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Antioxidant and anti-freezing peptides from salmon collagen hydrolysate prepared by bacterial extracellular protease
Accepted Manuscript Antioxidant and anti-freezing peptides from salmon collagen hydrolysate prepared by bacterial extracellular protease RiBang Wu, CuiLing Wu, Dan Liu, XingHao Yang, JiaFeng Huang, Jiang Zhang, Binqiang Liao, HaiLun He PII: DOI: Reference:
S0308-8146(17)31984-2 https://doi.org/10.1016/j.foodchem.2017.12.035 FOCH 22132
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
11 July 2016 26 September 2017 10 December 2017
Please cite this article as: Wu, R., Wu, C., Liu, D., Yang, X., Huang, J., Zhang, J., Liao, B., He, H., Antioxidant and anti-freezing peptides from salmon collagen hydrolysate prepared by bacterial extracellular protease, Food Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.12.035
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Antioxidant and anti-freezing peptides from salmon collagen hydrolysate prepared by bacterial extracellular protease
RiBang Wu1, CuiLing Wu1, Dan Liu 1, XingHao Yang1, JiaFeng Huang1, Jiang Zhang1, Binqiang Liao1, HaiLun He1* 1
School of Life Science, State Key Laboratory of Medical Genetics, Central South
University, Changsha 410013 ，China
*Corresponding author: Hailun He，Tel：+86-0731-82650230; Fax：+86-0731-82650230; E-mail address: [email protected]
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Abstract Extracted salmon skin collagen was hydrolysed with the free or immobilized extracellular protease of Vibrio sp. SQS2-3. The hydrolysate exhibited anti-freezing activity (> 3 kDa) and antioxidant activity (< 3000 Da) after ultrafiltration. The antioxidant peptide was further purified by size-exclusion chromatography and found to scavenge DPPH (73.29 ± 1.03%), ·OH (72.73 ± 3.34%,), and intracellular ROS in HUVECs; protect DNA against oxidation-induced damage; and have an ORAC of 2.78 ± 0.28 mmol TE/g. The antioxidant peptide fraction was identified using mass spectrometry, and nineteen salmon collagen-sourced peptides were obtained. Of these, the peptide Pro-Met-Arg-Gly-Gly-Gly-Gly-Tyr-His-Tyr is a novel sequence and was the major component; this peptide was shown to have antioxidant activity via the ORAC assay (2.51 ± 0.14 mmol TE/g). These results suggested that the protease from Vibrio sp. SQS2-3 is suitable for preparation of anti-freezing peptides and antioxidant peptides in a single step and represents a comprehensive use of fish skin collagen.
Keywords: Salmon skin collagen; bacterial extracellular protease; enzymatic hydrolysis; antioxidant peptide; anti-freezing
1. Introduction Reactive oxygen species (ROS), including hydroxyl radicals (·OH), superoxide anion radicals (O2· −), hydrogen peroxide (H2O2), peroxyl radicals (ROO· −), and
2
nitroxide radicals (NO·), are strongly related to aging and diseases, such as cancer, Alzheimer's disease, cardiovascular disease, diabetes and weakening of the immune system (Stohs, 1995; Diaz, 1997; Spolarics, 1998). Antioxidant substances (glutathione, β-carotene, vitamin C and vitamin E) and antioxidant enzymes (superoxide dismutase, glutathione S transferase and catalase) can remove excess free radicals to prevent oxidative stress. In addition, free radical-mediated oxidation in food also leads to potential toxicity, which is a concern of the food industry and consumers (Frankel, 2005). Therefore, synthetic antioxidants are utilized in the food industry, but their potential toxicity requires strict regulation; examples include butylated-hydroxytoluene
(BHT),
butylated-hydroxyanisole
(BHA),
tertbutylhydroquinone (TBHQ), and propyl gallate (PG). Peptides with antioxidant activity represent novel antioxidants that can replace synthetics. Many studies have reported that peptides released from parental protein by hydrolysis could be an effective way to obtain antioxidant peptides. Hydrogen bonds, electrostatic interactions, and disulphide bonds are the major forces that stabilize the structure of proteins (Benjakul, 2000). These bonds and interactions can be disrupted by a sharp temperature change during frozen storage (Benjakul, 2003; Benjakul, 2009). Cryoprotectant can protect proteins against freezing damage by inhibiting the generation of ice crystals (Alvarez, 2010), e.g., sorbitol and sucrose, which are common additives in the food storage industry (Jin, 2010). However, these cryoprotectants make food products sweet, which is often unacceptable to consumers (Ruttanapornvareesakul, 2006). It has been reported that a
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protein hydrolysate containing oligopeptides is able to inhibit protein denaturation, including peptides from blacktip shark skin (Kittiphattanabawon, 2012), gelatine from bovine hide (Wang, 2009) and sericin (Wu, 2015). Recent studies have shown that collagen hydrolysates have various biological activities, such as gelatin hydrolysis with anti-freezing activity from blacktip shark skin (Phanat, 2012), antioxidant collagen peptides from croceine croaker (Wang, 2013), and angiotensin I-converting enzyme-inhibitory antioxidant peptides from skate skin gelatin (Ngo, 2014). Skin from salmon contains by-products that are rich in collagen, which can create value-added products by enzymatic hydrolysis. However, most salmon skin is discarded and/or used as animal feed. Therefore, this study focused on value-added utilization of collagen from salmon skin through enzymatic hydrolysis. An antioxidant peptide from ultrafiltration fractions with a small molecule weight was identified; ultrafiltration fractions containing larger peptides that were identified as potential cryoprotectants.
2. Materials and methods 2.1 Materials Fresh salmon by-products with skin were purchased from a seafood market in Shanghai and stored at -20 °C prior to use. Soybean meal was purchased from a supermarket in Changsha, Hunan Province, China, and stored at -20 °C. The protease-producing strain Vibrio sp. SQS2-3 was isolated from the water of the intertidal zone in Hainan Province, China (GenBank: KF220487.1).
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Tryptone and yeast extract were purchased from Oxoid (Basingstoke, UK). 1,1-diphenyl-2-picrylhydrazyl (DPPH), fluorescein, 2,20-azobis (2-amidinopropane) dihydrochloride (AAPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich, Ltd. (Shanghai, China). Ninhydrin, 1,10-phenanthroline monohydrate was purchased from Aladdin® (Shanghai, China). Sephadex LH-20 was purchased from GE Healthcare Life Sciences (Uppsala, Sweden). RPMI 1640 medium and foetal bovine serum was purchased from Gibco® ThermoFisher Scientific Company (Grand Island, USA).
2.2 Methods 2.2.1 Preparation of salmon skin collagen Byproducts with skin were washed with flowing water for 5 min. The muscles were removed thoroughly. The skin was sheared into 5-mm pieces and washed with cold distilled water 5-7 times to remove fish oil. Salmon skin was mixed with distilled water at a ratio of 1:10 (w/v, g/ml), heated for 30 min at 75 °C, and centrifuged at 10,000 × g for 10 min to extract soluble collagen. The supernatant was dialysed using a dialysis bag (with 8 kDa to 14 k Da molecule weight cut-off) with 4 °C distilled water and lyophilized into collagen solids.
2.2.2 Preparation and Purification of bacterial extracellular protease Extracellular protease was obtained through bacterial fermentation according to the methods of Dan Liu (Liu, 2014) with some modifications. The protease-producing
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strain Vibrio sp. SQS2-3 was activated at 15 °C with shaking at 200 rpm for 24 h in 2216E medium. The activated strain was seeded into fermentation broth (0.5 % corn powder, 0.5 % bean powder, 0.25 % wheat bran, 0.1 % CaCl2, 0.4 % Na2HPO4, and 0.03 % KH2PO4, prepared with sea water) at a ratio of 1:50 (v/v, ml/ml) and cultured at 15 °C with shaking at 200 rpm. The supernatant was collected by centrifugation (10,000 × g, 4 °C, 20 min) after 120 h and dialysed in Tris-HCl (20 mM, pH 7.8) at 4 °C. The crude enzyme from Vibrio sp. SQS2-3 was purified in a HiTrap DEAE FF (5 ml, 16 × 25 mm) anion exchange column with an NGC chromatography system (Bio-Rad, Hercules, CA, USA). The chromatography column was equilibrated with 20 mM Tris-HCl (pH 7.8) at a flow rate of 2 ml/min for 10 min. The crude enzyme was loaded into the pre-equilibrated column at a flow rate of 2 ml/min and washed with 20 mM Tris-HCl (pH 7.8) for 10 min. The column was eluted with a linear gradient of 1 M NaCl (0-100%) at a flow rate of 2 ml/min for 12.5 min. Protein fractions were monitored at 280 nm. The protease activity and total protein content were measured by the Folin phenol assay (Anson, 1938) and BCA method, respectively.
2.2.3 Immobilization of extracellular protease Enzyme immobilization on chitosan was performed according to Singh et al. Chitosan powder was dissolved in 1.5% (v/v) glacial acetic acid to prepare the chitosan solution (1.5%, w/v). The solution was dropped into a 1 M KOH solution containing 25% (v/v) ethanol and incubated for 1 h to prepare chitosan beads. The
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beads were washed with 0.1 M sodium phosphate buffer (pH 7.8) and activated with 2% (v/v) glutaraldehyde at room temperature for 1 h. Subsequently, the activated chitosan beads were washed extensively with distilled water for complete removal of unreacted glutaraldehyde. For immobilization, the activated beads were incubated with purified protease at 4 °C for 12 h and washed with 0.1 M sodium phosphate buffer (pH 7.8) to remove unbound protease. The protease activity and total protein content were measured using the Folin phenol assay (Anson, 1938) and BCA method, respectively. The efficiency of enzyme immobilization was calculated as follows: immobilization ef icience % =
speci ic activity of immobilized protease × 100 speci ic activity of soluble protease
2.2.4 Enzymatic hydrolysis Salmon skin collagen was digested with the protease from Vibrio sp. SQS2-3 at an enzyme to substrate ratio of 1:10 (v/w, ml/mg) and temperature of 45 °C for 30, 60, 90, 120, and 150 min. The reaction was terminated at 95 °C for 10 min. The hydrolysate was collected and centrifuged at 10,000 × g at 4 °C for 10 min. The hydrolysis degree at various treatment times was analysed using the ninhydrin coloration method. The standard curve was determined using leucine at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0 mM.
2.2.5 Purification of bioactive fractions Ultrafiltration tubes with 3,000 Da MWCO (Amicon Ultra 14 ml 3K, Millipore, USA) were utilized for preliminary separation. The supernatant of the hydrolysate
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was added to the upper casings of the ultrafiltration tubes, which were subsequently centrifuged at 4500 × g and 4°C for 60 min. Fractions in the upper casings and lower casings after ultrafiltration were collected and named UF-1 and UF-2. UF-1, with larger molecule weight, was considered to be a potential anti-freezing agent. Distilled water was added to UF-1 to replace the salt-containing solution using ultrafiltration tubes with 3k Da MWCO at 4,500 × g and 4°C for 45 min. UF-2, which had smaller molecule size, was further purified by size exclusion liquid chromatography. The fraction was loaded (1 ml) onto a Sephadex LH-20 column (16 × 600 mm), equilibrated with distilled water and eluted with distilled water at a flow rate of 0.75 ml/min. Each fraction was monitored at 220 nm, collected at a volume of 5 ml and lyophilized. Each fraction peak was prepared at the same concentration (150 µg/ml), and the antioxidant activity was further detected in subsequent assays.
2.2.6 Measuring Ca2+-ATPase activity of myoglobulin after freeze-thawing cycles Fresh porcine muscle was washed with cold distilled water and sliced into pieces (5 mm × 5 mm). The muscle was homogenized in distilled water (2 °C) at a ratio of 1:10 (w/v, g/ml) by a homogenizer at a speed of 11,000 rpm for 1 min. The homogenate was then centrifuged at 10,000 × g for 30 min at 2 °C, and the precipitate was collected. To extract myoglobulin, the precipitate was further homogenized with 1.2 M KCl, pH 7.0 for 4 min at a ratio of 1:10 (w/v) in an ice bath. The extract was centrifuged at 5000 × g for 30 min at 2 °C. Myoglobulin was precipitated with three
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volumes of distilled water and collected by centrifuging at 5000 × g for 30 min at 2 °C. Then, myoglobulin was dissolved in an appropriate volume of KCl (1.2 M, pH 7.0) by stirring for 30 min at 2 °C. Myoglobulin was kept at 4 °C and was used within 12 h. The upper fraction from ultrafiltration was added to the myoglobulin solution at a ratio of 1:1 (v/v). The mixture was frozen at -20 °C for 20 min. The frozen sample was thawed at 37 °C in a water bath for 10 min. Freeze-thawing was performed for 0, 3, 6 and 9 cycles. Thawed samples were kept on ice until analysis. The upper fraction was replaced by distilled water in the blank. The Ca2+-ATPase activity of myoglobulin was determined using the method of Benjakul et al. (Benjakul, 1997) The freeze-thaw sample (25 µl) was mixed with 15 µl of 0.5 M Tris-maleate (pH 7.0), 25 µl of 0.1 M CaCl2 and 172.5 µl of distilled water. The mixture was mixed in a vortex mixer. ATP (20 mM, 12.5 µl) was added to initiate the reaction, which was incubated at room temperature for 10 min. The reaction was terminated by adding 125 µl of trichloroacetic acid (100 g/ml, 4 °C) and centrifuged at 5000 × g for 5 min. The inorganic phosphate liberated in the supernatant was measured by the method of Fiske and Subbarow (1925). The supernatant (50 µl) was added to 80 µl of 2.5% ammonium molybdate, 20 µl of 1% stannous chloride (prepared at a ratio of 1:10:25, SnCl2: HCl (conc.): 1 N H2SO4), and 900 µl of distilled water, and the mixture was incubated at room temperature for 10 min. The absorbance of the resulting mixture was measured at 720 nm by a multiplate reader. A blank solution was prepared by adding TCA prior to the addition of ATP. The
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Ca2+-ATPase activity of myoglobulin after freeze-thawing for 3, 6 and 9 cycles was reported as residual Ca2+-ATPase activity (%), which was calculated in comparison to the sample without freeze-thawing.
2.2.7 DPPH radical scavenging activity assay The DPPH radical scavenging activity assay was conducted according to the method of Shimada et al. (1992). DPPH solution (100 µl, 0.1 mM in 95% ethanol) was mixed with 20 µl of the purified fraction solution (100 µg/ml) and incubated at room temperature for 1 h. The absorbance of the resulting solution was measured at 517 nm using a microplate reader (Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). For the blank, the purified fraction was replaced with distilled water. Vitamin C at a concentration of 200 µg/ml was used as a positive control. The DPPH radical scavenging activity was calculated using the following formula: DPPH radical scavenging activity (%) = [1-Abs sample /Abs blank] × 100
2.2.8 Hydroxyl radicals scavenging activity assay The hydroxyl radical scavenging activity was measured according to the method developed by Wang et al. (2012) A FeSO4 solution (40 µl, 2 mM), 1, 10-phenanthroline (40 µl, 2 mM) and sample (80 µl) were mixed stepwise. The reaction was initiated by adding 40 µl of H2O2 (0.03% v/v). After incubation at 37 °C for 60 min, the absorbance of the resulting solution was measured at 536 nm using a microplate reader (Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). The
10
group without any antioxidant was used as the negative control, while the mixture without H2O2 was used as the blank. The hydroxyl radical scavenging activity (HRSA) was calculated as follows: HRSA (%) = [(As - An)/(Ab - An)] × 100 where As, An, and Ab are the absorbance values determined at 536 nm for the sample, negative control and blank after reaction, respectively.
2.2.9 Protection effect on oxidation-induced DNA damage The protection effect assay for oxidation-induced damage followed the method described by Qian et al. (2008). Plasmid DNA has different structures according to the degree of damage, which can be detected by agarose gel electrophoresis. The reaction system included 8 µl of pET-22b DNA, 2 µl of 2 mM FeSO4, 8 µl of antioxidant and 2 µl of 0.1 mM H2O2. The mixture was incubated at 37 °C for 10 min and analysed by 1% agarose gel electrophoresis.
2.2.10 Oxygen radical absorbance capacity (ORAC) assay The ORAC assay was measured according to the method of Alberto et al. (2004). The reaction was performed in 75 mM phosphate buffer (pH 7.4). The sample solution (20 µl) and fluorescein (100 µl, 96 nM) was added to a 96-well microplate, and the reaction was initiated by adding 30 µl of AAPH (120 mM). The fluorescence intensity was measured every 30 s for 180 cycles with excitation and emission wavelengths of 485 nm and 538 nm, respectively, at 37°C in a microplate reader
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(Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). Trolox was used as the positive control. The ORAC was defined as trolox equivalents (mmol TE/g peptide or mmol TE/mmol peptide) according to the area under the curve (AUC) and calculated as follows: ORAC = (AUCsample-AUCcontrol) / (AUCTrolox-AUCcontrol) × (MTrolox / Msample) where AUCsample, AUCcontrol and AUCTrolox were the integral areas under the fluorescence decay curve of the peptide with 75 mM PBS (pH 7.4) and Trolox, respectively. MTrolox and Msample were the concentrations of trolox and peptide, respectively.
2.2.11 Cell culture and cytotoxicity determination Human umbilical vein endothelial cells (HUVECs) were grown in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% (v/v) foetal bovine serum (FBS), 100 µg/ml penicillin-streptomycin and 5% CO2 at 37 °C. The cytotoxicity levels of samples on cells were measured using the MTT method. HUVECs were treated with 0.25% trypsin containing 0.02% EDTA and re-suspended with RPMI 1640 medium containing 10% (v/v) FBS. Cells (1 × 105 cells/ml) were seeded into 96-well plates and incubated for 24 h with peptides (20 and 100 µg/ml，respectively). After various treatments, the medium was removed and cells were incubated in a solution of 1 mg/ml MTT at 37 °C for 4 h. The supernatant was removed, and 150 µl of DMSO was added to solubilize the formed formazan salt. The concentration of formazan salt was determined by measuring the absorbance at 490 nm using a
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microplate reader (Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). The viability of cells was quantified as a percentage compared to the blank (peptides replaced with 0.01 M phosphate buffer saline).
2.2.12 Determination of ROS production Intracellular formation of ROS was determined by employing the oxidation sensitive dye 2’,7’-dichloro-fluorescence diacetate (DCFH-DA) as a substrate. HUVECs (1 × 105 cells/ml) were seeded into a 24-well plate and grown in RPMI 1640 medium containing 10% (v/v) FBS, 100 µg/ml penicillin-streptomycin and 5% CO2 at 37 °C for 12 h. The medium was replaced by RPMI 1640 medium (without FBS, but with added glucose), and the cells were incubated for 24 h. Subsequently, 10 µM DCFH-DA (prepared with RPMI 1640 medium) was added to each well and incubated for 1 h at 37 °C. Excess DCFH-DA was removed and cells were washed with RPMI 1640 medium three times. Treated cells were immersed in 400 µl of 0.01 M phosphate buffer saline, and images of stimulated HUVECs were collected using a Nikon ECLIPSE TE2000-U with a digital CCD camera (DS-U2, Nikon, Japan) under fluorescence.
2.2.13 Identification of antioxidant fraction and solid phase synthesis of peptides The active peak (F5) was further analysed by liquid chromatography-mass spectrometry to identify the amino acid sequences and parent proteins’ peptide composition (analysed by Sangon Biotech Co., Ltd). Peptides were synthetized
13
selectively through solid phase synthesis (synthetized by China Peptides Co., Ltd.) according to the amino acid sequences determined by liquid chromatography-mass spectrometry.
3. Results and discussion 3.1 Purification and immobilization of extracellular protease As some of the most important industrial enzymes, proteases have long been used for industrial purposes, and the use of immobilized enzymes in industry is a fascinating area of research. In this study, proteases were purified using anion exchange chromatography. The specific activity of purified protease was 2586.97 ± 67.35 U/mg, while the specific activity of crude enzyme was 902.38 ± 30.32 U/mg. Proteases were immobilized on chitosan beads via covalent binding. The specific activity of immobilized proteases was up to 1700.42 ± 32.31 U/mg, with an immobilization of 65.73 ± 1.38%. Thus, the protease from Vibrio sp. SQS2-3 in its immobilized form could potentially be applied in industrial processes.
3.2 Preparation and biological activity of salmon skin collagen hydrolysate The hydrolysate of salmon skin collagen was obtained after digestion with the protease of Vibrio sp. SQS2-3. As depicted in Fig. 1a, collagen from salmon skin can be digested by the protease. After 150 min of treatment, the hydrolysis degree did not significantly increase, which indicated that the hydrolysis had reached its maximum. To completely utilize collagen hydrolysate, it was subsequently fractionated into two
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parts (designated as UF-1 and UF-2) by ultrafiltration.
Research has shown that collagen can be a source of anti-freezing peptides. For example, Phanat et al. found peptides from the hydrolysate of blacktip shark skin that exhibited a cytoprotective effect on surimi subjected to various freeze-thaw cycles (Phanat, 2012). UF-1 was considered to be a potential anti-freezing fraction. As shown in Fig. 2, the residual Ca2+-ATPase activity of myoglobin decreased obviously after 3, 6, and 9 freeze-thaw cycles with distilled water and UF-2. When UF-1 was added to myoglobin, the residual Ca2+-ATPase activity decreased significantly slower than those with distilled water or UF-2 after freeze-thaw cycles. During freeze-thaw cycles, myosin may denature, likely because ice crystals are generated and the ionic strength increases, which destroys the original protein structure (Benjakul & Sutthipan, 2009). Peptides released from collagen commonly contain hydrophilic amino acid residues, which can bind water to prevent ice crystal formation. In addition, small, short peptides are considered to have lower anti-freezing activity because small peptides prefer to localize in the aqueous phase (Phanat, 2012). Peptides with more amino acid residues can form α-helical structures more easily. Jeong et al. reported that type I anti-freezing peptides with Ala and Thr exist in an α-helical conformation to form antiparallel homo-dimers. Thus, type I anti-freezing peptides could enhance their ice-binding and antifreeze activity by increasing the amount of active sites. (Jeong, 2013). Damodaran also reported that the -Gly-Pro-X- or -Gly-X-X- tripeptide repeat sequences in collagen peptides likely
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make great contributions to the inhibition of ice crystal generation (Damodaran, 2007).
Fraction UF-1 (> 3 kDa) displayed persistent low DPPH scavenging activity during hydrolysis, while the DPPH scavenging activity of the small fraction UF-2 (< 3 kDa) increased gradually with an improvement in the degree of hydrolysis (Fig. 1b). After 150 min of treatment, the antioxidant activity of the product reached a maximum (51.27 ± 1.97% in DPPH scavenging activity), close to that of the 180-240 min treatment, which indicated that the hydrolysis had reached a near-steady state.
3.3 Purification of antioxidant peptides from UF-2 and detection of antioxidant activity To obtain the antioxidant peptide, the smaller peptide fraction from ultrafiltration (UF-2) was further purified using size exclusion liquid chromatography. As shown in Fig. 3a, seven peptides fractions (F1-F7) were isolated from the smaller fraction of ultrafiltration (UF-2) after UF-2 was purified by Sephadex LH-20. The antioxidant activity assay showed that F5 exhibited much higher DPPH radical and hydroxyl radical scavenging activities (73.29 ± 1.03% and 72.73 ± 3.34%, respectively) than other isolated fractions (shown in Fig. 3b and 3c). The smallest fraction, F7, displayed the second strongest antioxidant activity, which was obviously lower than that of F5. From these results, we concluded that although F7 was the smallest peptide fraction and displayed a certain degree of antioxidant activity, F5 possibly contained more
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effective antioxidant peptides that could convert DPPH radicals and hydroxyl radicals into stable products and terminate the radical chain reaction. Both F5 and F7 were smaller than F1-F4, which proved that small peptides are more likely to exhibit antioxidant activity. To accurately evaluate the antioxidant activity of F5, two other assays were performed, including the ORAC assay and an assay to evaluate the protective effect against
oxidation-induced
DNA
damage.
The
protective
effect
against
oxidation-induced DNA damage was analysed to elucidate the positive role of the antioxidant peptide. In this study, the hydroxyl radical-generating system was based on the Fenton reaction (Fe2+ + H2O2). The running rate of plasmid DNA in agarose gel electrophoresis was in the order supercoiled DNA > linear DNA > open circular DNA due to the differences in the spatial structures. In addition, the degree of DNA damage was in the order linear DNA > open circular DNA > supercoil DNA. As shown in Fig. 4, most supercoiled DNA was converted into the open circular form due to hydroxyl radical damage. The purified fraction F5 had a significant protective effect on oxidation-induced DNA damage. The ORAC assay was used to detect the antioxidant activity of the peptide to quench peroxyl radicals (Elias, 2006). The protective effect of an antioxidant can be measured by assessing the fluorescence decay curve of the sample compared to a blank in which no antioxidant is present (Sheih, 2009). The time-dependent fluorescence decay curves induced by AAPH are shown in Fig. 5. Fig. 5a shows that the fluorescence intensity of the PBS group decreased rapidly, while F5 showed a
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dose-dependent increase in the inhibition of fluorescence decay. The ORAC assay was initiated by peroxyl radicals (LOO·) that also formed during lipid peroxidation. Radical chain reactions caused by peroxyl radicals were blocked by the antioxidant donating hydrogen. After that, the hydrogen donor formed a stable structure or became a more stable radical.
3.4 Cytotoxicity test of purified peptides on human umbilical vein endothelial cell The purified peptide fraction F5 was tested for its effect on the viability of HUVECs using the MTT assay. Fraction F5 did not display significant cytotoxicity at 20 and 100 µg/ml (Fig. 6). In addition, it showed even higher cell viability in test groups. This confirmed that the purified peptide fraction is safe towards HUVECs in terms of cytotoxicity and can be used to evaluate the intracellular radical-scavenging activity.
3.5 Intracellular radical-scavenging effects of peptides in human umbilical vein endothelial cells Hyperglycaemia is a factor that induces endothelial dysfunction in diabetes due to oxidative stress (Hemang, 2013). To investigate intracellular radical-scavenging effects, HUVECs were labelled with DCFH-DA. DCFH-DA can penetrate cells freely, where it is hydrolysed by an intracellular esterase to DCFH, which is then trapped inside cells. DCFH is non-fluorescent but is oxidized by intracellular ROS to form fluorescing DCF. As shown in Fig. 7, cells treated with RPMI 1640 medium
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containing 35 mM glucose displayed a stronger DCF-fluorescence intensity than cells in normal RPMI 1640 medium, which indicated that glucose at a high concentration could induce oxidative stress in HUVECs. Cells treated with different concentrations of purified peptide fraction F5 displayed less fluorescence intensity than the PBS-treated control group, suggesting that the purified peptide fractions could scavenge intracellular ROS to protect HUVECs from radical-mediated damage in hyperglycaemia.
3.6 Identification and verification of antioxidant peptide Purified fraction F5 was considered the most important contributor to the antioxidant activity of collagen hydrolysate. The peptide components of F5 were analysed using liquid chromatography mass spectrometry. Nineteen peptides were found in F5 (shown in Table. 1), and the peptide PMRGGGGYHY accounted for more than 86% of the content of F5. The sequence alignment showed that the peptide (PMRGGGGYHY) was part of the alpha-3 chain of collagen type I. The peptide was synthetized according to the sequence, and its antioxidant activity was detected using the ORAC assay. As shown in Fig. 5b, the synthetized peptide displayed dose-dependent antioxidant activity. Compared to the antioxidant activity of F5, as shown in Fig. 5c, the peroxyl radical scavenging activity of PMRGGGGYHY (2.51 ± 0.14 mmol TE/g) was lower than the antioxidant activity of F5 (2.78 ± 0.28 mmol TE/g), indicating that PMRGGGGYHY may be one of the major peptides that contributes to the antioxidant activity of F5.
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However, many other antioxidant peptides have not yet been identified. Notably, this peptide contains several amino acid residues, including methionine, tyrosine and histidine. Previous studies have reported that methionine residues constitute an important antioxidant defence mechanism. Exposed methionine residues work as a reactive site that scavenges oxidants through the formation of a sulfoxide structure after oxidation to stop free-radical chain reactions (Rodney, 1996). The methionine in PMRGGGGYHY was detected as oxidized, indicating that it may be an active site of this antioxidant peptide. In addition, aromatic amino acids, such as tyrosine, also play an important role in antioxidant activity as hydrogen donors (Wang, 2014). Tyrosine residues remove free radicals and change them into phenoxy radicals, which are much more stable. This is because the reactive activity of unpaired electrons is decreased by the formation of an electron-conjugated system, so the propagation of the radical-mediated peroxidizing chain reaction could be inhibited (Sheih, 2009). Histidine has been reported to be an important active site in antioxidant peptides due to its imidazole characteristics; it can also function as a proton donor, hydrogen donor, and lipid peroxyl radical trap (Niranjan, 2005; Je, 2007; Li, 2007). In addition, Glycine is also considered to be a potential target site of free radicals by supporting protons (Zhang, 2009). The backbone of peptides containing glycine are more flexible, exposing functional groups to free radicals (Wu, 2015).
4. Conclusion Many studies have shown that collagen is an ideal low-value protein resource that
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can be used in bioactive peptide preparations. However, new bacterial proteases have seldom been applied in previous studies. In addition, it is worth studying how to use these protein resources comprehensively. This study aimed to explore the potential application of bacterial extracellular protease in preparing a bioactive peptide. This study investigated whether bacterial extracellular protease can be used to hydrolyse collagen to prepare antioxidant and anti-freezing peptides in a single process. These peptides were considered for use in reducing oxidative damage and as cryoprotectants in protein storage. The characteristics and molecular mechanism will be further studied in the future.
Acknowledgements The work was supported by the National Natural Science Foundation of China (31370104, 21205142), National Sparking Plan Project (2013GA770009), Opening Foundation of the Chinese National Engineering Research Center for Control and Treatment of Heavy Metal Pollution (No. 2015CNERC-CTHMP-07), Open-End Fund for Valuable and Precision Instruments of Central South University (CSUZC201640), and the Fundamental Research Funds for the Central Universities of Central South University (2015zzts273, 2017zzts351, 2017zzts076).
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Figure legends Figure 1. (a) Hydrolysis degree of collagen treated for 30, 60, 90, 120, 150, 180, 210 and 240 min. (b) DPPH radical scavenging activity of the ultrafiltration fraction of collagen hydrolysates treated for 30, 60, 90, 120, 150, 180, 210 and 240 min. Values are expressed as the mean ± SD (n=3). Figure 2. Anti-freezing activity of UF-1-protecting Ca2+-ATPase activity of myoglobin against freeze-thawing circles. Values are expressed as the mean ± SD (n=3). Figure 3. (a) Size-exclusion liquid chromatography of the UF-2 fraction from ultrafiltration on a Sephadex LH-20 column. The F1 - F7 fractions represent purified fractions; (b) DPPH radical scavenging activity of the purified fraction from size exclusion liquid chromatography; (c) hydroxyl radical scavenging activity of the purified fraction from size exclusion liquid chromatography. Values are expressed as the mean ± SD (n=3). Figure 4. Protective effect of purified peptide on hydroxyl radical-induced oxidation of plasmid DNA. Lane 1, pET-22b plasmid DNA; Lane 2, Purified fraction F5; Lane 3, FeSO4 and H2O2 treatment (as DNA damage group); Lanes 4-5, FeSO4 and H2O2 treatment in the presence of purified fraction F5 at concentrations of 100 µg/ml and 150 µg/ml, respectively. Figure 5. Peroxyl radical scavenging activity (ORAC assay) of (a) purified fraction F5 and (b) synthesized peptide PMRGGGGYHY. (c) Comparison of the peroxyl radical scavenging activity of purified fraction F5 and synthesized peptide PMRGGGGYHY. Figure 6. Cell viability determined by MTT assay. Cytotoxicity of purified peptides from collagen hydrolysates on HUVECs was compared to non-treated group (blank). Values are expressed as the mean ± SD (n=3). Figure 7. Intracellular ROS in HUVECs indicated as green fluorescence by 28
DCFH-DA.
29
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Table 1
Table 1. Peptides identified through liquid chromatography mass spectrum Amino acid sequence
Source
PMRGGGGYHY (Oxidation @Met)
alpha 3 type I collagen [Oncorhynchus mykiss]
PHTGIFNVPMEGHY
EMILIN-2-like [Salmo salar]
PGTIVGSAAFN
sodium/calcium exchanger 2-like [Salmo salar]
VGKVTNGGYH
unnamed protein product [Oncorhynchus mykiss]
GITGPAGPRGPAGPHGPPGKDGRAGGH
alpha 2 type I collagen [Oncorhynchus keta]
GESGPAGPAGPNGPA
alpha 3 type I collagen [Oncorhynchus mykiss]
GIAGPAGPRGPSGPA
alpha 1 type I collagen [Oncorhynchus mykiss]
SGPAGPAGPNGPA
alpha 3 type I collagen [Oncorhynchus mykiss]
AGARSGADVDEGME
unnamed protein product [Oncorhynchus mykiss]
GARGADGSTGPAGPAGPL
type I collagen alpha 2 chain [Oncorhynchus keta]
LGFTQGSAAAGGG
tumor necrosis factor receptor associated factor 2
[Oncorhynchus mykiss]
GPAGPAGPPGPKGPVGPPGPA
unnamed protein product [Oncorhynchus mykiss]
GEGLLAVQITDPEGK
unnamed protein product [Oncorhynchus mykiss]
GLNGPSGPRGPHGS
alpha 3 type I collagen [Oncorhynchus mykiss]
DGRAGGHGAIGPVGH
alpha 2 type I collagen [Oncorhynchus keta]
IAGPAGPRGPSGPA
alpha 1 type I collagen [Oncorhynchus mykiss]
GPAGPRGPAGPHGPPG
alpha 2 type I collagen [Oncorhynchus keta]
GSVGIAGGPGHQGPG
alpha 2 type I collagen [Oncorhynchus keta]
AGGGYDQSGGYD
alpha 2 type I collagen [Oncorhynchus keta]
Highlights Salmon skin collagen was hydrolysed using immobilized/free marine bacterial collagenase The hydrolysates showed excellent anti-freezing (>3 kDa) and antioxidant activity (< 3 kDa). Activities of the purified peptides fraction included scavenging free radicals, protecting DNA from oxidative damage, and reducing oxidative stress, based on measures using ORAC and HUVEC. 19 novel peptides sequences were identified using mass spectrum.
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S0308-8146(17)31984-2 https://doi.org/10.1016/j.foodchem.2017.12.035 FOCH 22132
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
11 July 2016 26 September 2017 10 December 2017
Please cite this article as: Wu, R., Wu, C., Liu, D., Yang, X., Huang, J., Zhang, J., Liao, B., He, H., Antioxidant and anti-freezing peptides from salmon collagen hydrolysate prepared by bacterial extracellular protease, Food Chemistry (2017), doi: https://doi.org/10.1016/j.foodchem.2017.12.035
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Antioxidant and anti-freezing peptides from salmon collagen hydrolysate prepared by bacterial extracellular protease
RiBang Wu1, CuiLing Wu1, Dan Liu 1, XingHao Yang1, JiaFeng Huang1, Jiang Zhang1, Binqiang Liao1, HaiLun He1* 1
School of Life Science, State Key Laboratory of Medical Genetics, Central South
University, Changsha 410013 ，China
*Corresponding author: Hailun He，Tel：+86-0731-82650230; Fax：+86-0731-82650230; E-mail address: [email protected]
1
Abstract Extracted salmon skin collagen was hydrolysed with the free or immobilized extracellular protease of Vibrio sp. SQS2-3. The hydrolysate exhibited anti-freezing activity (> 3 kDa) and antioxidant activity (< 3000 Da) after ultrafiltration. The antioxidant peptide was further purified by size-exclusion chromatography and found to scavenge DPPH (73.29 ± 1.03%), ·OH (72.73 ± 3.34%,), and intracellular ROS in HUVECs; protect DNA against oxidation-induced damage; and have an ORAC of 2.78 ± 0.28 mmol TE/g. The antioxidant peptide fraction was identified using mass spectrometry, and nineteen salmon collagen-sourced peptides were obtained. Of these, the peptide Pro-Met-Arg-Gly-Gly-Gly-Gly-Tyr-His-Tyr is a novel sequence and was the major component; this peptide was shown to have antioxidant activity via the ORAC assay (2.51 ± 0.14 mmol TE/g). These results suggested that the protease from Vibrio sp. SQS2-3 is suitable for preparation of anti-freezing peptides and antioxidant peptides in a single step and represents a comprehensive use of fish skin collagen.
Keywords: Salmon skin collagen; bacterial extracellular protease; enzymatic hydrolysis; antioxidant peptide; anti-freezing
1. Introduction Reactive oxygen species (ROS), including hydroxyl radicals (·OH), superoxide anion radicals (O2· −), hydrogen peroxide (H2O2), peroxyl radicals (ROO· −), and
2
nitroxide radicals (NO·), are strongly related to aging and diseases, such as cancer, Alzheimer's disease, cardiovascular disease, diabetes and weakening of the immune system (Stohs, 1995; Diaz, 1997; Spolarics, 1998). Antioxidant substances (glutathione, β-carotene, vitamin C and vitamin E) and antioxidant enzymes (superoxide dismutase, glutathione S transferase and catalase) can remove excess free radicals to prevent oxidative stress. In addition, free radical-mediated oxidation in food also leads to potential toxicity, which is a concern of the food industry and consumers (Frankel, 2005). Therefore, synthetic antioxidants are utilized in the food industry, but their potential toxicity requires strict regulation; examples include butylated-hydroxytoluene
(BHT),
butylated-hydroxyanisole
(BHA),
tertbutylhydroquinone (TBHQ), and propyl gallate (PG). Peptides with antioxidant activity represent novel antioxidants that can replace synthetics. Many studies have reported that peptides released from parental protein by hydrolysis could be an effective way to obtain antioxidant peptides. Hydrogen bonds, electrostatic interactions, and disulphide bonds are the major forces that stabilize the structure of proteins (Benjakul, 2000). These bonds and interactions can be disrupted by a sharp temperature change during frozen storage (Benjakul, 2003; Benjakul, 2009). Cryoprotectant can protect proteins against freezing damage by inhibiting the generation of ice crystals (Alvarez, 2010), e.g., sorbitol and sucrose, which are common additives in the food storage industry (Jin, 2010). However, these cryoprotectants make food products sweet, which is often unacceptable to consumers (Ruttanapornvareesakul, 2006). It has been reported that a
3
protein hydrolysate containing oligopeptides is able to inhibit protein denaturation, including peptides from blacktip shark skin (Kittiphattanabawon, 2012), gelatine from bovine hide (Wang, 2009) and sericin (Wu, 2015). Recent studies have shown that collagen hydrolysates have various biological activities, such as gelatin hydrolysis with anti-freezing activity from blacktip shark skin (Phanat, 2012), antioxidant collagen peptides from croceine croaker (Wang, 2013), and angiotensin I-converting enzyme-inhibitory antioxidant peptides from skate skin gelatin (Ngo, 2014). Skin from salmon contains by-products that are rich in collagen, which can create value-added products by enzymatic hydrolysis. However, most salmon skin is discarded and/or used as animal feed. Therefore, this study focused on value-added utilization of collagen from salmon skin through enzymatic hydrolysis. An antioxidant peptide from ultrafiltration fractions with a small molecule weight was identified; ultrafiltration fractions containing larger peptides that were identified as potential cryoprotectants.
2. Materials and methods 2.1 Materials Fresh salmon by-products with skin were purchased from a seafood market in Shanghai and stored at -20 °C prior to use. Soybean meal was purchased from a supermarket in Changsha, Hunan Province, China, and stored at -20 °C. The protease-producing strain Vibrio sp. SQS2-3 was isolated from the water of the intertidal zone in Hainan Province, China (GenBank: KF220487.1).
4
Tryptone and yeast extract were purchased from Oxoid (Basingstoke, UK). 1,1-diphenyl-2-picrylhydrazyl (DPPH), fluorescein, 2,20-azobis (2-amidinopropane) dihydrochloride (AAPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich, Ltd. (Shanghai, China). Ninhydrin, 1,10-phenanthroline monohydrate was purchased from Aladdin® (Shanghai, China). Sephadex LH-20 was purchased from GE Healthcare Life Sciences (Uppsala, Sweden). RPMI 1640 medium and foetal bovine serum was purchased from Gibco® ThermoFisher Scientific Company (Grand Island, USA).
2.2 Methods 2.2.1 Preparation of salmon skin collagen Byproducts with skin were washed with flowing water for 5 min. The muscles were removed thoroughly. The skin was sheared into 5-mm pieces and washed with cold distilled water 5-7 times to remove fish oil. Salmon skin was mixed with distilled water at a ratio of 1:10 (w/v, g/ml), heated for 30 min at 75 °C, and centrifuged at 10,000 × g for 10 min to extract soluble collagen. The supernatant was dialysed using a dialysis bag (with 8 kDa to 14 k Da molecule weight cut-off) with 4 °C distilled water and lyophilized into collagen solids.
2.2.2 Preparation and Purification of bacterial extracellular protease Extracellular protease was obtained through bacterial fermentation according to the methods of Dan Liu (Liu, 2014) with some modifications. The protease-producing
5
strain Vibrio sp. SQS2-3 was activated at 15 °C with shaking at 200 rpm for 24 h in 2216E medium. The activated strain was seeded into fermentation broth (0.5 % corn powder, 0.5 % bean powder, 0.25 % wheat bran, 0.1 % CaCl2, 0.4 % Na2HPO4, and 0.03 % KH2PO4, prepared with sea water) at a ratio of 1:50 (v/v, ml/ml) and cultured at 15 °C with shaking at 200 rpm. The supernatant was collected by centrifugation (10,000 × g, 4 °C, 20 min) after 120 h and dialysed in Tris-HCl (20 mM, pH 7.8) at 4 °C. The crude enzyme from Vibrio sp. SQS2-3 was purified in a HiTrap DEAE FF (5 ml, 16 × 25 mm) anion exchange column with an NGC chromatography system (Bio-Rad, Hercules, CA, USA). The chromatography column was equilibrated with 20 mM Tris-HCl (pH 7.8) at a flow rate of 2 ml/min for 10 min. The crude enzyme was loaded into the pre-equilibrated column at a flow rate of 2 ml/min and washed with 20 mM Tris-HCl (pH 7.8) for 10 min. The column was eluted with a linear gradient of 1 M NaCl (0-100%) at a flow rate of 2 ml/min for 12.5 min. Protein fractions were monitored at 280 nm. The protease activity and total protein content were measured by the Folin phenol assay (Anson, 1938) and BCA method, respectively.
2.2.3 Immobilization of extracellular protease Enzyme immobilization on chitosan was performed according to Singh et al. Chitosan powder was dissolved in 1.5% (v/v) glacial acetic acid to prepare the chitosan solution (1.5%, w/v). The solution was dropped into a 1 M KOH solution containing 25% (v/v) ethanol and incubated for 1 h to prepare chitosan beads. The
6
beads were washed with 0.1 M sodium phosphate buffer (pH 7.8) and activated with 2% (v/v) glutaraldehyde at room temperature for 1 h. Subsequently, the activated chitosan beads were washed extensively with distilled water for complete removal of unreacted glutaraldehyde. For immobilization, the activated beads were incubated with purified protease at 4 °C for 12 h and washed with 0.1 M sodium phosphate buffer (pH 7.8) to remove unbound protease. The protease activity and total protein content were measured using the Folin phenol assay (Anson, 1938) and BCA method, respectively. The efficiency of enzyme immobilization was calculated as follows: immobilization ef icience % =
speci ic activity of immobilized protease × 100 speci ic activity of soluble protease
2.2.4 Enzymatic hydrolysis Salmon skin collagen was digested with the protease from Vibrio sp. SQS2-3 at an enzyme to substrate ratio of 1:10 (v/w, ml/mg) and temperature of 45 °C for 30, 60, 90, 120, and 150 min. The reaction was terminated at 95 °C for 10 min. The hydrolysate was collected and centrifuged at 10,000 × g at 4 °C for 10 min. The hydrolysis degree at various treatment times was analysed using the ninhydrin coloration method. The standard curve was determined using leucine at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0 mM.
2.2.5 Purification of bioactive fractions Ultrafiltration tubes with 3,000 Da MWCO (Amicon Ultra 14 ml 3K, Millipore, USA) were utilized for preliminary separation. The supernatant of the hydrolysate
7
was added to the upper casings of the ultrafiltration tubes, which were subsequently centrifuged at 4500 × g and 4°C for 60 min. Fractions in the upper casings and lower casings after ultrafiltration were collected and named UF-1 and UF-2. UF-1, with larger molecule weight, was considered to be a potential anti-freezing agent. Distilled water was added to UF-1 to replace the salt-containing solution using ultrafiltration tubes with 3k Da MWCO at 4,500 × g and 4°C for 45 min. UF-2, which had smaller molecule size, was further purified by size exclusion liquid chromatography. The fraction was loaded (1 ml) onto a Sephadex LH-20 column (16 × 600 mm), equilibrated with distilled water and eluted with distilled water at a flow rate of 0.75 ml/min. Each fraction was monitored at 220 nm, collected at a volume of 5 ml and lyophilized. Each fraction peak was prepared at the same concentration (150 µg/ml), and the antioxidant activity was further detected in subsequent assays.
2.2.6 Measuring Ca2+-ATPase activity of myoglobulin after freeze-thawing cycles Fresh porcine muscle was washed with cold distilled water and sliced into pieces (5 mm × 5 mm). The muscle was homogenized in distilled water (2 °C) at a ratio of 1:10 (w/v, g/ml) by a homogenizer at a speed of 11,000 rpm for 1 min. The homogenate was then centrifuged at 10,000 × g for 30 min at 2 °C, and the precipitate was collected. To extract myoglobulin, the precipitate was further homogenized with 1.2 M KCl, pH 7.0 for 4 min at a ratio of 1:10 (w/v) in an ice bath. The extract was centrifuged at 5000 × g for 30 min at 2 °C. Myoglobulin was precipitated with three
8
volumes of distilled water and collected by centrifuging at 5000 × g for 30 min at 2 °C. Then, myoglobulin was dissolved in an appropriate volume of KCl (1.2 M, pH 7.0) by stirring for 30 min at 2 °C. Myoglobulin was kept at 4 °C and was used within 12 h. The upper fraction from ultrafiltration was added to the myoglobulin solution at a ratio of 1:1 (v/v). The mixture was frozen at -20 °C for 20 min. The frozen sample was thawed at 37 °C in a water bath for 10 min. Freeze-thawing was performed for 0, 3, 6 and 9 cycles. Thawed samples were kept on ice until analysis. The upper fraction was replaced by distilled water in the blank. The Ca2+-ATPase activity of myoglobulin was determined using the method of Benjakul et al. (Benjakul, 1997) The freeze-thaw sample (25 µl) was mixed with 15 µl of 0.5 M Tris-maleate (pH 7.0), 25 µl of 0.1 M CaCl2 and 172.5 µl of distilled water. The mixture was mixed in a vortex mixer. ATP (20 mM, 12.5 µl) was added to initiate the reaction, which was incubated at room temperature for 10 min. The reaction was terminated by adding 125 µl of trichloroacetic acid (100 g/ml, 4 °C) and centrifuged at 5000 × g for 5 min. The inorganic phosphate liberated in the supernatant was measured by the method of Fiske and Subbarow (1925). The supernatant (50 µl) was added to 80 µl of 2.5% ammonium molybdate, 20 µl of 1% stannous chloride (prepared at a ratio of 1:10:25, SnCl2: HCl (conc.): 1 N H2SO4), and 900 µl of distilled water, and the mixture was incubated at room temperature for 10 min. The absorbance of the resulting mixture was measured at 720 nm by a multiplate reader. A blank solution was prepared by adding TCA prior to the addition of ATP. The
9
Ca2+-ATPase activity of myoglobulin after freeze-thawing for 3, 6 and 9 cycles was reported as residual Ca2+-ATPase activity (%), which was calculated in comparison to the sample without freeze-thawing.
2.2.7 DPPH radical scavenging activity assay The DPPH radical scavenging activity assay was conducted according to the method of Shimada et al. (1992). DPPH solution (100 µl, 0.1 mM in 95% ethanol) was mixed with 20 µl of the purified fraction solution (100 µg/ml) and incubated at room temperature for 1 h. The absorbance of the resulting solution was measured at 517 nm using a microplate reader (Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). For the blank, the purified fraction was replaced with distilled water. Vitamin C at a concentration of 200 µg/ml was used as a positive control. The DPPH radical scavenging activity was calculated using the following formula: DPPH radical scavenging activity (%) = [1-Abs sample /Abs blank] × 100
2.2.8 Hydroxyl radicals scavenging activity assay The hydroxyl radical scavenging activity was measured according to the method developed by Wang et al. (2012) A FeSO4 solution (40 µl, 2 mM), 1, 10-phenanthroline (40 µl, 2 mM) and sample (80 µl) were mixed stepwise. The reaction was initiated by adding 40 µl of H2O2 (0.03% v/v). After incubation at 37 °C for 60 min, the absorbance of the resulting solution was measured at 536 nm using a microplate reader (Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). The
10
group without any antioxidant was used as the negative control, while the mixture without H2O2 was used as the blank. The hydroxyl radical scavenging activity (HRSA) was calculated as follows: HRSA (%) = [(As - An)/(Ab - An)] × 100 where As, An, and Ab are the absorbance values determined at 536 nm for the sample, negative control and blank after reaction, respectively.
2.2.9 Protection effect on oxidation-induced DNA damage The protection effect assay for oxidation-induced damage followed the method described by Qian et al. (2008). Plasmid DNA has different structures according to the degree of damage, which can be detected by agarose gel electrophoresis. The reaction system included 8 µl of pET-22b DNA, 2 µl of 2 mM FeSO4, 8 µl of antioxidant and 2 µl of 0.1 mM H2O2. The mixture was incubated at 37 °C for 10 min and analysed by 1% agarose gel electrophoresis.
2.2.10 Oxygen radical absorbance capacity (ORAC) assay The ORAC assay was measured according to the method of Alberto et al. (2004). The reaction was performed in 75 mM phosphate buffer (pH 7.4). The sample solution (20 µl) and fluorescein (100 µl, 96 nM) was added to a 96-well microplate, and the reaction was initiated by adding 30 µl of AAPH (120 mM). The fluorescence intensity was measured every 30 s for 180 cycles with excitation and emission wavelengths of 485 nm and 538 nm, respectively, at 37°C in a microplate reader
11
(Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). Trolox was used as the positive control. The ORAC was defined as trolox equivalents (mmol TE/g peptide or mmol TE/mmol peptide) according to the area under the curve (AUC) and calculated as follows: ORAC = (AUCsample-AUCcontrol) / (AUCTrolox-AUCcontrol) × (MTrolox / Msample) where AUCsample, AUCcontrol and AUCTrolox were the integral areas under the fluorescence decay curve of the peptide with 75 mM PBS (pH 7.4) and Trolox, respectively. MTrolox and Msample were the concentrations of trolox and peptide, respectively.
2.2.11 Cell culture and cytotoxicity determination Human umbilical vein endothelial cells (HUVECs) were grown in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% (v/v) foetal bovine serum (FBS), 100 µg/ml penicillin-streptomycin and 5% CO2 at 37 °C. The cytotoxicity levels of samples on cells were measured using the MTT method. HUVECs were treated with 0.25% trypsin containing 0.02% EDTA and re-suspended with RPMI 1640 medium containing 10% (v/v) FBS. Cells (1 × 105 cells/ml) were seeded into 96-well plates and incubated for 24 h with peptides (20 and 100 µg/ml，respectively). After various treatments, the medium was removed and cells were incubated in a solution of 1 mg/ml MTT at 37 °C for 4 h. The supernatant was removed, and 150 µl of DMSO was added to solubilize the formed formazan salt. The concentration of formazan salt was determined by measuring the absorbance at 490 nm using a
12
microplate reader (Enspire 2300, Multimode Plate Reader, Perkin Elmer, USA). The viability of cells was quantified as a percentage compared to the blank (peptides replaced with 0.01 M phosphate buffer saline).
2.2.12 Determination of ROS production Intracellular formation of ROS was determined by employing the oxidation sensitive dye 2’,7’-dichloro-fluorescence diacetate (DCFH-DA) as a substrate. HUVECs (1 × 105 cells/ml) were seeded into a 24-well plate and grown in RPMI 1640 medium containing 10% (v/v) FBS, 100 µg/ml penicillin-streptomycin and 5% CO2 at 37 °C for 12 h. The medium was replaced by RPMI 1640 medium (without FBS, but with added glucose), and the cells were incubated for 24 h. Subsequently, 10 µM DCFH-DA (prepared with RPMI 1640 medium) was added to each well and incubated for 1 h at 37 °C. Excess DCFH-DA was removed and cells were washed with RPMI 1640 medium three times. Treated cells were immersed in 400 µl of 0.01 M phosphate buffer saline, and images of stimulated HUVECs were collected using a Nikon ECLIPSE TE2000-U with a digital CCD camera (DS-U2, Nikon, Japan) under fluorescence.
2.2.13 Identification of antioxidant fraction and solid phase synthesis of peptides The active peak (F5) was further analysed by liquid chromatography-mass spectrometry to identify the amino acid sequences and parent proteins’ peptide composition (analysed by Sangon Biotech Co., Ltd). Peptides were synthetized
13
selectively through solid phase synthesis (synthetized by China Peptides Co., Ltd.) according to the amino acid sequences determined by liquid chromatography-mass spectrometry.
3. Results and discussion 3.1 Purification and immobilization of extracellular protease As some of the most important industrial enzymes, proteases have long been used for industrial purposes, and the use of immobilized enzymes in industry is a fascinating area of research. In this study, proteases were purified using anion exchange chromatography. The specific activity of purified protease was 2586.97 ± 67.35 U/mg, while the specific activity of crude enzyme was 902.38 ± 30.32 U/mg. Proteases were immobilized on chitosan beads via covalent binding. The specific activity of immobilized proteases was up to 1700.42 ± 32.31 U/mg, with an immobilization of 65.73 ± 1.38%. Thus, the protease from Vibrio sp. SQS2-3 in its immobilized form could potentially be applied in industrial processes.
3.2 Preparation and biological activity of salmon skin collagen hydrolysate The hydrolysate of salmon skin collagen was obtained after digestion with the protease of Vibrio sp. SQS2-3. As depicted in Fig. 1a, collagen from salmon skin can be digested by the protease. After 150 min of treatment, the hydrolysis degree did not significantly increase, which indicated that the hydrolysis had reached its maximum. To completely utilize collagen hydrolysate, it was subsequently fractionated into two
14
parts (designated as UF-1 and UF-2) by ultrafiltration.
Research has shown that collagen can be a source of anti-freezing peptides. For example, Phanat et al. found peptides from the hydrolysate of blacktip shark skin that exhibited a cytoprotective effect on surimi subjected to various freeze-thaw cycles (Phanat, 2012). UF-1 was considered to be a potential anti-freezing fraction. As shown in Fig. 2, the residual Ca2+-ATPase activity of myoglobin decreased obviously after 3, 6, and 9 freeze-thaw cycles with distilled water and UF-2. When UF-1 was added to myoglobin, the residual Ca2+-ATPase activity decreased significantly slower than those with distilled water or UF-2 after freeze-thaw cycles. During freeze-thaw cycles, myosin may denature, likely because ice crystals are generated and the ionic strength increases, which destroys the original protein structure (Benjakul & Sutthipan, 2009). Peptides released from collagen commonly contain hydrophilic amino acid residues, which can bind water to prevent ice crystal formation. In addition, small, short peptides are considered to have lower anti-freezing activity because small peptides prefer to localize in the aqueous phase (Phanat, 2012). Peptides with more amino acid residues can form α-helical structures more easily. Jeong et al. reported that type I anti-freezing peptides with Ala and Thr exist in an α-helical conformation to form antiparallel homo-dimers. Thus, type I anti-freezing peptides could enhance their ice-binding and antifreeze activity by increasing the amount of active sites. (Jeong, 2013). Damodaran also reported that the -Gly-Pro-X- or -Gly-X-X- tripeptide repeat sequences in collagen peptides likely
15
make great contributions to the inhibition of ice crystal generation (Damodaran, 2007).
Fraction UF-1 (> 3 kDa) displayed persistent low DPPH scavenging activity during hydrolysis, while the DPPH scavenging activity of the small fraction UF-2 (< 3 kDa) increased gradually with an improvement in the degree of hydrolysis (Fig. 1b). After 150 min of treatment, the antioxidant activity of the product reached a maximum (51.27 ± 1.97% in DPPH scavenging activity), close to that of the 180-240 min treatment, which indicated that the hydrolysis had reached a near-steady state.
3.3 Purification of antioxidant peptides from UF-2 and detection of antioxidant activity To obtain the antioxidant peptide, the smaller peptide fraction from ultrafiltration (UF-2) was further purified using size exclusion liquid chromatography. As shown in Fig. 3a, seven peptides fractions (F1-F7) were isolated from the smaller fraction of ultrafiltration (UF-2) after UF-2 was purified by Sephadex LH-20. The antioxidant activity assay showed that F5 exhibited much higher DPPH radical and hydroxyl radical scavenging activities (73.29 ± 1.03% and 72.73 ± 3.34%, respectively) than other isolated fractions (shown in Fig. 3b and 3c). The smallest fraction, F7, displayed the second strongest antioxidant activity, which was obviously lower than that of F5. From these results, we concluded that although F7 was the smallest peptide fraction and displayed a certain degree of antioxidant activity, F5 possibly contained more
16
effective antioxidant peptides that could convert DPPH radicals and hydroxyl radicals into stable products and terminate the radical chain reaction. Both F5 and F7 were smaller than F1-F4, which proved that small peptides are more likely to exhibit antioxidant activity. To accurately evaluate the antioxidant activity of F5, two other assays were performed, including the ORAC assay and an assay to evaluate the protective effect against
oxidation-induced
DNA
damage.
The
protective
effect
against
oxidation-induced DNA damage was analysed to elucidate the positive role of the antioxidant peptide. In this study, the hydroxyl radical-generating system was based on the Fenton reaction (Fe2+ + H2O2). The running rate of plasmid DNA in agarose gel electrophoresis was in the order supercoiled DNA > linear DNA > open circular DNA due to the differences in the spatial structures. In addition, the degree of DNA damage was in the order linear DNA > open circular DNA > supercoil DNA. As shown in Fig. 4, most supercoiled DNA was converted into the open circular form due to hydroxyl radical damage. The purified fraction F5 had a significant protective effect on oxidation-induced DNA damage. The ORAC assay was used to detect the antioxidant activity of the peptide to quench peroxyl radicals (Elias, 2006). The protective effect of an antioxidant can be measured by assessing the fluorescence decay curve of the sample compared to a blank in which no antioxidant is present (Sheih, 2009). The time-dependent fluorescence decay curves induced by AAPH are shown in Fig. 5. Fig. 5a shows that the fluorescence intensity of the PBS group decreased rapidly, while F5 showed a
17
dose-dependent increase in the inhibition of fluorescence decay. The ORAC assay was initiated by peroxyl radicals (LOO·) that also formed during lipid peroxidation. Radical chain reactions caused by peroxyl radicals were blocked by the antioxidant donating hydrogen. After that, the hydrogen donor formed a stable structure or became a more stable radical.
3.4 Cytotoxicity test of purified peptides on human umbilical vein endothelial cell The purified peptide fraction F5 was tested for its effect on the viability of HUVECs using the MTT assay. Fraction F5 did not display significant cytotoxicity at 20 and 100 µg/ml (Fig. 6). In addition, it showed even higher cell viability in test groups. This confirmed that the purified peptide fraction is safe towards HUVECs in terms of cytotoxicity and can be used to evaluate the intracellular radical-scavenging activity.
3.5 Intracellular radical-scavenging effects of peptides in human umbilical vein endothelial cells Hyperglycaemia is a factor that induces endothelial dysfunction in diabetes due to oxidative stress (Hemang, 2013). To investigate intracellular radical-scavenging effects, HUVECs were labelled with DCFH-DA. DCFH-DA can penetrate cells freely, where it is hydrolysed by an intracellular esterase to DCFH, which is then trapped inside cells. DCFH is non-fluorescent but is oxidized by intracellular ROS to form fluorescing DCF. As shown in Fig. 7, cells treated with RPMI 1640 medium
18
containing 35 mM glucose displayed a stronger DCF-fluorescence intensity than cells in normal RPMI 1640 medium, which indicated that glucose at a high concentration could induce oxidative stress in HUVECs. Cells treated with different concentrations of purified peptide fraction F5 displayed less fluorescence intensity than the PBS-treated control group, suggesting that the purified peptide fractions could scavenge intracellular ROS to protect HUVECs from radical-mediated damage in hyperglycaemia.
3.6 Identification and verification of antioxidant peptide Purified fraction F5 was considered the most important contributor to the antioxidant activity of collagen hydrolysate. The peptide components of F5 were analysed using liquid chromatography mass spectrometry. Nineteen peptides were found in F5 (shown in Table. 1), and the peptide PMRGGGGYHY accounted for more than 86% of the content of F5. The sequence alignment showed that the peptide (PMRGGGGYHY) was part of the alpha-3 chain of collagen type I. The peptide was synthetized according to the sequence, and its antioxidant activity was detected using the ORAC assay. As shown in Fig. 5b, the synthetized peptide displayed dose-dependent antioxidant activity. Compared to the antioxidant activity of F5, as shown in Fig. 5c, the peroxyl radical scavenging activity of PMRGGGGYHY (2.51 ± 0.14 mmol TE/g) was lower than the antioxidant activity of F5 (2.78 ± 0.28 mmol TE/g), indicating that PMRGGGGYHY may be one of the major peptides that contributes to the antioxidant activity of F5.
19
However, many other antioxidant peptides have not yet been identified. Notably, this peptide contains several amino acid residues, including methionine, tyrosine and histidine. Previous studies have reported that methionine residues constitute an important antioxidant defence mechanism. Exposed methionine residues work as a reactive site that scavenges oxidants through the formation of a sulfoxide structure after oxidation to stop free-radical chain reactions (Rodney, 1996). The methionine in PMRGGGGYHY was detected as oxidized, indicating that it may be an active site of this antioxidant peptide. In addition, aromatic amino acids, such as tyrosine, also play an important role in antioxidant activity as hydrogen donors (Wang, 2014). Tyrosine residues remove free radicals and change them into phenoxy radicals, which are much more stable. This is because the reactive activity of unpaired electrons is decreased by the formation of an electron-conjugated system, so the propagation of the radical-mediated peroxidizing chain reaction could be inhibited (Sheih, 2009). Histidine has been reported to be an important active site in antioxidant peptides due to its imidazole characteristics; it can also function as a proton donor, hydrogen donor, and lipid peroxyl radical trap (Niranjan, 2005; Je, 2007; Li, 2007). In addition, Glycine is also considered to be a potential target site of free radicals by supporting protons (Zhang, 2009). The backbone of peptides containing glycine are more flexible, exposing functional groups to free radicals (Wu, 2015).
4. Conclusion Many studies have shown that collagen is an ideal low-value protein resource that
20
can be used in bioactive peptide preparations. However, new bacterial proteases have seldom been applied in previous studies. In addition, it is worth studying how to use these protein resources comprehensively. This study aimed to explore the potential application of bacterial extracellular protease in preparing a bioactive peptide. This study investigated whether bacterial extracellular protease can be used to hydrolyse collagen to prepare antioxidant and anti-freezing peptides in a single process. These peptides were considered for use in reducing oxidative damage and as cryoprotectants in protein storage. The characteristics and molecular mechanism will be further studied in the future.
Acknowledgements The work was supported by the National Natural Science Foundation of China (31370104, 21205142), National Sparking Plan Project (2013GA770009), Opening Foundation of the Chinese National Engineering Research Center for Control and Treatment of Heavy Metal Pollution (No. 2015CNERC-CTHMP-07), Open-End Fund for Valuable and Precision Instruments of Central South University (CSUZC201640), and the Fundamental Research Funds for the Central Universities of Central South University (2015zzts273, 2017zzts351, 2017zzts076).
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Figure legends Figure 1. (a) Hydrolysis degree of collagen treated for 30, 60, 90, 120, 150, 180, 210 and 240 min. (b) DPPH radical scavenging activity of the ultrafiltration fraction of collagen hydrolysates treated for 30, 60, 90, 120, 150, 180, 210 and 240 min. Values are expressed as the mean ± SD (n=3). Figure 2. Anti-freezing activity of UF-1-protecting Ca2+-ATPase activity of myoglobin against freeze-thawing circles. Values are expressed as the mean ± SD (n=3). Figure 3. (a) Size-exclusion liquid chromatography of the UF-2 fraction from ultrafiltration on a Sephadex LH-20 column. The F1 - F7 fractions represent purified fractions; (b) DPPH radical scavenging activity of the purified fraction from size exclusion liquid chromatography; (c) hydroxyl radical scavenging activity of the purified fraction from size exclusion liquid chromatography. Values are expressed as the mean ± SD (n=3). Figure 4. Protective effect of purified peptide on hydroxyl radical-induced oxidation of plasmid DNA. Lane 1, pET-22b plasmid DNA; Lane 2, Purified fraction F5; Lane 3, FeSO4 and H2O2 treatment (as DNA damage group); Lanes 4-5, FeSO4 and H2O2 treatment in the presence of purified fraction F5 at concentrations of 100 µg/ml and 150 µg/ml, respectively. Figure 5. Peroxyl radical scavenging activity (ORAC assay) of (a) purified fraction F5 and (b) synthesized peptide PMRGGGGYHY. (c) Comparison of the peroxyl radical scavenging activity of purified fraction F5 and synthesized peptide PMRGGGGYHY. Figure 6. Cell viability determined by MTT assay. Cytotoxicity of purified peptides from collagen hydrolysates on HUVECs was compared to non-treated group (blank). Values are expressed as the mean ± SD (n=3). Figure 7. Intracellular ROS in HUVECs indicated as green fluorescence by 28
DCFH-DA.
29
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Table 1
Table 1. Peptides identified through liquid chromatography mass spectrum Amino acid sequence
Source
PMRGGGGYHY (Oxidation @Met)
alpha 3 type I collagen [Oncorhynchus mykiss]
PHTGIFNVPMEGHY
EMILIN-2-like [Salmo salar]
PGTIVGSAAFN
sodium/calcium exchanger 2-like [Salmo salar]
VGKVTNGGYH
unnamed protein product [Oncorhynchus mykiss]
GITGPAGPRGPAGPHGPPGKDGRAGGH
alpha 2 type I collagen [Oncorhynchus keta]
GESGPAGPAGPNGPA
alpha 3 type I collagen [Oncorhynchus mykiss]
GIAGPAGPRGPSGPA
alpha 1 type I collagen [Oncorhynchus mykiss]
SGPAGPAGPNGPA
alpha 3 type I collagen [Oncorhynchus mykiss]
AGARSGADVDEGME
unnamed protein product [Oncorhynchus mykiss]
GARGADGSTGPAGPAGPL
type I collagen alpha 2 chain [Oncorhynchus keta]
LGFTQGSAAAGGG
tumor necrosis factor receptor associated factor 2
[Oncorhynchus mykiss]
GPAGPAGPPGPKGPVGPPGPA
unnamed protein product [Oncorhynchus mykiss]
GEGLLAVQITDPEGK
unnamed protein product [Oncorhynchus mykiss]
GLNGPSGPRGPHGS
alpha 3 type I collagen [Oncorhynchus mykiss]
DGRAGGHGAIGPVGH
alpha 2 type I collagen [Oncorhynchus keta]
IAGPAGPRGPSGPA
alpha 1 type I collagen [Oncorhynchus mykiss]
GPAGPRGPAGPHGPPG
alpha 2 type I collagen [Oncorhynchus keta]
GSVGIAGGPGHQGPG
alpha 2 type I collagen [Oncorhynchus keta]
AGGGYDQSGGYD
alpha 2 type I collagen [Oncorhynchus keta]
Highlights Salmon skin collagen was hydrolysed using immobilized/free marine bacterial collagenase The hydrolysates showed excellent anti-freezing (>3 kDa) and antioxidant activity (< 3 kDa). Activities of the purified peptides fraction included scavenging free radicals, protecting DNA from oxidative damage, and reducing oxidative stress, based on measures using ORAC and HUVEC. 19 novel peptides sequences were identified using mass spectrum.
30