialyzed for 12 h at 4℃ with a 500 Da molecular-weight cut-off semipermeable membrane (Beijing Chemical Reagent Co., Ltd.) to remove the NaCl in the collected solutions. During the diafiltration process, distilled water was replaced every 2 hours to ensure the effect of desalination. Finally, each fraction was freeze-dried for further analysis. 2.4. Analysis of amino acid composition Amino acid composition of peptide isolates was determined by high performance liquid chromatography using phenylthiocarbamyl (PITC), pre-column derivatization, according to the method of Vasanits and Molnár-Perl (1999), with some modifications. Triplicate samples were hydrolyzed in a glass tube fitted with a cap with 6 N HCl at 110℃ for 24 h. Amino acid analysis was conducted on a Shimadzu LC-15C HPLC system after precolumn derivatization with PITC 5

ACCEPTED MANUSCRIPT and triethylamine. The derivative amino acid solutions were filtered through a 0.22 μm microfiltration membrane, and the filtrates were injected onto the RP-HPLC analysis system equipped with a HPLC column (ZORBAX SB-C18, 4.6 mm i.d. × 250 mm, 5μm, Agilent

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Technologies, USA) and detected at 254 nm. Amino acid standard solutions were used as a standard and the contents of individual amino acids were expressed as g/100 g of the total amino

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acids in each sample. 2.5. Analysis of molecular weight distribution

The molecular weight distribution of casein hydrolysate was analyzed by size exclusion with

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a HPLC (SE-HPLC) using the Shimadzu LC-15C HPLC system (Shimadzu Scientific Instruments, Kyoto, Japan) as described by Xie, Liu, Wang, and Li (2014), with some modifications. Samples

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were dissolved in buffer (45% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid), and aliquots of 10 μL samples (2 mg/mL) were filtered with a 0.22 μm microfiltration membrane that

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were loaded onto a TSK gel G2000 SWXL column (7.8 × 300 mm, TOSOH, Tokyo, Japan).

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Samples were eluted with the same buffer at a flow rate of 0.5 mL/min, and monitored at 214 nm. Bacitracin (1,423 Da), WPWW (tetrapeptide, 674 Da), NCS (tripeptide, 322 Da), and Gly-Sar

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(146 Da) were used as standards to obtain a molecular weight calibration curve, which was established between retention times and logs of molecular masses of the standards. 2.6. In vitro simulated gastrointestinal (GI) digestion

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Casein hydrolysate fractions were digested enzymatically with pepsin and pancreatin, according to the method of Wang, Li, Wang, and Xie (2015), with some modifications. The samples were diluted to 10 mg/ml with 0.01 mol/L HCl (pH 2.0). Using a ratio of enzyme to substrate of 1:50 (w/w), pepsin was added and the mixture was incubated in a shaking platform for 2 h at 37°C. The pH was first adjusted to 5.3 with 0.9 mol/L NaHCO3 and, subsequently, adjusted to pH 7.5 after adding 2 mol/L NaOH. Pancreatin (enzyme to substrate ratio 1:25, w/w); the mixture was incubated in a water bath for 2 h at 37°C under constant stirring. Finally, the aliquots were submerged in boiling water for 10 min to terminate the enzymatic digestion. The pH of the digest was adjusted to 7.0 with 0.01M HCl. Then the digest was centrifuged at 8000 g for 10 min at 4°C. The supernatants were collected, freeze-dried, and stored at -80°C. Casein and GSH were used as the control samples during the in vitro simulated gastrointestinal digestion. 2.7. Caco-2 cell absorption 6

ACCEPTED MANUSCRIPT The cell culture of Caco-2 was carried out as described previously by Ohsawa et al. (2008), with a slight modification. Caco-2 cells were cultured in tissue culture flasks (Nunc, 25 cm2) in Dulbecco’s Modified Eagle’s Medium (DMEM) that contained 20% (v/v) fetal bovine serum

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(FBS), 1% (v/v) nonessential amino acid, penicillin (100 units/mL), and streptomycin (100 μg/mL) in a fully humidified (90% relative humidity) atmosphere with 5% CO2 at 37°C. Cell culture

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medium was replaced every other day. The cells used were at passages 30-40. Caco-2 cells (1.5 mL of 1×105 cells/mL) were seeded onto cell culture inserts (0.4 μm pore size, 4.2 cm2 grown surface, Nunc) in a 6-well culture companion plate, which was used for the

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cell permeability study. Cell culture medium was changed carefully every other day for at least 21 days until the Caco-2 cells were differentiated fully as monolayers. A Millicell Voltohmmeter

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(Millipore Corp., Bedford, Massachusetts,USA) was used to measure the transepithelial electrical resistance (TEER), which can reflect the integrity of the monolayer. Only the Caco-2 monolayers

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with a TEER of at least 900 Ω were chosen for the transport study (Samaranayaka, Kitts &

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Li-Chan, 2010). Cell permeability measurements were conducted in triplicate. As Kotzé et al. (1997) described, fully differentiated cell monolayers were washed gently

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twice with pre-warmed Hank’s buffered saline solution (HBSS) at pH 7.2, and the cells were equilibrated under the pH condition at 37°C for 30 min. Then, the apical HBSS was removed, and aliquots of 1.5 mL of peptide digest (10 mg/mL) in HBSS were added on the apical side. Solvent

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blanks without peptide digest also were added to the apical side. The basolateral side contained 2.5 mL fresh pre-warmed HBSS. The cell culture plates were incubated in a fully humidified (90% relative humidity) atmosphere with 5% CO2 at 37°C for 2 h. The absorbed permeates on the basolateral side were collected and freeze-dried for further assays. 2.8. Antioxidant activity assay 2.8.1. Antioxidant activity of the Trolox equivalent The ABTS·+ radical scavenging activities of samples were assayed using the method reported by You et al. (2010), with a slight modification (Wang et al., 2015). GSH was used as a positive control and the activities of samples were interpolated to calculate the concentration in trolox equivalent (TE). 2.8.2. Oxygen radical antioxidant capacity The ORAC assay, developed by Cao, Alessio, & Cutler (1993), and Dávalos, 7

ACCEPTED MANUSCRIPT Gómez-Cordovés, & Bartolomé (2004), was used in this study with a slight modification. Fluorescein (FL) was used as a probe, and black 96-well microplates and a Tecan Infinite M200 microplate reader equipped with Tecan i-Control software were used as reported by Wang et al.

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(2015). The ORAC values of the samples were expressed as μmol/l Trolox equivalents (μmol/l TE).

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2.8.3. Residual ratio of antioxidant activity during Caco-2 cell absorption

The stability of peptide fractions during simulated gastrointestinal digestion and Caco-2 cell absorption was determined by the residual ratio of antioxidant activity (RRAA). RRAA was

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expressed as follows: RRAA= (RAA/IAA)×100, where RAA and IAA are the remaining

2.9. Determination of bioavailability

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antioxidant activity and the initial antioxidant activity, respectively.

Bioavailability (BA) of casein fractions was measured by the amount of peptide nitrogen

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(PN), as reported by Xie, Wang, Ao, and Li (2013). The PN analysis was based on amino group

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content measured according to the TNBS methods (Wang et al., 2015). The content of PN was calculated as the difference between total nitrogen and amino acid nitrogen. For measuring free

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amino acid content, samples were dissolved in 3.5% sulfosalicylic acid, and aliquots of the solutions were centrifuged at 8000 ×g for 10 min; the supernatant was collected and used for the TNBS assay. To determine total nitrogen content, samples were hydrolyzed previously with 6 M

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HCl at 110 °C for 12 h. The fully hydrolyzed sample, containing free amino acids, was used for the TNBS assay.

As Xie et al. (2013) reported, the BA of each peptide fraction was expressed as BA= (RPN/IPN) × 100, where RPN and IPN are the remaining peptide nitrogen after intestinal absorption and the initial peptide nitrogen before intestinal absorption, respectively. 2.10. Peptide sequence analysis by nanoESI-QTOF-MS/MS A Waters Corporation NanoAcquity liquid chromatograph interfaced to a Waters Corporation QTOF mass spectrometer Q-TOF-2 was used for the identification of the peptides in the digest and absorbate of F1. A Waters 75 μm ×250 mm BEH C18 column with 1.7 μm particle was used for the separation of the fraction. Peptides were eluted with eluent A (distilled water containing 0.1% methanoic acid), eluent B (Acetonitrile containing 0.1% methanoic acid), and eluent C (distilled water containing 0.1% methanoic acid). After 5 minutes, desalination with eluent C in the 8

ACCEPTED MANUSCRIPT pre-column at a flow rate of 200 nL/min was used with the following gradient: 10% eluent B at 5 min, increasing to 85% eluent B at 65 min, and maintained at 85% eluent B for 20 min. Mass range of 350-1500 and 50-2000 were scanned for the MS and MS/MS procedures, respectively.

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Interpretations of spectra (MS/MS) were made with Mascot Science Search software. 2.11. Statistical analysis

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All experiments were conducted in triplicate. Data were expressed as means ± standard deviation (SD). The data were analyzed by ANOVA and Duncan’s new multiple range test using SPSS 18.0 (SPSS Inc, Chicago, IL, USA). Statistical significance was set at p < 0.05.

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3. Results and discussion

3.1. Characteristics of casein hydrolysate and casein peptide fractions

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A total of four main fractions (F1- 4) were obtained after the separation of casein hydrolysate on the cation exchange column (Fig. 1A). According to the principles of cation exchange

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chromatography, positively charged peptides bind well to the negatively charged matrix, so that

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the negatively charged fractions 1 and 2 were eluted first using the equilibration buffer. Then, positively charged fractions (F3 and F4) were eluted afterward with equilibration buffer that

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contained 0.5 M NaCl. Although the methods of preparation and separation of casein hydrolysate were similar to that reported by Ao and Li (2013), the observed chromatographic profiles in our research and theirs were a little different due to the differences in samples, the amount of sampling,

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volumes of resin, and the elution program. In the chromatographic profile obtained by Ao and Li (2013), the casein hydrolysate was separated into six fractions clearly on SP Sephadex C-25 column，while the corresponding fractions 1 and 2, as well as fractions 4 and 5, were not separated clearly in our results. 3.1.1. Molecular weight distributions The casein hydrolysate was analyzed for molecular weight distribution (Fig.1B). Chromatographic data indicated that the casein hydrolysate was composed of low molecular weight peptides whose major peaks were located at 500–1000 Da (77.58%) and 1000–3000 Da (19.90%). The fraction with a molecular weight lower than 500 in casein hydrolysate was 2.32%, which would have been filtered out by the 500 Da molecular-weight semipermeable membranes during the diafiltration process with salt at the end of separation procedure by SP sephadex C-25. 3.1.2. Amino acid composition 9

ACCEPTED MANUSCRIPT The amino acid composition of casein hydrolysate (H) and the four casein peptide fractions (F1, F2, F3, and F4) are presented in Table 1. All samples contained low levels of cysteine, but they were all rich in glutamic acid, which was due to large amounts of glutamic acid in casein

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(Agudelo, Gauthier, Pouliot, Marin, & Savoie, 2004). F1 contained Asp and Glu at approximately 42.64%, which was the largest amount of acidic amino acid among the four fractions, followed by

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F2 (33.84%). Conversely, there were more basic amino acids (Arg, His, and Lys) in F3 and F4 at approximately 15.33% and 37.90%, respectively. From F1 to F4, the content of acidic amino acids decreased gradually, and the content of basic amino acids increased gradually. The results were

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consistent with the charge characteristics of each fraction. 3.1.3. Antioxidant activities

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Previous reports showed that alcalase is capable of producing antioxidant peptides from numerous protein sources (Li, Jiang, Zhang, Mu, & Liu, 2008). Both casein hydrolysate (H) and

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casein peptide fractions (F1-F4) were tested for TEAC and ORAC values as showed in Fig.1C and

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D, respectively. The TEAC and ORAC values of H were higher than that of the four fractions (F1-F4) (p＜0.05). The result might be due to the loss of antioxidant activities in the four fractions

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during the separation, desalting, and concentration process. In F1-F4, the fractions with more acidic amino acids (F1 and F2) had lower TEAC and ORAC values than the fractions with more basic amino acids (F3 and F4) (p＜0.05). Reduced glutathione (GSH) and alcalase were used as

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control, respectively. TEAC values of H (5 mg/mL) were equivalent to the values of GSH with a concentration of 1mg /mL (p＞0.05), but the ORAC values of H were significantly lower than that of GSH with the same concentration (p＜0.05). Compared to casein peptides, alcalase with the same concentration showed little antioxidant activities. 3.2. Degradation of different peptide fractions during digestion and absorption The change in content of PN was used as an indicator to reflect the degree of degradation of peptide fractions in the digestive process. With the action of digestive enzymes, the original peptide bonds of peptides are broken and small peptides or free amino acids are formed. The more the polypeptides are degraded, the higher the amino nitrogen content should be, and the lower should be the content of PN. The content of PN is quantified by the difference between the total nitrogen content and the amino nitrogen content. After the simulated GI digestion, the PN content of each fraction had significant losses (P < 10

ACCEPTED MANUSCRIPT 0.05) as compared to the fractions without digestion (Fig.2 (A)), but the degree of reduction in PN was different among the fractions. The PN reduction in F1 was least at 3.11%, followed by F4 (21.03%) and H (24.1%). The reductions of PN in F2 and F3 were pronounced at 33.64% and

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33.49%, respectively. During in vitro GI digestion, the fraction (F1) with the highest content of acidic amino acids showed the greatest resistance to GI digestive enzymes. This result was

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consistent with the report of Ao & Li (2013), who obtained the same result by detecting the molecular weight distributions of the digest. After permeating by Caco-2 cells, the PN content in the absorbates of F1 was the highest, followed by F3, F2, and F4 (Fig.2 (B)).

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Compared to the absorbates of casein peptide fractions, the PN content in the absorbates of casein and casein hydrolysate was lower, although the PN content of casein and casein

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hydrolysate before Caco-2 cell absorption was higher than casein peptide fractions. Apparently, the PN reduction during cell absorption was significantly higher than that during GI digestion. The

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PN reduction in F1 was least at 77.3% after Caco-2 cell absorption, and the reduction in casein

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was the largest.

Several factors can lead to the reduction of PN in proteins or peptides during absorption.

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Degradation by the intestinal peptidases and the cellular permeability of Caco-2 cells for different peptide fractions had the greatest influence. The intestinal peptidases play an important role in the degradation process of peptide to oligopeptide and free amino acids (Samaranayaka et al., 2010),

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which can lead to a sharp drop of PN in transit. Moreover, based on the length of peptides and the amino acid composition, the cellular permeability of different peptide fractions was different. Low cellular permeability may make a great difference for the drop of PN in transit (Miguel et al., 2008; Shimizu, Tsunogai, & Arai, 1997). 3.3. Antioxidant activities of different peptide fractions during digestion and absorption In the presence of peptidase, peptides are hydrolyzed into different fragments with different antioxidant activities. Therefore, the changes in antioxidant activities could be used as another indicator to evaluate the resistance of the peptides’ digestive enzymes. After the simulated GI digestion, the TEAC and ORAC values of F1-F4 increased to some extent compared to that of the undigested fractions (Fig. 2C). However, the digest of fractions with more acidic amino acids (F1 and F2) had lower TEAC and ORAC values than those with more basic amino acids (F3 and F4) due to the lower initial activities (p＜0.05). The antioxidant 11

ACCEPTED MANUSCRIPT activities of digests were affected not only by the degradation, but also by the amino acid composition. Basic amino acid residues, such as His, Arg, and Lys, promote the antioxidative activity of bioactive peptides (Chen, Muramoto, &Yamauchi, 1995). For the acidic fractions,

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hydrolysis by pepsin and pancreatin resulted in the formation of more oligopeptides that displayed higher radical, scavenging activity (Ajibola, Fashakin, Fagbemi, & Aluko, 2011). Basic fractions

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were degraded during the entire GI digestion and may be part of the oligopeptides were degraded into free amino acids. Except for the antioxidant ability of basic amino acids, amino acids with aromatic residues can donate protons to electron deficient radicals and have radical-scavenging

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properties (Rajapakse et al., 2005). The ORAC value of basic fractions that increased in the digestion might have been due to the contribution of some free amino acids to the antioxidative

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activity (Clausen, Skibsted, & Stagsted, 2009).

As presented in Fig. 2D, after Caco-2 cell absorption, the antioxidant activities of absorbates

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shifted to different extents. TEAC values of each fraction were reduced significantly, but no

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significant reduction of ORAC values were observed in F1 and F2. The antioxidant activities of absorbate in F1 or F2, including TEAC and ORAC values, exhibited higher values than that of

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GSH (p<0.05).

3.4. Residual ratio of antioxidant activity After we assayed fractions with TEAC and ORAC, the residual ratios of antioxidant activities

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(RRAA) of casein peptide fractions via absorption were similar among F1 to F4 (Fig.3 (A-1) and (A-2)). The RRAA of the fractions with more acidic amino acids (F1 and F2) were significantly higher than that of the fractions with more basic amino acids (F3 and F4) (p＜0.05), which was similar to the change in PN. According to the structure-activity relationship of peptides, the change in activity reflects the change in the peptide’s structure. In this study, we focused on the structure-peptidase resistance relationship. The fractions with more acidic amino acids (F1 and F2) were more resistant to peptidase during Caco-2 cell absorption. Therefore, the antioxidant activities of the fractions with more acidic amino acids remained stable easily during Caco-2 cell absorption, and the antioxidant activities of the fractions with more basic amino acids (F3 and F4) were more susceptible to absorption by Caco-2 cells due to peptidase degradation. During the process of absorption, polypeptides may be degraded into small peptides and free amino acids if they are susceptible to intestinal peptidases. 12

ACCEPTED MANUSCRIPT 3.5. Bioavailability of casein peptide fractions Bioavailability is a key concept for nutritional efficiency of food and food formulas, irrespective of the type of food being considered (functional or not) (Blenford, 1995). To

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understand the amounts of peptides that humans can actually utilize, bioavailability of casein peptide fractions was calculated based on the amount of peptide nitrogen. Among the four

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fractions (F1-F4), bioavailability (BA) of F1 was highest with 23.14%, BA of F4 was lowest with 10.25% (p < 0.05), and no significant differences existed between F2 and F3 (p > 0.05) (Fig. 3B). Because bioavailability of casein and casein peptides was measured based on peptide

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nitrogen, the factors that can lead to the drop of PN during absorption will have a negative effect on bioavailability. Namely, the difference in BA of the casein peptide fraction may be caused by

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the resistance of peptides to digestive enzymes and the cellular permeability of Caco-2 cells. Rojanasakul et al. (1992) reported that paracellular transport, which is a type of transport in which

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a substance may be absorbed, is connected closely to the charge property of peptides. This is

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because the space in tight junctions exhibits an electrostatic field with a negative net charge and the paracellular flux of peptides may be influenced by the charge-charge interactions. According

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to the literature report (Rojanasakul et al., 1992), basic components of peptides with a positive net charge are transported more easily by electrostatic attraction along the paracellular pathway. However, the most favorable charge for peptides for the paracellular transport is still inconclusive.

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In the present study, F1, the negatively charged fraction, exhibited better permeability for the paracellular route than F4, the positively charged fraction, which resulted in different bioavailability. Rubas et al. (1995) also concluded that the paracellular transport of a series of peptides with a net charge of -1 to -2 exhibited optimum permeability, which was further confirmed in our study. 3.6. Characterization of peptide sequences surviving the in vitro gastrointestinal digestion and Caco-2 cell absorption The fraction (F1) with the highest bioavailability was selected to identify the intact absorbed peptides (IAP) that are resistant to peptidase. For peptide identification, the peptides in the GI digestion solution and the basolateral-chamber solution after 120 min incubation by Caco-2 cells were analyzed by an analytical C18 HPLC and then subjected to nano-electrospray ionization-Q-time of flight MS/MS. According to the total ion chromatograms, peptides in the 13

ACCEPTED MANUSCRIPT digest and the absorbate had some ion peaks with similar mass-to-charge ratios, such as 728.42, 756.34 (756.42), 768.40 (768.36), and 879.45 (879.44) (Fig.4 (A-1) and (A-2)). Interpretation of the MS/MS spectra was performed using Mascot Science Search software.

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By sequence comparison with the casein amino acid sequence (bovine) in NCBI, a total of 26 peptides were identified in the F1 GI digest, while a total of 35 peptides were identified in the

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absorbate (Table 2). By comparing the identified peptide sequences, 12 IAPs were obtained (Table 2). The numbers of amino acid residues of these peptides were different, and varied from 5 to 20. However, it is well known that the intestinal absorption of natural di- and tripeptide is

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mainly an active, transporter-mediated process by Pep T1 (Daniel, 2004). Shorter sequences less than 4 amino acid residues may be more bioavailable than the longer sequences which were not

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identified in the present manuscript. Among the 12 IAPs, only one peptide sequence was from αs1-CN (αs1 -CN 174-186), the others were all from β-CN; Many of these peptides were rich in

D

acidic amino acids. One of the IAPs (PPFLQPE) with a molecular weight of 827 Da is illustrated

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in Fig.4 (B). The MS/MS spectrograms of PPFLQPE in GI digestion solution and in basolateral-chamber solution after 120 min of incubation of fraction F1 are shown in Fig.4 (B-1)

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and (B-2), respectively. The IAPs identified in this research were a little different with the result reported by Picariello et al. (2010), due to the difference in samples and methods. The amino acid residues at the N-terminal and C-terminal of the IAPS were analyzed.

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Proline (P), valine (V) and threonine (T) were the amino acid residues at the N-terminus, and valine and glutamic acid (E) were the main amino acid residues at the C-terminal. This may be related to the relatively high content of these four amino acids in the peptide fractions. However, the most convincing reason for this result might be the specific cleavage sites of peptidases in the intestinal epithelium. In the present study, the Caco-2 cell monolayer model was used to simulate the intestinal absorption. Caco-2 cells have been used widely to study the absorption of nutrients for their ability to differentiate into enterocyte-like cells. Differentiation in Caco-2 cells is characterized by the formation of tight junctions and the appearance of an apical brush border on which many hydrolases are expressed, such as aminopeptidase N, P, W, dipeptidyl peptidase Ⅳ(DPP-Ⅳ), endopeptidase-24.11, γ-glutamyl transpeptidase, microsomal dipeptidase, and peptidyl dipeptidase A (Chantret et al., 1988; Howell, Kenny & Turner, 1992). These enzymes are members of a group 14

ACCEPTED MANUSCRIPT of cell-surface peptidases, which are responsible for the degradation of peptides before absorption into the blood stream. Barret, Rawlings and Woessner, (2004) reported that the aminopeptidases can be divided

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into leucine aminopeptidase, valine aminopeptidase, phenylalanine aminopeptidase, and proline aminopeptidase, depending on the most responsive substrate. Different aminopeptidases have

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different abilities to hydrolyze amino acid residues, while the same aminopeptidase has a different ability to hydrolyze different amino acid residues at the N-terminus. The amino acid residues P, V, and T at the N-terminal may be more resistant to peptidase. Furthermore, previous studies have

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reported that proline-containing peptides were resistant to intestinal proteolysis (Cardillo et al., 2003; Mizuno, Nishimura, Matsuura, Gotou, & Yamamoto, 2004). In this study, most of the

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identified IAPs were proline-rich peptides, which was consistent with the previous report. It is notable that IAPs also contain a high content of valine in their sequences. However, whether the

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high content of valine contributes to the resistance to peptidase degradation is inconclusive.

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4. Conclusions

Studies have identified a great number of peptide sequences with specific bioactivities in the

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major milk proteins and also the conditions for their release. However, estimates of their bioavailability are reported rarely. In this study, in vitro bioactivity and bioavailability of casein, casein hydrolysate and peptide fractions were investigated. The results revealed that casein

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hydrolysate improved bioavailability significantly compared to casein. Furthermore, amino acid composition of peptides affected the resistance of peptides to digestive enzymes and intestinal peptidases. Although the fractions with more basic amino acids had higher antioxidant activity, the acidic fractions had higher bioavailability and a higher residual ratio of antioxidant activity based on

TEAC

and

ORAC.

Twelve

intact

absorbed

peptides

were

identified

by

nano-ESI-QTOF-MSMS.Some of the IAPs were reported to exhibit bioactivities. For example, TQTPVVVPPFLQPE had antioxidant activity that was identified from yoghurt peptides (Farvin, Baron, Nielsen, Otte, & Jacobsen, 2010); TEDELQDKIHP had ACE-inhibitory activity (Hayes, Stanton, Slattery, O'Sullivan, Hill, et al., 2007). The results add further support to the feasibility of casein peptide as a provider of functional ingredients. Furthermore, because the characteristics of amino acid residues at the N-terminal were obvious, the results highlight the structural properties of IAPs. Although it seems that peptides at the N-terminal that are rich in proline and valine are 15

ACCEPTED MANUSCRIPT somehow resistant to peptidases, an experiment using defined synthetic peptides

should be

conducted in the future. Acknowledgments

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This research was supported by National Natural Science Foundation of China (NSFC, No. 31271846) and the Program for New Century Excellent Talents in the University of Ministry of

AC

CE P

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D

MA

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Education of China (Grant No. 2010JS078).

16

ACCEPTED MANUSCRIPT References Agudelo, R. A., Gauthier, S. F., Pouliot, Y., Marin, J., & Savoie, L. (2004). Kinetics of peptide fraction release during in vitro digestion of casein. Journal of the Science of Food and

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T

Agriculture, 84 (4), 325-332.

Ajibola, C. F., Fashakin, J. B., Fagbemi, T. N., & Aluko, R. E. (2011). Effect of peptide size on

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simulated gastrointestinal digestion in vitro: Charge properties of peptides affect digestive stability. Food Research International, 52 (1), 334-341.

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Barret, A. J., Rawlings, N. D., & Woessner, J. F. (2004). Serine and threonine peptidases. Handbooks of Proteolytic Enzymes, 2, 1417-1447.

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Blenford, D. (1995). Bioavailability is key to nutrient effectiveness. Food Ingredients and

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Processing International, 17, 28-30.

Cao, G., Alessio, H. M., & Cutler, R. G. (1993). Oxygen-radical absorbance capacity assay for

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Organic & biomolecular chemistry, 1(9), 1498-1502. Chen,H.,Muramoto, K., & Yamauchi, F. (1995). Structural analysis of antioxidative peptides from soybean.beta.-conglycinin. Journal of Agricultural and Food Chemistry, 43(3), 574–578. Chantret, I., Barbat, A., Dussaulx, E., Brattain, M. G., & Zweibaum, A. (1988). Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer research, 48(7), 1936-1942. Clausen, M. R., Skibsted, L. H., & Stagsted, J. (2009). Characterization of major radical scavenger species in bovine milk through size exclusion chromatography and functional assays. Journal of Agricultural and Food Chemistry, 57 (7), 2912–2919. Daniel, H. (2004). Molecular and integrative physiology of intestinal peptide transport. Annual Review of Physiology, 66, 361-384. Dávalos, A., Gómez-Cordovés, C., & Bartolomé, B. (2004). Extending applicability of the oxygen 17

ACCEPTED MANUSCRIPT radical absorbance capacity (ORAC_fluorescein) assay. Journal of Agricultural and Food Chemistry, 52 (1), 48–54. del Mar Contreras, M., Carrón, R., Montero, M. J., Ramos, M., & Recio, I. (2009). Novel

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Kim, E.K., Lee, S.J., Jeon, B.T., Moon, S.H., Kim, B.k., Park, T.K., Han, J.S., & Park, P.J. (2009). Purification and characterisation of antioxidative peptides from enzymatic hydrolysates of venison protein. Food Chemistry, 114, 1365-1370. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: production and functionality. International Dairy Journal, 16 (9), 945–960. Kotzé, A. F., de-Leeuw, B. J., Lueßen, H. L., de-Boer, A. G., Verhoef, J. C., & Junginger, H. E. (1997). Chitoans for enhanced delivery of therapeutic peptides across intestinal epithelia: in vitro evaluation in Caco-2 cell monolayers. International Journal of Pharmaceutics, 159 (2), 24-253. Li, B., Chen, F., Wang, X., Ji, B. P., & Wu, Y. N. (2007). Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromato-graphy and electrospray ionization-mass spectrometry. Food Chemistry, 102, 1135-1143. 18

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digestibility of antihypertensive β-Casein tripeptides, Val-Pro-Pro and Ile-Pro-Pro, in the gastrointestinal tract: analyses using an in vitro model of mammalian gastrointestinal

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Biological and toxicological implications. Journal of Chromatography B, 878, 295-308. Rajapakse N., Mendis E., Byun H. G., & Kim, S. K. (2005). Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems. Journal of Nutritional Biochemistry, 16 (9), 562-569. Rival, S. G., Fornaroli, S., Boeriu, C. G., & Wichers, H. J. (2001). Caseins and casein hydrolysates. 1. Lipoxygenase inhibitory properties. Journal of Agricultural and Food Chemistry, 49 (1): 287-294. Rojanasakul, Y., Wang, L. Y., Bhat, M., Glover, D. D., Malanga, C. J., & Ma, J. K. (1992). The transport barrier of epithelia: a comparative study on membrance permeability and charge selectivity in the rabbit. Pharmaceutical Research, 9 (8), 1029-1024. Rubas, W., Villagran, J., Cromwell, M., Mcleod, A., Wassenberg, J., & Mrsny, R. (1995). Correlation of solute flux across Caco-2 monolayers and colonic tissue in vitro. STP Pharma 19

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human intestinal cell, Caco-2. Peptides, 18 (5), 681-687.

Silva, S. V., & Malcata, F. X. (2005). Caseins as source of bioactive peptides. International Dairy

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Tomita, M., Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., & Kawase, K. (1991).

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Potent Antibacterial Peptides Generated by Pepsin Digestion of Bovine Lactoferrin. Journal

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of Dairy Science, 74 (12), 4137-4142. Vasanits, A., & Molnár-Perl, I. (1999). Temperature, eluent flow-rate and column effects on the

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retention and quantitation properties of phenylthiocarbamyl derivatives of amino acids in reversed-phase high-performance liquid chromatography. Journal of Chromatography A, 832 (1), 109–122.

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Wang, C., Li, B., Wang. B., & Xie, N. N. (2015). Degradation and antioxidant activities of peptides and zinc–peptide complexes during in vitro gastrointestinal digestion. Food Chemistry, 173, 733-740. Xie, N. N., Liu, S. S., Wang, C., & Li, B. (2014). Stability of casein antioxidant peptide fractions during in vitro digestion/Caco ‑ 2 cell model: characteristics of the resistant peptides. European Food Research and Technology, 239 (4), 577-586. Xie, N. N., Wang, C., Ao, J., & Li, B. (2013). Non-gastrointestinal-hydrolysis enhances bioavailability and antioxidant efficacy of casein as compared with its in vitro gastrointestinal digest. Food Research International, 51 (1), 114-122. You, L. J., Zhao, M. M., Regenstein, J. M., & Ren, J. Y. (2010). Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion. Food Chemistry, 120 (3), 810–816. 20

ACCEPTED MANUSCRIPT Zhang, J., Zhang, H., Wang, L., Guo, X., Wang, X., & Yao, H. (2009). Antioxidant activities of the rice endosperm protein hydrolysate: identification of the active peptide. European Food

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Research and Technology , 229 (4), 709-719.

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ACCEPTED MANUSCRIPT

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Figure 1

22

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D

MA

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IP

T

ACCEPTED MANUSCRIPT

AC

CE P

Figure 2

23

D

MA

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IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

Figure 3

24

D

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NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

Figure 4

25

ACCEPTED MANUSCRIPT Figure captions Fig.1. (A) Chromatographic profile obtained by fractionation of Casein-Alcalase hydrolysate on

IP

T

SP Sephadex C-25 cation exchange column. The casein hydrolysate was separated into 4 fractions

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(F1-F4) on SP Sephadex C-25 column. F1 and F2 were eluted by mobile phase A (20 mM, pH 4.0 sodium acetate buffer), F3 and F4 were eluted by mobile phase B (mobile phase A + 0.5 M NaCl). (B) Molecular weight distribution of Casein-Alcalase hydrolysate: Cchromatogram of

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Casein-Alcalase hydrolysate separated using TSK gel G2000 SWXL column. (C) Trolox

MA

equivalent antioxidant activity (TEAC) of casein hydrolysate, and casein peptide fractions isolated by SP Sephadex C-25 cation exchange column with GSH and Alcalase as control. (D) Oxygen

D

radical antioxidant capacity (ORAC) of casein hydrolysate, and casein peptide fractions isolated

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by SP Sephadex C-25 cation exchange column with GSH and Alcalase as control. The data with

CE P

different lowercase letters in test are significantly different (p < 0.05).

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Fig.2. (A) Changes of Ppeptide nitrogen content and antioxidant activitychanges of casein, casein hydrolysate, and casein peptide fractions during simulated gastrointestinal digestion and Caco-2 cell absorption. Peptide nitrogen content before and after simulated gastrointestinal digestion (A), and before and after Caco-2 cell absorption (B). (B) Peptide nitrogen changes of gastrointestinal digestes of casein, casein hydrolysate, and casein peptide fractions after Caco-2 cell absorption. (C) Antioxidant activity before and after simulated gastrointestinal digestion detected by TEAC (C-1) and ORAC (C-2), and before and after Caco-2 cell absorption detected by TEAC (D-1) and ORAC (D-2).changes of casein, casein hydrolysate, and casein peptide fractions by TEAC (C-1) and ORAC (C-2) after simulated gastrointestinal digestion. (D) Antioxidant activity changes of 26

ACCEPTED MANUSCRIPT casein, casein hydrolysate, and casein peptide fractions by TEAC (D-1) and ORAC (D-2) after simulated gastrointestinal digestion. The data with different lowercase letters in test are

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significantly different (p < 0.05), and the lowercase with and without superscript represented the

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comparison within the different groups.

Fig.3. (A) Residual ratio of antioxidant activities (RRAA) of casein, casein hydrolysate, and

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casein peptide fractions detected by TEAC (A-1) and ORAC (A-2) after in vitro digestion and

MA

absorption. (B) Bioavailability of casein, casein hydrolysate, and casein peptide fractions. The

D

data with different lowercase letters in test are significantly different (p < 0.05).

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Fig.4. (A) The total ion chromatograms of gastrointestinal digestion solution (A-1) and the

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basolateral-chamber solution after 120 min incubation (A-2) of fraction (F1). The ion peaks with similar mass-to-charge ratios in the chromatograms were pointed by arrow symbol. (B) The

AC

MS/MS spectrograms of resistant peptide PPFLQPE (one of the IAPs) in gastrointestinal digestion solution (B-1) and basolateral-chamber solution after 120 min incubation (B-2).

27

ACCEPTED MANUSCRIPT Table 1 Amino acid compositions of the casein hydrolysates (H) and the casein peptide fractions (F1-F4) isolated by SP Sephadex C-25 cation exchange column

F4

4.93±0.32

5.00±0.30

17.55±1.47

12.70±0.18

F1

F2

F3

Asp

6.67±0.41

7.14±0.76

8.98±0.04

Glu

20.91±1.14

35.50±3.01

24.86±0.78

Ser

5.23±0.27

11.73±0.99

Gly

1.78±0.11

1.77±0.12

His

2.98±0.16

1.15±0.07

Thr

3.85±0.24

4.17±0.32

Ala

2.96±0.20

Pro

IP

H

SC R

T

Relative amount (g/100 g)

2.52±0.23

2.14±0.05

1.82±0.02

2.03±0.17

1.16±0.04

0.89±0.01

4.36±0.41

6.27±0.12

5.23±0.06

2.97±0.26

1.57±0.06

3.23±0.26

3.17±0.03

1.77±0.15

1.52±0.03

9.88±0.68

5.72±0.35

11.27±0.11

15.42±1.50

6.27±0.38

Arg

3.82±0.23

1.26±0.11

1.05±0.01

3.84±0.36

11.39±0.26

Tyr

5.54±0.42

2.83±0.25

4.30±0.04

5.48±0.51

7.27±0.14

Val

6.14±0.44

5.39±0.40

6.72±0.04

7.34±0.64

4.09±0.13

Met

2.97±0.19

2.30±0.14

3.15±0.03

4.10±0.35

2.11±0.08

Cys

＜0.1

0

0

0

0

Ile

5.25±0.34

6.86±0.50

4.78±0.03

5.32±0.46

5.03±0.12

Leu

8.95±0.67

6.12±0.42

8.83±0.06

10.47±0.95

6.33±0.07

Phe

5.13±0.37

1.99±0.05

5.70±0.07

4.77±0.47

6.91±0.10

MA

D

TE

CE P

Lys

NU

5.63±0.04

7.93±0.46

2.84±0.17

3.61±0.02

7.13±0.66

20.24±0.48

a

Acidic amino acid

27.58

42.64

33.84

22.48

17.70

b

14.73

5.25

5.55

15.33

37.90

100

100.00

100.00

100.00

100.00

AC

Basic amino acid Total a b

Sum of Asp and Glu,

Sum of Arg, His and Lys

28

ACCEPTED MANUSCRIPT Table 2 The identification of peptides surviving the in vitro gastrointestinal digestion and Caco-2 cell absorption by nanoESI-QTOF-MSMS from fraction F1 Protein fragment

Amino acid sequence

Protein fragment

NVPGE

β-CN 7-11

VPPFLQPE

β-CN 84-91

PGEIV

β-CN 9-13

PPFLQPE

β-CN 85-91

TEDELQ

β-CN 41-46

PPFLQPEV

β-CN 85-92

TEDELQDK

β-CN 41-48

VPYPQ

β-CN 178-182

TEDELQDKIHP

β-CN 41-51

QGLPQEVLN

αs1-CN 9-17

DELQD

β-CN 43-47

TQTPVVVPPFLQPE

β-CN 78-91

TQTPVVVPPFLQPEV

β-CN 78-92

PVVVPPFLQPE

T

Amino acid sequence

NU

SC R

IP

Peptides in GI-digested solution

αs1-CN 172-178

YTDAPS

αs1-CN 173-178

TDAPS

αs1-CN 174-178

β-CN 81-91

TDAPSFSDIPNPI

αs1-CN 174-186

PVVVPPFLQPEV

β-CN 81-92

NQFLPYP

κ-CN 53-59

VVPPFLQPE

β-CN 83-91

VIESPPEIN

κ-CN 152-160

β-CN 83-92

ESPPE

κ-CN 154-158

β-CN 83-93

ESPPEIN

κ-CN 154-160

VVPPFLQPEVM

D TE

VVPPFLQPEV

MA

QYTDAPS

CE P

Peptides in Basolateral-chamber solution

β-CN 7-13

PPFLQPE

β-CN 85-91

PGEIV

β-CN 9-13

PPFLQPEV

β-CN 85-92

QTEDEL

β-CN 40-45

MGVSKVKEAMAPKHKEMPFP

β-CN 93-112

TEDELQDK

β-CN 41-48

HKEMPFPKYPVEPF

β-CN 106-119

TEDELQDKIHP

β-CN 41-51

MPFPKYPVEPF

β-CN 109-119

VYPFPGPIHN

β-CN 58-68

HLPLPL

β-CN 134-139

NIPPLTQTPVVVPPFL

β-CN 73-88

SWMHQPHQPLP

β-CN 142-152

NIPPLTQTPVVVPPFLQPE

β-CN 73-91

YQEPVLGPVRGPFPIIV

β-CN 193-209

NIPPLTQTPVVVPPFLQPE-V

β-CN 73-92

PVLGPVRGPFPIIV

β-CN 196-209

TQTPVVVPPF

β-CN 78-86

VLGPVRGPFPIIV

β-CN 197-209

TQTPVVVPPFLQPE

β-CN 78-91

GPVRGPFPIIV

β-CN 199-209

TQTPVVVPPFLQPEV

β-CN 78-92

PVRGPFPIIV

β-CN 200-209

TPVVVPPFLQPE

β-CN 80-91

DVPSE

αs1-CN 85-89

TPVVVPPFLQPEV

β-CN 80-92

SDIPNPI

αs1-CN 180-186

PVVVPPFLQPE

β-CN 81-91

TDAPSFSDIPNPI

αs1-CN 174-186

PVVVPPFLQPEV

β-CN 81-92

IISQE

αs2-CN 14-18

AC

NVPGEIVE

29

ACCEPTED MANUSCRIPT VVPPFLQPE

β-CN 83-91

VVPPFLQPEV

β-CN 83-92

AVALAKNTME

αs2-CN 1-5

β-CN 85-91

Intact absorbed peptides in F1 β-CN 9-13

PPFLQPE

VVPPFLQPEV

β-CN 83-92

PPFLQPEV

TEDELQDK

β-CN 41-48

TEDELQDKIHP

β-CN 41-51

PVVVPPFLQPE

β-CN 81-91

TQTPVVVPPFLQPEV

β-CN 78-92

PVVVPPFLQPEV

β-CN 81-92

TQTPVVVPPFLQPE

β-CN 78-91

VVPPFLQPE

β-CN 83-91

TDAPSFSDIPNPI

αs1-CN 174-186

SC R

IP

T

PGEIV

β-CN 85-92

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Note: Interpretations of spectra (MS/MS) were made with Mascot Science Search software (Masslynx 4.1, Waters.com) by sequence comparison with casein amino acid sequence (bovine) in NCBI. Underscore amino acid

AC

CE P

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D

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sequence is signal sequence in primary structure of αs2-Casein.

30

ACCEPTED MANUSCRIPT Highlights > Casein hydrolysate improved significantly bioavailability compared to casein

IP

T

> Amino acid composition of peptides affected the resistance to intestinal peptidases

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> Acidic peptide fractions had higher bioavailability and residual antioxidant activity > Twelve intact-absorbed peptides (IAP) were identified

AC

CE P

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> The amino acid residues at the N-terminus of IAP are Pro, Val and Thr

31