Isolation and identification of antioxidant peptides from crocodile meat hydrolysates using silica gel chromatography

Materials

The crocodile (Crocodylus siamensis) meat was purchased from the Huarun supermarket, Guangzhou, China. The meat was brought in an ice box to the laboratory and refrigerated at −20°C before use. The silica gel and silica gel G plates (thickness 0.25 mm, 100 × 100 mm or 200 × 100 mm) were purchased from Qingdao Marine Chemicals Co. (Qingdao, China). Protein marker is purchased from Takara Co. (Shanghai, China). Coomassie brilliant blue R-250 and G250, DPPH (diphenyl picryl hydrazinyl radical), ABTS [2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)], and BCA (bicinchoninic acid) were purchased from Qihuashen Co. (Guangzhou, China). Papain (2.0 × 105 U/g), pepsin (2.3 × 105 U/g), trypsin (1.5 × 105 U/g) and alcalase (3.0 × 105 U/g) were purchased from Qihuashen Co. (Guangzhou, China). Other chemical and biochemical reagents are analytical or biochemical grade and were purchased from local suppliers.

Preparation of crocodile meat proteins and hydrolysates

Degrease

The crocodile (Crocodylus siamensis) meat was cut into small pieces, homogenized at room temperature for half an hour and then defatted as described by Jang et al.23. The fat in the homogenate of crocodile meat was removed by stirring with isopropanol (1:4, w/v) for 50 min at room temperature. After centrifugation at 700g for 30 min, the supernatant was discarded, and the precipitate was lyophilized in a freeze dryer as the defatted crocodile meat.

protein extraction

Five grams of the defatted crocodile meat powder were extracted with 20 ml distilled water and stirred for 10, 20, 40 or 80 min, at room temperature or on boiling water bath, and then centrifuged at 1600g for 20 min. The supernatant was taken as protein extracts for enzymatic digestion.

Protein hydrolyzation

The crocodile proteins were hydrolyzed according to the previously described procedures24 with slight modifications. The protein extracts equivalent to 5 g of crocodile meat was diluted to 100 mL with distilled water and zymolized for 2 h at their corresponding optimal pH and temperature conditions by papain (pH 6.2, 37 °C), pepsin (pH 2.0, 37 °C ), trypsin (pH 7.6, 37°C) and alcalase (pH 8.0, 50°C). The addition of protease was 2%. After reaction, the hydrolysates were adjusted to pH 7.0 and heated at 97°C for 10 min to deactivate the enzymes. The hydrolysates were obtained by centrifugation at 1600g for 20 min. The supernatant was freeze-dried and stored at −20°C.

Protein analysis

Protein content

The protein contents were determined using the Bradford assay25.

Degree of hydrolysis (DH)

The degree of hydrolysis of the hydrolysates was determined using the TNBS (trinitro benzene sulfonic acid) method outlined in Maux et al.26.

SDS-PAGE and Tricine-SDS-PAGE analyzes of proteins and peptides

Crocodile meat proteins were analyzed using SDS-PAGE method according to Babini et al.27. Stacking and separation gels were 5% and 12%, respectively. All samples with 2% (v/v) 2-mercaptoethanol were heated for 5 min in a boiling water bath. Ten microliter of sample solution was loaded into sample well and electrophoresed at constant voltage of 80 V for stacking gel and 120 V for separating gel. The gel was stained using Coomassie Brilliant Blue G-250.

The hydrolysates were analyzed using Tricine-SDS-PAGE method, according to the method of Li et al.28. Stacking and separation gels were 4% and 20%, respectively. All samples with 2% (v/v) 2-mercaptoethanol were heated for 5 min in a boiling water bath. Ten microliter of sample solution was loaded and electrophoresed at constant voltage of 30 V for stacking gel and 100 V for separating gel. The gel was stained using Coomassie Brilliant Blue G-250.

Determination of the antioxidant activities

The DPPH radical-scavenging activity was determined according to the method of Santos et al.29 with some modification. Samples were diluted to 3, 6, 9, 12 and 15 mg/mL with 0.01 mol/L phosphate buffer (pH 7.0). To a 160 μL aliquot of each sample, 640 μL of 0.1 mmol/L DPPH in ethanol (DPPH was dissolved in 95% ethanol) was added. The solution was well mixed and left at ambient temperature for 30 min in the dark. After that, the absorbance of the solution was spectrophotometrically measured at 517 nm. The absorbance value of the sample mixed with DPPH in ethanol was presented as Asample. The absorbance value of sample mixed with 95% ethanol solution was presented as Acontrol. The absorbance value of DPPH in ethanol alone was presented as Ablank. The percentage of DPPH scavenging activity was calculated as: Radical scavenging activity (%) =[1 − (Asample − Acontrol)/Ablank]×100.

The ABTS radical scavenging activity was measured according to the method of Zhang et al.30. Briefly, 7 mmol/L ABTS solution was well mixed with 2.45 mmol/L potassium persulfate solution in equal volume, and then the solution was left overnight (12–16 h) in the dark at room temperature. The above solution was diluted with 5 mmol/L phosphate buffer (pH 7.4) to make the absorbance by spectrophotometer at 734 nm be 0.7 ± 0.02. Twenty microliters of sample solution with 3, 6, 9, 12 and 15 mg/mL, was added to 2 mL diluted ABTS reagent, and the mixture was left at ambient temperature for 6 min accurately. Absorbance was then recorded at 734 nm. The blank was prepared in the same way, except that sample was replaced with deionized water. The percentage of ABTS radical scavenging activity was calculated as follows: Radical scavenging activity (%) = (Ablank− Asample)/HASblank×100.

TLC-bioautography

Following the chromatographic separation and evaporation of the mobile phase, the silica gel plates were sprayed equably by chromogenic reagent according to the method of Krüger et al.31. Spraying ninhydrin alcohol solution (0.5%, w/v), peptide bands emerged red in 30 min at 105 °C; spraying DPPH alcohol solution (0.1%, w/v), antioxidant bands emerged white over night at room temperature; spraying copper sulfate (1%, w/v), BCA aqueous solution (5%, w/v), protein bands emerged blue over night at room temperature.

Separation and purification of peptides

Molecular weight fractionation

The hydrolysates were fractionated by using ultrafiltration membranes according to Abdelhedi et al.32 with molecular weight cut-off (MWCO) membranes of 3 kDa and 30 kDa. The fractions obtained were < 3 kDa, 3–30 kDa, > 30 kDa. The collected fractions were freeze dried, stored at −20°C.

Separation of active peptides by silica gel column chromatography

Active fraction of MW < 3 kDa was dissolved in 1 ml distilled water and loaded onto a silica column (Φ2.7 × 40 cm). The column was eluted sequentially with solvents of 1,2-dichloroethane/methanol of 7/3, 6/4, 5/5, 4/6, 3/7, 2/8, 1/9, 0/10, and methanol /water of 9/1, 50/50 and 0/10), each for one bed volume, at a flow rate of 2.0 ml/min. The fractions were concentrated and stored at −20°C until used.

Analysis and purification of antioxidant peptide by TLC

The samples or active fractions from silica column chromatography were analyzed or purified by TLC. Silica gel TLC plates were developed/washed with methanol and then activated at 105 °C for 30 min, before used for sample separation. After loading the samples, the TLC plates were developed with solvents of 1,2-dichloroethane: methanol: ammonia 1:9:0.2 (V/V).

Identification and synthesis of the purified peptides

Purified peptides were identified by HPLC–MS in the Wininnovate Company (Shenzhen, China). Identified active peptides were synthesized by NJ peptide Company (Nanjing, China) for activity assay.

Statistical analysis of the data

All quantitative analyzes were repeated at least three times. The averages plus standard errors were presented.

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