PHILIPP AGRIC SCIENTIST Vol. 94 No. 3, 258-263 September 2011
ISSN 0031-7454
Amino Acid Composition, Antioxidant Property and Angiotensin I-Converting Enzyme (ACE) Inhibitory Activity of Micropeptides in Skim Milk Powder Lei Cui1,2, Mini Huang1, Daxian Zhao3, Yun Deng1,2, Bingjun Qian1,2,*, Li Zhang1, Yali Luo1, Qingxiao Wu1 and Mengzhang Wu1 1
Department of Food Science and Engineering, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, P. R. China 2 SJTU-Bor Luh Food Safety Center, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China 3 State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. China * Author for correspondence; e-mail:
[email protected]; Tel: + 86-21-34206613, Fax: + 86-21-34206918 The study evaluated the amino acid composition, and the antioxidant and antihypertensive characteristics of micropeptides in skim milk powder. Milk proteins were slightly destroyed during the production of skim milk powder based on results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The skim milk powder was rehydrated and salted-out. After centrifugation, the supernatants were subjected to microfiltration and then separated with Superdex30G column fractionation, producing three fractions (PF-I, PF-II and PF-III). The molecular weight of fraction PF-III was about 1.0 kDa with a yield of 10.9 ± 0.73 mg g-1 skim milk powder. Fraction PF-III at 10 mg mL-1 showed modest antioxidant activity with a 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging rate of 36.69 ± 0.92%. The inhibitiory rate of the angiotensin I-converting enzyme (ACE) activity of PF-III at a concentration of 3 mg mL-1 was about 20.12 ± 7.59%. Fraction PF-III was found to be rich in Gly, Phe, Leu, Glu and Asp, and its total hydrophobic amino acid content was as high as 50.53%, which may have contributed to the antioxidant and ACE-inhibitory activities.
Key Words: amino acid composition, angiotensin I-converting enzyme, antioxidant, micropeptide, skim milk Abbreviations: ACE – angiotensin I-converting enzyme, AOA – antioxidant activity, BHT – 2,6-di-tert-butyl-4methylphenol, DPPH – 1.1-diphenyl-2-picrylhydrazyl, HA – hippuric acid, HHL – hippuryl-1-histidyl-1-leucine, IPP – Ile-Pro-Pro, PBS – phosphate buffer solution, RP-HPLC – reversed phase-high performance liquid chromatography, SDS-PAGE – sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SEC – size exclusion chromatography, THAA – total hydrophobic amino acid
INTRODUCTION Milk proteins are precursors of many different bioactive peptides defined as specific protein fragments that have a wide range of positive impact on body functions or conditions and may ultimately influence health through their antihypertensive, antioxidant and other properties (Meisel 2004). These peptides, which are inactive within the sequence of the milk protein, can be released and activated by fermentation or ripening during food processing. Previous studies have suggested that skim milk fermented by Lactobacillus helveticus showed beneficial effects in spontaneously hypertensive rats (SHR). The blood pressure lowering effect, allowed by the tripeptides Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP), was based on angiotensin-converting enzyme (ACE) inhibition (Nakamura et al. 1995, 1996). Virtanen et al.
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(2007) demonstrated that fermentation of milk with Lactobacillus jensenii (ATCC 25258) or Lactobacillus acidophilus (ATCC 4356) generated antioxidant activity in the whey fraction, allowed by peptides derived from the proteolysis of whey. Besides fermentation, other food processing technologies may have a denaturation effect on proteins. Wolkers et al. (1998) reported that small droplets of the poly-L-lysine solution adapt an extended β-sheet conformation when slow-dried at 25 °C over a period of several hours. Shi et al. (2008) concluded that the denaturation of proteins was induced in the drying process of horse mackerel using a heat-pump dehumidifier with an initial temperature of 20 °C and a cycle time of 3.5 h. Forbes et al. (2007) showed that spray-drying of trypsin perturbs the secondary structure of the solid protein, resulting in the loss of enzymatic activity. The Philippine Agricultural Scientist Vol. 94 No.3 (September 2011)
Amino Acid Composition and Bioactivity of Micropeptides in Skim Milk Powder
Protein degradation usually accompanies denaturation. Heating and surface tension, as physical factors causing protein denaturation, are involved in sterilization and the spray drying process in milk powder production, respectively. However, few reports have mentioned their destructive effect on proteins in milk powder or evaluated the bioactivities of protein degradation during milk powder production. This study investigated the amino acid composition, and the antioxidant and the antihypertensive properties of micropeptide production in skim milk, induced by sterilization and spray dry processing.
MATERIALS AND METHODS Materials Skim milk powder (≥32.4% protein, ≤0.8% fat) was obtained from Fonterra Co-operative Group (Auckland, New Zealand). Vitamin C, 2,6-di-tert-butyl-4methylphenol (BHT) and disodium ethylenediaminetetraacetate (EDTA) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Atocopherol (Vitamin E, VE) was purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radical was purchased from A Johnson Matthey Company (Malvern, USA). Bacidin, bovine serum albumin (BSA), hippuryl-l-histidyl-l-leucine (HHL) and hippuric acid (HA) were obtained from Sigma Chemical Co. (Louis, USA). Synthetic peptides VPP (Val-Pro-Pro) and IPP (Ile-Pro-Pro) were synthesized by GL BIOCHEM (Shanghai) Ltd. (Shanghai, China). Milk Protein Composition Analysis by SDS-PAGE To identify the integrity and composition of milk protein in skim milk powder, 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis was applied (Yuka et al. 2009). Skim milk powder (150 mg) was dissolved in ice-cold distilled water to obtain a final volume of 100 mL. This protein solution was further diluted into a series of concentrations for electrophoresis with Mini-Protean Tetra Electrophoresis System (Biorad, USA). Separation of Micropeptide Fractions from Skim Milk Powder The rehydrated skim milk was prepared by dissolving 12 g of skim milk powder in 100 mL of ice-cold distilled water and mixed gently. Ammonium sulfate was added to the rehydrated skim milk solution at a ratio of 1:5 (w/v) to salt out the proteins with large molecular weight by stirring on ice-bath. The treated samples were centrifuged at 12,000 rpm at 4 °C for 15 min. The supernatant containing the micropeptide fractions was filtered using 0.22 μm microfiltration. The Philippine Agricultural Scientist Vol. 94 No.3 (September 2011)
Lei Cui et al.
Separation of Peptidic Fractions by Size Exclusion Chromatography (SEC) The filtered samples were separated on a Superdex 30 prep grade (2.6×65 cm) column by using KTA purifier 10 (GE Laboratories, Inc., USA). Phosphate buffer solution (PBS) (10 mM, pH 7.4) was used to equilibrate the column and to elute the peptides at a flow rate of 1.0 mL min-1 and pressure of 0.2–0.4 MPa. A constant amount of sample (1.0 mL) was applied to the column, and the fraction with a molecular weight of less than 1.0 kDa from several runs was collected and lyophilized and then stored at -80 °C for further analysis. The absorbance of the effluent was measured at 215 nm using KTA purifier 10 (GE Laboratories, Inc., USA). Bacidin (1.4 kDa) was used as molecular standard. The peptide content was measured using the standard curve of the concentration and the peak area of standard peptide bacidin constructed by size exclusion chromatography (SEC), as described above. DPPH Radical Scavenging Activity Radical scavenging activity was determined using DPPH free radical scavenging assay with some modifications (Larrauri et al. 1998). One-milliliter samples (10 mg mL-1) from SEC peptidic fraction PF-III was mixed with 4 mL of 75 µM-methanolic DPPH free radical. Mixtures were vortexed for 30 s to homogenize, and left to react sufficiently for 60 min in the dark. Finally, absorbance was measured at 516 nm using 75 µM-methanolic DPPH free radical solutions as blank. The scavenging activity of the DPPH radical was expressed using the equation: Scavenging activity (%) =100 × (A0 - A1)/A0 where A0 is the absorbance of the methanolic DPPH free radical blank and A1 is the absorbance of the final reaction mixture with peptides. Results were compared with the activity of BHT and VE as positive control and deionized water as negative control. Determination of ACE Inhibitory Activity Evaluation of ACE inhibitory activity was assayed using reversed phase-high performance liquid chromatography (RP-HPLC) described by Wu and Ding (2002), based on the hydrolysis of hippuryl-1-histidyl-1-leucine (HHL) by ACE to hippuric acid (HA) and 1-histidyl-1-leucine (HL) as products. The release of HA from HHL is directly related to ACE activity. Briefly, 30 μL of ACE crude extracts were mixed with 75 μL of 3.0 mg mL-1 peptide fraction PF-III. The mixture was incubated at 37 °C for 10 min. Then, 200 µL of 5 mM HHL solution substrate were added and the reaction mixture was incubated at 37 °C for 40 min. The reaction was stopped by addition of 250 µL of 1 M HCl; 10 µL of solution was analyzed by RP-HPLC, and a semi-preparative C18 column (80 , 5 μm, 4.6 mm × 250 mm, Agilent) was selected. The column was eluted with 20% acetonitrile (in water, v/v) 259
