The effect of molecular weights on the survivability of casein-derived antioxidant peptides after the simulated gastrointestinal digestion

Innovative Food Science and Emerging Technologies 16 (2012) 341–348

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The effect of molecular weights on the survivability of casein-derived antioxidant peptides after the simulated gastrointestinal digestion Min Chen a, Bo Li a, b,⁎ a b

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China Key Laboratory of Functional Dairy, Ministry of Education, Beijing 100083, China

a r t i c l e

i n f o

Article history: Received 10 May 2012 Accepted 31 July 2012 Editor Proof Receive Date 29 August 2012 Keywords: Antioxidant peptide Gastrointestinal digestion Molecular weight Amino acid composition

a b s t r a c t A two-stage in vitro gastrointestinal (GI) digestion model was used to assess the survivability of antioxidant peptide fractions with different molecular weights (MW). Research on MW distribution showed that peptides above 3000 Da were more easily digested by gastric digestion than those below 3000 Da. And peptides tended to be more hydrolyzed by intestinal digestion than by gastric digestion. Determination of free amino groups suggested that peptide bonds in the peptides above 3000 Da were more inclined to be cut off during the GI digestion. However, the amount of free amino acid (AA) increased as the MW decreased and accounted for 30%–40% (w/w) of the GI digests. The peptides below 1000 Da exhibited the best initial and surviving antioxidant activities in 2, 2′-azinobis (3-ethylbenzothiszoline-6-sulphonic acid) diammonium salt (ABTS•+), hydroxyl radical scavenging and ORAC value. In conclusion, casein-derived antioxidant peptides below 1000 Da were a promising antioxidant administered orally. Industrial revelance: Numerous products are already on the market or under development by food companies, exploiting the potential of food-derived bioactive peptides. To exert physiological effects in vivo, bioactive peptides must survive the gastrointestinal (GI) digestion, and then reach their target sites after absorption. So far, only a few among the numerous bioactive peptides displaying activities in vitro have been proven effective in vivo. The real bioavailability of quite an amount of marketed products containing bioactive peptides is unclear. However, our research conducted further examination of bio-accessibility of casein-derived antioxidant peptides and assessed the effect of MW on the survivability of casein-derived antioxidant peptides after the simulated GI digestion. The survivability of peptides was above 60% after the GI digestion. And the casein-derived peptides below 1000 Da exhibited the best initial and surviving antioxidant activities. The enriched antioxidant peptide fraction above could be a promising candidate for dietary supplements and nutraceuticals with good GI survivability and bio-accessibility. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Accumulating evidence indicates that oxidative stress is closely related to the pathogenesis of various disorders and diseases, such as cardiovascular diseases, diabetes mellitus, cancer and neurological disorders (Valko et al., 2007). One of the most effective defenses against various diseases is removing free radicals and ROS. Thus, the role of natural antioxidants has received much attention. In addition to polyphenols, flavones and vitamins, food-protein hydrolysate and isolated peptides have been proved to exhibit promising antioxidant activity in vitro, including peptides from whey protein (Peng, Xiong, & Kong, 2009), soybean (Chen, Muramoto, & Yamauchi, 1995), collagen (Li, Chen, Wang, Ji, & Wu, 2007) and sunflower protein (Megías et al., 2008). However, it is possible that some antioxidants are undermined during the gastrointestinal (GI) digestion before they ⁎ Corresponding author at: P.O. Box 294, Qinghua East Road 17, Haidian District, Beijing 100083, People's Republic of China. Tel./fax: +86 10 62736243. E-mail address: [email protected] (B. Li). 1466-8564/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ifset.2012.07.009

play the antioxidant role. For example, hydrolysis of various digestive enzymes in the human digestive tract to polypeptides may lead to a change of bioactivity. Furthermore, the GI tract is known to be a major oxidation site where various free radicals are generated in the digestion process (Srigiridhar, Nair, Subramanian, & Singotamu, 2001; You, Zhao, Regenstein, & Ren, 2010). Thus, one of the greatest challenges is proving the efficacy of their bioactive components in vivo when these antioxidant peptides are ingested orally. Though animal studies and human clinical trials are the best ways to study the bioactivity and bioavailability of functional ingredients, in vitro simulated GI digestion is being extensively used. It allows for rapid and inexpensive study of bioactive compounds for assessment of their efficacy in vivo (Cinq-Mars, Hu, Kitts, & Li-Chan, 2008). In the research of bioactive peptides, lots of papers investigated the potential ACE inhibitory activity of protein hydrolysate and peptide sequences in a simulated GI digestion. It is inferred that the bioactivity of short-chain peptides may be preserved during the GI process, whereas the longer molecules need to be protected (Roufik, Gauthier, & Turgeon, 2006). Quirós, Contreras, Ramos, Amigo, and Recio (2009) evaluated the impact of a simulated

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gastrointestinal digestion on the stability of eight synthetic peptides only to find peptide with sequence LHLPLP was resistant to digestive enzymes. Megías et al. (2008) reported that protein hydrolysate produced by hydrolysis with the pepsin plus pancreatin exhibited better tolerance than that produced by hydrolysis with microbial protease alcalase. In addition, a recent study showed that peptides containing more neutral and basic amino acid (AA) underwent faster degradation than those with more acidic AA (Picariello et al., 2010). The above research indicated that molecular weights (MW), AA composition and sequence affected the efficacy of these peptides. Researches on bioavailability of antioxidant peptides were mainly carried out on protein hydrolysate (Orsini Delgado, Tironi, & Añón, 2011; You et al., 2010; Zhu, Chen, Tang, & Xiong, 2008) and proved that these hydrolysates exhibited good tolerance to the GI digestion. However, protein hydrolysate presents a broad MW distribution and includes large quantities of non-active peptides and free amino acids. Thus, we cannot figure out whether and how bioactive peptides were hydrolyzed or generated after the GI digestion. Little research is done on this. The object of this study was to assess the effect of MW on the survivability of antioxidant peptides after the simulated GI digestion. A two-stage simulated GI digestion model in vitro was used to simulate the process of human GI digestion. Casein hydrolysate was separated by ultrafiltration and gel filtration chromatography. Three peaks with antioxidant activity were collected and lyophilized. The MW distribution, AA composition and radical scavenging activities of different antioxidant peptide fractions were determined. Besides, the MW distribution, amount of free amino groups, free AA composition and radical scavenging activities of different GI digests were investigated. Degradation rules of antioxidant peptides were explored and the effect of MW on the GI survivability of antioxidant peptides was evaluated. This research conducted further examination of bioavailability of bioactive peptides and provided guidance to the control of MW for oral functional products and antioxidants. 2. Materials and methods 2.1. Materials Skim milk powder was purchased from Anchor milk products (Fonterra Brands NZ Ltd., Manurewa, New Zealand). Aqueous solution of skim milk powder was adjusted to pH 4.6 with 0.2 M acetic acid/sodium acetate buffer and centrifuged at 5000 ×g for 10 min. The casein precipitation was collected, washed with deionized water, lyophilized and finally stored in a desiccator for further use. Alcalase 2.4 L (EC3.4.21.14, ≥2.4 U/g, U: Anson units, AU) was purchased from Novozyme Co. (Bagsvered, Denmark), pepsin (EC3.4.23.1, 1:10,000, 800–2500 units/mg) and chymotrypsin (EC3.4.21.1, ≥40 U/ mg) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and trypsin (EC3.4.21.4, 1:250, 250 NF U/mg) was purchased from Amresco (AMRESCO Inc., USA). Bacitracin (MW 1450 Da), Gly-Gly-Tyr-Arg (MW 451 Da), AsnCys-Ser (MW 322 Da), Gly-Gly-Gly (MW 189 Da), 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS•+), 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), phenyl isothiocyanate (PITC), triethylamine (TFA), 2, 2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), fluorescein sodium salt and amino acid standard solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-Deoxy-D-ribose was purchased from Inalco (Spa Milano, Italy) and L-glutathione (reduced) from Japan. All other chemicals used were of analytical grade and purchased from Beijing Chemical Reagent Co., Ltd. (Beijing, China). 2.2. Preparation of casein hydrolysate Casein and alcalase were dissolved in HAc–NaAc buffer solution (pH 8.0) to give a starting protein concentration of 5% (w/v) and

the ratio of enzyme to substrate was 0.192 AU/g Pr. The hydrolysis was conducted at pH 8.0 in a water bath shaker at 55 °C for 4 h, which was stopped by heating in boiling water for 15 min. After the hydrolysate was cooled to room temperature and centrifuged at 10,000 ×g for 15 min at 4 °C, the supernatants were then collected, freeze-dried, and stored at − 20 °C for further use. 2.3. Preparation of antioxidant peptides with different MW 30 mL of the lyophilized hydrolysate dissolved in distilled water (50 mg/mL) was added in the ultrafiltration cup. It was connected to the nitrogen cylinder and the pressure was kept at 0.5 atm. The hydrolysate went through the MWCO of ultrafiltration membranes for 10 kDa. The filtrate was lyophilized and separated by gel filtration chromatography on a Sephadex G-25 column (1.6 × 100 cm) at a flow rate of 1.0 mL/min and monitored at 220 nm. The mobile phase was distilled water. Three antioxidant peptide peaks with antioxidant activity were collected, lyophilized, and stored at −20 °C for further use. Three fractions were named high MW fraction (FH), intermediate MW fraction (FI) and the low MW fraction (FL), respectively. 2.4. In vitro simulated GI digestion The digestion of casein-derived antioxidant peptides was performed as described by Vassilopoulou et al. (2006) and Quirós et al. (2009) using an in vitro gastric digestion with pepsin, followed by intestinal digestion in the presence of a model bile salt mix, trypsin and chymotrypsin. Peptides were redissolved (3%, wt/vol) in 0.01 M HCl and adjusted to pH 2.0 with 1.0 M HCl. The sample was firstly hydrolyzed with pepsin (2% w/w, protein basis) for 90 min at 37 °C at pH 2.0. Then the pH was adjusted to 5.3 with 0.9 M NaHCO3 solution and further to pH 7.5 with 1.0 M NaOH solution followed by hydrolysis with trypsin (4% w/w, protein basis) and chymotrypsin (4% w/w, protein basis) at pH 7.5 and 37 °C for 240 min. The test tubes were kept in boiling water for 10 min for enzyme inactivation. Then the GI digests were cooled to room temperature and centrifuged at 11,000 ×g for 15 min. The supernatant was lyophilized and stored at − 20 °C for further analysis. Aliquots of gastric digests (10.0 mL) were removed during the in vitro digestion to investigate the changes in antioxidant activities of casein-derived peptides during the simulated GI digestion. 2.5. Determination of free amino groups The amount of free amino groups was determined using the TNBS method according to Spellman, McEvoy, O'Cuinn, and FitzGerald (2003). Samples (0.25 mL each) were pipetted into test tubes containing 2.0 mL of sodium phosphate buffer (0.2125 M, pH 8.2), and 2 mL of TNBS reagent (0.1%) was added, followed by mixing and incubation at 50 °C for 60 min in a covered water bath (to exclude light). At the end of the incubation, the reaction was terminated by the addition of 4 mL of 0.1 M HCl to each tube. The solutions were cooled to room temperature, and the absorbance was measured at 420 nm. L-Leucine (0–2.5 mM) was used to generate a standard curve. The contents of free amino groups in the digest samples were expressed as Leu amino equivalents (mg/g), based on the equation of Leu standard curve generated. 2.6. Determination of MW distributions Molecular weight distribution of the initial peptide fractions and their digests were determined according to Ma, Xiong, Zhai, Zhu, and Dziubla (2010) by using a low-pressure size exclusion chromatography with a Sephadex G-15 column (1.6 × 100 cm) with a UV detector and a software workstation. 1.0 mL of sample (2 mg/mL,

M. Chen, B. Li / Innovative Food Science and Emerging Technologies 16 (2012) 341–348

dissolved in distilled water) was filtered by the 0.22 μm membrane filter. The filtrate was then eluted by distilled water at 1.0 mL/min at room temperature and the absorbance (220 nm) of the eluents was measured. The column was calibrated using Bacitracin (MW 1450 Da), Gly-Gly-Tyr-Arg (MW 451 Da), Asn-Cys-Ser (MW 322 Da) and Gly-Gly-Gly (MW 189 Da). The evolution volume (mL) of blue dextran was used to establish the void volume of the column. Molecular weight (MW) distribution of the individual sample was estimated from an MW calibration curve generated from the elution volume of the above standards (LgMW ¼ −0:0138  þ4:592, R 2 = 0.9925). For quantitative determinations, peak areas in the sample chromatograms were manually integrated by using Origin8.0 expressed as the percentage area of its chromatogram peak. 2.7. Amino acid analysis 2.7.1. Total amino acid Total amino acid (TAA) composition of the antioxidant peptide fractions was determined by pre-column derivatization high performance liquid chromatography according to the method of Vasanits and Molnár-Perl (1999) with some modifications. Triplicate samples were hydrolyzed in sealed, evacuated glass tubes with 6 N HCl at 110 °C for 24 h. Then each hydrolysate was brought to volume (10 mL) with deionized water. After filtration through two layers of filter paper, 1.0 mL of filtrate was transferred into a 25.0 mL beaker and dried in a vacuum drier. The dry sample was redissolved in 1.0 mL of HCl (pH 2.0) for amino acid analysis. Determination was carried out using a C18 column (250 × 4.6 mm, 5 μm) on an LC-15C modular system (Shimadzu) for amino acid separation. Pre-column reaction with PITC yielded amino acid derivatives. The concentration of each kind of amino acid was determined from their respective absorption intensity, which was calibrated to the concentrations of amino acid standards (0.125 μmol/mL, 0.5 μmol/mL, 1.0 μmol/mL, 2.0 μmol/mL, 2.5 μmol/mL). 2.7.2. Free amino acid analysis The method described by Wu, Chen, and Shiau (2003) was used with slight modifications. The digest samples were precipitated with 10% cold trichloroacetic acid for 2 h and then centrifuged at 11,000 g for 15 min. The pH of the supernatant was adjusted to 2.0, and the solution was passed through a membrane (0.22 μm). Then the filtrate was subjected to RP-HPLC analysis (Agilent HP1100) after precolumn derivatizing with PITC as indicated above. 2.8. Determination of radical scavenging activity 2.8.1. ABTS• + scavenging activity assay ABTS• + radical scavenging activities of peptide fractions and their GI digests were determined as described by You et al. (2010). 2.8.2. Hydroxyl radical scavenging activity assay The ability of peptide fractions and their GI digests to scavenge hydroxyl radical was determined according to Halliwell, Gutteridge, and Aruoma (1987) with some modifications. The reaction mixture contained 200 μL of a premixed 10 mM FeCl2, and 10 mM EDTA (1:1, v/v) solution, 1.2 mL of 50 mM NaH2PO4–Na2HPO4 buffer (pH 7.4), 200 μL of 10 mM 2-deoxy-D-ribose (dissolved in 50 mM NaH2PO4– Na2HPO4 buffer, pH 7.4), 200 μL of sample (containing 10 mg/mL) and 200 μL of 10 mM H2O2. Tubes were vortexed and incubated at 37 °C for 60 min. Thereafter, 1.0 mL of 28.0 g/L TCA was added to each tube followed by 1.0 mL of 10.0 g/L TBA. The samples were vortexed, heated in a water-bath at 100 °C for 20 min and then incubated in an ice-water bath for 5 min. The extent of oxidation was estimated from the absorbance at 532 nm. Equivalent of (1.0, 2.0, 5.0, 8.0 and 10.0 mg/mL) GSH solution instead of a sample were determined for a standard curve. Results of hydroxyl radical scavenging

343

activity (HRSA) were expressed as percent of 2-deoxy-D-ribose degradation (%): HRSA ¼ ½ðA0 −AÞ=A0   100% where A is the absorbance observed at 532 nm when the sample was added, and A0 is the absorbance of the control (equivalent distilled water instead of the sample). 2.8.3. ORAC assay The determination was totally referred to a method described by Dávalos et al. (2004). For the ORAC assay, the 485-P excitation and 520-P emission filters were used. The reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 μL. Black 96-well microplates (96F Untreated Microwell, Nunc, Denmark) were used. Antioxidant (20 μL) and fluorescein (120 μL; 70 μM, final concentration) solutions were placed in the well of the microplate. The mixture was preincubated for 15 min at 37 °C. AAPH solution (60 μL; 12 mM, final concentration) was added rapidly using a multichannel pipet. The microplate was immediately placed in the reader and the fluorescence recorded every minute for 80 min at 37 °C. The microplate was automatically shaken prior each reading. A blank (FL AAPH) using phosphate buffer instead of the antioxidant solution and eight calibration solutions using Trolox (1–8 μM, final concentration) as antioxidants were also carried out in each assay. Raw data were exported from the Fluostar Galaxy software to an Excel (Microsoft, Roselle, IL) sheet for further calculations. ORAC values were expressed as Trolox equivalents by using the standard curve calculated for each assay. Final results were in μM of Trolox equivalent/mg of protein content for peptide fraction and their digests. 2.9. Statistical analysis All the tests were conducted in triplicate. The results obtained were subjected to one-way analysis of variance (ANOVA). Duncan's new multiple range test was performed to determine the significant difference between samples within the 95% confidence interval using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Characteristics of antioxidant peptides with different MW Fig. 1 was the gel filtration spectrum of separation of FH, FI and FL. These three antioxidant peptide fractions had different MW distribution. 3.1.1. MW distributions MW distributions of the three antioxidant fractions with different MW (FH, FI and FL) were present in Table 1. The fraction of 189–1000 Da stood for oligopeptides in each sample. The fraction of 189–500 Da mainly containing dipeptides and tripeptides can be absorbed intact into the epithelial cell. On the contrary, high MW of peptides above 3000 Da probably resulted in incomplete absorption by the intestine (Pauletti et al., 1996). In this research, FH was mainly composed of antioxidant peptides with MW above 3000 Da (78.61%). 88.53% of FI distributed in the range of 1000–3000 Da, and 92.43% of FL was below 1000 Da and rich in the 500–1000 Da fraction (71.00%). 3.1.2. AA composition As shown in Table 2, FH, FI and FL had high amounts of Glu and Leu, which were due to large amounts of the two kinds of AA in casein (Agudelo, Gauthier, Pouliot, Marin, & Savoie, 2004). FH contained the largest amount of basic AA (18.8%) among the three fractions with different MW (P b 0.05). In addition, FI contained the most acidic AA (27.1%), FL contained the most hydrophobic AA among three peptide fractions (47.7% of the TAA). The amounts of Tyr, Cys, Leu and Phe in

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A (mV)

ABTS•+ radical scavenging activity FL

14

100

FI

80

A (mV)

10

70

FH

8

60

6

50

4

40 30

2

20

0 -2

1

6

11

16

21

26

31

36

41

46

51

56

61

66

71

Number of tubes

ABTS•+ radical scavenging activity(%)

90

12

10 0

Fig. 1. Spectrum of gel filtration chromatogram: separation of three antioxidant peptide fractions. Peptide fractions included the high MW fraction (FH), the intermediate MW fraction (FI) and the low MW fraction (FL).

FL were respectively 144.6 mg/g, 7.3 mg/g, 126.6 mg/g and 118.8 mg/g, evidently more than those of FH and FI. This might be contributed to its stronger antioxidant capacity since these hydrophobic amino acids were found to be important to the antioxidant activity of peptides (Chen et al., 1995).

higher antioxidant activity than the other two fractions and its ORAC value is 7.58 ± 0.04 μM/mg protein. In a word, the antioxidant peptide fraction, mainly distributed below 1000 Da, exhibited the best antioxidant activity. 3.2. Changes in MW distribution after digestion

3.1.3. Antioxidant activities A broad variety of in vitro techniques has been developed for the detection of antioxidants on the basis of different antioxidative mechanisms under variable conditions (Pihlanto, 2006). In our study, ABTS• +, hydroxyl radical scavenging activities and ORAC value of the three fractions with different MW were determined respectively. Scavenging activity for the ABTS• + radical, expressed as TEAC, was shown in Fig. 2(A). The TEAC values of the three fractions followed the order: FL > FI > FH (P b 0.05), and FL exhibited the strongest antioxidant activity (2.08 ± 0.05 mM, TEAC). As shown in Fig. 2(B), the lower IC50 (concentration of peptides to scavenge 50% of radicals) showed the higher antioxidant capacity. FL exhibited the strongest hydroxyl radical scavenging activity among three fractions, IC50 value was 1.32 ±0.02 mg/mL. The IC50 of three fractions were all stronger than that of GSH (IC50 = 5.84± 0.27 mg/mL). According to the mechanism of Fenton reaction (Fe2+ + H2O2 → Fe3+ + •OH + OH−), the oxidation of •OH could be restrained by either Fe2+ chelating or •OH stabilization. Hence, the strong hydroxyl radical scavenging activity of the casein-derived antioxidant peptide fractions, notably that below 1000 Da, can be attributed to the removal of pro-oxidant (free Fe2+) and stabilization of radicals through hydrogen or electron donation (Ma, Xiong, Zhai, Zhu, & Dziubla, 2010). In the ORAC assay, the higher ORAC values suggested stronger antioxidant activity. As shown in Fig. 2(C), the ORAC values of FI and FH had no significant difference, yet FL showed significantly (P b 0.05)

Fig. 3 was the gel filtration spectrum of determination of MW distribution. Table 1 showed the MW distribution of the three antioxidant fractions (FH, FI and FL) and residual peptides in their digests. The gastric digests were FH-GD, FI-GD and FL-GD and GI digests were FH-GID, FI-GID and FL-GID. After the simulated gastric digestion, contents of the peptides above 3000 Da in FH were drastically (P b 0.05) degraded from 78.61% to 18.97%. 58.51% of the residual peptides in FH-GD were below 2000 Da. However, only 11.42% of the peptides (distributed in 1000–3000 Da) in FI and 31.2% of peptides (distributed in 500–1000 Da) in FL were digested. These results showed that antioxidant peptides above 3000 Da were easier to be influenced by pepsin and the low pH than those below 3000 Da. After being subjected to the subsequent intestinal digestion, about half of the residual peptides in FH-GID were oligopeptides. As for FI, the peptides distributed in 1000–3000 Da were significantly reduced (P b 0.05) from 77.11% to 33.96%, and 63.03% of the residual peptides in FI-GID were oligopeptides. When it turned to FL, the peptides distributed in 500–1000 Da were decreased by 74.15% (P b 0.05), and nearly 80% of the residual peptides in FL-GID were distributed in 189–500 Da, and existed as dipeptides, tripeptides and tetrapeptides. These results suggested that the antioxidant peptides below 3000 Da tended to be digested in the intestine. After the simulated GI digestion, 10.10% of the residual peptides in FH-GID were above 3000 Da,

Table 1 Molecular weight distribution of different antioxidant peptide fractionsa and residual peptides in their digestsb. Sample

FH FH-GD FH-GID FI FI-GD FI-GID FL FL-GD FL-GID a b

Molecular weight distribution (%) 10,000–3000 Da

3000–2000 Da

2000–1000 Da

1000–500 Da

500–189 Da

78.61 18.97 10.10 5.53 0.63 0.28 0.00 0.00 0.00

15.65 22.51 11.40 43.65 29.64 9.88 0.57 0.47 0.26

5.75 32.57 31.24 44.88 47.47 26.81 7.00 2.34 0.52

0.00 16.57 26.60 5.94 10.34 26.62 71.00 48.83 18.35

0.00 9.37 20.67 5.94 11.92 36.41 21.43 48.36 80.88

Peptide fractions included the high MW fraction (FH), the intermediate MW fraction (FI) and the low MW fraction (FL). Digests contained the gastric digests (GD) and gastrointestinal digests (GID).

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Table 2 Total amino acid composition of three antioxidant peptide fractionsa and free amino acid analysis of three GI digestsb. Amino acid

Asp (Asn) Glu (Gln) Ser Gly His Thr Ala Pro Arg Tyr Val Met Cys Ile Leu Phe Lys Sum Basice AA Acidicf AA Hydrophobicg AA a b c d e f g

TAAc (mg/g Protein)

FAAd (mg/g GI digest)

FAA/TAA (%)

FH

FI

FL

FH-GID

FI-GID

FL-GID

FH/FH-GID

FI/FI-GID

FL/FL-GID

56.8 158.2 40.1 21.7 39.3 37.6 25.8 84.0 37.5 39.6 53.9 30.1 3.5 45.5 51.8 37.7 81.9 845.0 158.9 214.6 328.7

68.0 164.9 42.6 15.7 24.5 38.9 30.0 69.4 36.4 52.8 49.4 41.4 4.1 42.3 82.6 33.4 63.8 860.1 124.7 233.1 348.3

47.3 117.4 41.8 15.5 24.4 37.3 33.0 50.8 25.4 144.6 48.4 39.2 7.3 35.0 126.6 118.8 35.4 948.0 85.3 165.0 452.2

8.8 23.2 8.4 6.4 7.7 8.6 7.1 13.4 7.4 36.1 18 26.6 0.8 9.4 19.0 18.0 16.1 235.0 31.1 32.1 111.5

9.9 25.6 10.6 7.9 10.9 11.3 9.3 17.3 10.2 48.0 26.6 36.2 0.7 12.0 22.8 21.3 19.5 300.1 29.8 30.8 106.9

20.5 49.9 16.5 11.5 11.6 15.2 12.5 25.2 12.2 56.7 20.4 13.7 1.1 17.2 39.1 30.2 25.7 379.1 37.5 32.7 143.1

15.5 14.7 20.9 29.5 19.6 22.9 27.5 16.0 19.7 91.2 33.4 88.4 22.9 20.7 36.7 47.7 19.7 27.8 18.8 14.3 32.5

14.6 15.5 24.9 50.3 44.5 29.0 31.0 24.9 28.0 90.9 53.8 87.4 17.1 28.4 27.6 63.8 30.6 34.9 30.1 14.0 41.1

43.3 42.5 39.5 74.2 47.5 40.8 37.9 49.6 48.0 39.2 42.1 34.9 15.1 49.1 30.9 25.4 72.6 40.0 58.2 42.7 35.0

Peptide fractions included the high MW fraction (FH), the intermediate MW fraction (FI) and the low MW fraction (FL). The GI digests included FH-GID, FI-GID and FL-GID. TAA means total amino acid composition of three antioxidant peptide fractions. FAA means free amino acid composition of the GI digests of different peptide fractions. Basic AA contains His, Arg and Lys. Acidic AA contains Asp and Glu. Hydrophobic AA includes Ala, Pro, Tyr, Val, Met, Leu and Phe.

which seemed to be less absorbable compared to those peptides below 500 Da. 3.3. Contents of free amino groups Fig. 4 showed the amounts of free amino groups of different antioxidant peptide fractions and their digests. Gastric digestion lasted for the first 1.5 h and the subsequent intestinal digestion lasted for 4 h. After the gastric digestion, for FH, the amounts of free amino groups had the biggest increase (27.4 mg/g), however, the increases in free amino groups for FI and FL were 20.2 mg/g and 21.4 mg/g, respectively. The above results were consistent with the study of MW distribution. That is to say, the antioxidant peptides above 3000 Da were more sensitive to the pepsin and low pH during the gastric digestion. After the subsequent intestinal digestion, the increases in free amino groups for FI and FL were 38.1 mg/g and 36.2 mg/g, respectively, two times of their increase by gastric digestion. From the results we inferred that peptides tended to be more hydrolyzed by intestinal digestion than by gastric digestion, especially those below 3000 Da. After the GI digestion, the increases in free amino groups for FH, FI and FL were 61.7 mg/g, 58.3 mg/g and FL 57.6 mg/g, respectively. Peptide bonds in the peptides with higher MW were more inclined to be cut off. The results were consistent with the research of Ruiz, Ramos, and Recio (2004). 3.4. FAA composition after GI digestion As Table 2 showed, after the simulated GI digestion, FAA in the digests of different antioxidant peptide fractions accounted 27.8% of FH-GID, 34.9% of FI-GID and 40.0% of FL-GID. FL-GID included the most FAA. Basic AA, acidic AA and hydrophobic AA are close to the properties of peptides (Chen et al., 1995; Megías et al., 2008; Saiga, Tanabe, & Nishimura, 2003). In all GI digests, hydrophobic AA were more than basic AA, and basic AA were more than acidic AA, which was related to the specificity of digestive enzymes.

When we compared FH-GID, FI-GID and FL-GID with each other, the composition of FAA exhibited differently. The fraction of FI was mainly distributed in 1000–3000 Da, and FI-GID contained the most hydrophobic AA (41.1%) among the three GI digests. However, the fraction of FL was mainly distributed below 1000 Da, and Fl-GID had the most basic AA (58.2%) and acidic AA (42.7%). These results showed that hydrophobic AA in peptides above 1000 Da were easier to be hydrolyzed, but basic AA and acidic AA in peptides below 1000 Da were easier to be digested. 3.5. Changes in antioxidant activities after digestion TEAC values of the fraction FH, FI and FL and their gastric and GI digests were shown in Fig. 2(A). Changes in antioxidant capacity of three peptide fractions with different MW during in vitro gastrointestinal digestion were similar on ABTS•+ radical scavenging activity. All showed a decrease during the gastric digestion and then increased during the subsequent intestinal digestion, the conclusion of which was consistent with You et al. (2010) and Zhu et al. (2008). After the simulated GI digestion, the TEAC values of FH increased to two-fold from 0.60 ± 0.07 mM to 0.37± 0.01 mM, which indicated that antioxidant peptides with stronger activity were produced during the GI digestion. No significant change happened to the antioxidant activity of FI. The TEAC value of FL decreased by 28% (P b 0.05), which was probably because plenty of FAA without antioxidant activities were produced. However, FL-GID exhibited the highest ABTS• + scavenging activity (1.51± 0.02 mM) among the three GI digests. Hydroxyl radical scavenging activity of three antioxidant peptide fractions and their gastric and GI digests were shown in Fig. 2(B). After the gastric digestion, all the IC50 values of the three fractions increased significantly(P b 0.05), which indicated that the antioxidant activities of the three fractions decreased. After the subsequent intestinal digestion, the IC50 values of FH-GD and FI-GD decreased again, however, there was no significant change in the IC50 value of FL-GD. After the whole GI digestion, compared to the samples before digestion, the IC50 value of FH increased by 32%, the IC50 value of FI

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FH

A

FI

FL

2.5

a

TEAC(mM)

2.0

d

1.0

d e

f,g

Initial Fractions

Gastric Digests

FH

B Hydroxyl radical scavenging activity IC50(mg/mL)

6.00 5.00

FI

FL

a

b c

d

e

2.00

f

f

g

3.00

h

1.00 Initial Fractions

Gastric Digests FH

a

8.00

Trolox Equivalent(µM/mg)

GI Digests

4.00

0.00

C

e,f

g

0.5 0.0

b

c

1.5

FI

7.00

GI Digests

FL b c

6.00 5.00 4.00 d

3.00

d e

2.00

e,f f

1.00 0.00

g

Initial Fractions

Gastric Digests

GI Digests

Fig. 2. Antioxidant activities of three antioxidant fractions and their gastric and gastrointestinal (GI) digests. ABTS•+ radical scavenging activity was showed in (A), hydroxyl radical scavenging activity (IC50) was showed in (B) and ORAC values were showed in (C). The data with different lowercase letters in the test are significantly different (P b 0.05).

increased by 55% and the IC50 value of FL increased by 130%. These results indicated hydroxyl radical scavenging activity of all the three fractions declined. Among three GI digests, the hydroxyl radical scavenging activity of FL-GID was the strongest, and the IC50 value of FL-GID was 3.03 ± 0.024 mg/mL. ORAC values of the three antioxidant peptide fractions and their gastric and GI digests were shown in Fig. 2(C). After the gastric digestion, ORAC values of the antioxidant peptides with different MW declined significantly, FH-GD almost showed no antioxidant activity. However, after the subsequent intestinal digestion, the ORAC value of FH rebounded significantly (P b 0.05), the ORAC value of FI had no significant difference, and the ORAC value of FL declined again. Compared with the fractions before digestion, the antioxidant activity of FH and FI only left 27.8% and 40.0%, however, FL kept 72.2% of its initial activity. After the GI digestion, FL-GID exhibited the highest ORAC value (5.47 ± 0.57 μM/mg Trolox) and had the strongest antioxidant activity. Generally speaking, the casein-derived antioxidant peptides below 1000 Da exhibited the best surviving antioxidant activity. At present, food-derived bioactive peptides are considered as prominent candidates for dietary supplements and nutraceuticals in various

health-promoting functional foods due to their different beneficial physiological effects (Hartmann & Meisel, 2007). Numerous products are already on the market or under development by food companies, exploiting the potential of food-derived bioactive peptides, such as Calpis, Cysteine Peptide and Vivinal Alpha etc. (Korhonen, 2009). To exert physiological effects in vivo, bioactive peptides must survive the GI digestion, and then reach their target sites after absorption. So far, only a few among the numerous bioactive peptides displaying activities in vitro have been proven effective in vivo. The real bioavailability of quite an amount of marketed products containing bioactive peptides is unclear. However, our research conducted further examination of bio-accessibility of casein-derived antioxidant peptides and assessed the effect of MW on the survivability of casein-derived antioxidant peptides after the simulated GI digestion. The survivability of peptides was above 60% after the GI digestion. And the casein-derived peptides below 1000 Da exhibited the best initial and surviving ABTS• +, hydroxyl radical scavenging activity and ORAC value. The enriched antioxidant peptide fraction above could be a promising candidate for dietary supplements and nutraceuticals with good GI survivability and bio-accessibility. Our results could provide guidance for the preparation of oral functional food products containing bioactive peptides.

M. Chen, B. Li / Innovative Food Science and Emerging Technologies 16 (2012) 341–348

A

FH 3k Da 2k Da

1k Da

500 Da

Absorbance at 220 nm (mV)

FL

240

189 Da

a b

24

200

21 18 15 12 9 6 3 0 0 -3

20

40

60

80

min 100 120 140 160 180 200 220

30 27

3k Da 2k Da

1k Da

500 Da

c e

d

d,e

160

g

f 120

h 80 40 0

B

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

Time (h)

189 Da

Fig. 4. Amounts of the amino groups of three antioxidant fractions and their gastric and gastrointestinal digests. The data with different lowercase letters in the same test are significantly different (P b 0.05).

24 21 18 15 12 9 6 3 0 -3

Absorbance at 220 nm (mV)

FI

27

Leu Equivalent (mg/g)

Absorbance at 220 nm (mV)

30

C

347

min 0

20

40

60

30

80 100 120 140 160 180 200 220

3k Da 2k Da

1k Da

500 Da

189 Da

27

increased as the MW decreased, and accounted for 30%–40% of the GI digests. For all the three fractions, hydrophobic AA and basic AA were more easily hydrolyzed than acidic AA, which was consistent with the specificity of digestive enzymes. The peptides below 1000 Da exhibited the best initial and surviving ABTS• +, hydroxyl radical scavenging activity and ORAC value. In conclusion, the casein-derived antioxidant peptides below 1000 Da were a prominent candidate as dietary supplements with good GI survivability and bio-accessibility for the preparation of oral functional food products.

Acknowledgments This work is financially supported by the National Natural science Foundation Committee of the People's Republic of China (no. 20976205) and the Chinese Research Projects of Young Teachers (no. 2010JS078).

24 21 18 15 12

References

9 6 3 0 0 -3

20

40

60

min 80 100 120 140 160 180 200 220

Fig. 3. The gel filtration spectrum of different antioxidant peptide fractions and their gastric digests (GD) and gastrointestinal digests (GID). A for FH, B for FI and C for FL ( Initial Fractions, GD, GID).

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