Anti-Oxidative And Anti-Aging Activities Of Porcine By-Product Collagen Hydrolysates Produced Part 2

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2.2. Ultrafiltration Effect on Collagen Hydrolysate Properties

An ultrafiltration process may be a useful, industrially advantageous method for producing small peptide fractions with a desired molecular size and high bioactivity, depending on the composition of the starting hydrolysate and the activity being studied [19]. Solubility, free amino group content, and the yield of CHs after lyophilization is shown (Table 1). The solubility and free amino group content of the control were 11.95% and 0.79%, respectively. After enzymatic hydrolysis and ultrafiltration with 3 kDa molecular weight cut-off, the highest solubility (21.17%), and free amino group content (14.17%)were observed in CH-Alcalase<3 kDa. However, CH-Alcalase<3 kDa was the lowest yield (12.42%)observed. Therefore, although ultrafiltration is useful in separating CHs with a low molecular weight it may cause a reduction in yield.

The average molecular weight of protein hydrolysates is an important factor that determines their biological properties [19]. Generally, an average fraction with MW<3 kDa represents a collagen hydrolysate; an average fraction with MW>50 kDa represents gelatin, and an average fraction with MW>300 kDa represents collagen [20,21]. The relative molecular weight distribution of the control (pretreatment sample), CH-Alcalase, and CH-Alcalase<3 kDa is depicted in Figure 4. The molecular weight distribution was over 20,100 Da for the control, which did not include the collagen hydrolysate (MW<3 kDa). This could not be numerically provided in this study, as the detection limit of the index detector system only ranged from 106 to 20,100 Da. However, CH-Alcalase showed detectable values in a higher range of relative molecular weight distribution, which ranged from 20,100 Da to 4270 Da (maximum peak:12,600 Da). In CH-Alcalase<3 kDa, the molecular weight distribution mainly showed three peaks: one with an MW of approximately 4270 Da (maximum peak), one with an MW of approximately 424 Da, and one with an MW of approximately 222 Da and 102 Da. Ultrafiltration is an effective purification method used to obtain low molecular weight peptides from crude hydrolysates. Results indicated that enzymatic hydrolysis by Alcalase clearly reduced the high MW of the control (either collagen or gelatin), and that ultrafiltration was an effective purification method that can be used to obtain low molecular weight peptides(<3 kDa) from crude collagen hydrolysates. Reportedly, low molecular weight peptides (2-20 amino acids) are more biologically active compared to their parent polypeptide/proteins, which are larger [22].

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Amino acid composition in the CHs is shown (Table 2). The CHS by different proteases had different amino acid compositions and antioxidant properties [23]. The amino acid composition of collagen was rich in glycine(Gly), proline (Pro), and glutamic acid (Glu). The amino acid content of CHs(CH-Alcalase and the CH-Alcalase<3 kDa) increased more than that of the control, following enzymatic hydrolysis with or without ultrafiltration. In particular, the content of Gly, Pro, and Glu was much higher in CH-Alcalase<3 kDa(Gly 218 mg/g, Pro 152 mg/g, and Glu 120 mg/g) than CH-Alcalase(Gly149 mg/g, Pro 95 mg/g, and Glu78 mg/g). An increase in the content of these amino acids is strongly related to enhanced antioxidant capabilities [16,20]. Gly and Pro contain hydrophobic amino acid groups, and Glu contains negatively charged amino acid groups. cistanche stem These amino acids have been reported to enhance antioxidant activity because of their increased solubilities in lipids or via free radical reactions [1,24].

2.3. Antioxidant and Anti-Aging Activities of Collagen Hydrolysates

Antioxidant activities of the CHs were measured using 2,2'-and-bis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)radical-scavenging activity assay and reducing power assay. The ABTS radical-scavenging activity of the control (collagen suspension), CH-Alcalase, and CH-Alcalase <3 kDa at different concentrations is shown (Figure 5A).ABTS radical scavenging activity of peptides assay is important to exclusively measure the ability of an antioxidant peptide to induce a hydrogen atom transfer [25]. ABTS radical-scavenging effects of all treatments increased in a concentration-dependent manner (p<0.05). CH-Alcalase and CH-Alcalase<3 kDa showed much higher ABTS radical-scavenging activity values, with 41.4%-88.2% compared to the control(<8.5%. Both CH-Alcalase and CH-Alcalase<3 kDa had high ABTSradical-scavenging abilities of ~60% when concentrations were greater than 2.5 mg/mL. CH-Alcalase<3 kDa showed a significantly higher ABTS radical-scavenging activity value than CH-Alcalase. cistanche salsa extract In general, the antioxidant activity of peptides may be influenced by their amino acid sequences, the number of free amino acids present, the degree of hydrolysis, and the molecular weight of peptides [26,27]. In particular, low molecular weight hydrolysates possessed stronger antioxidant properties compared to high molecular weight hydrolysates [2,26].

Figure 5. Antioxidant activities of collagen hydrolysates (CH) in 2,2'-and-bis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)radical scavenging(A)and reducing power (B) assays. Data denoted by different letters (a-c) show statistically significant differences depending on different treatments (p<0.05). cistanche tubulosa benefits and side effects Data denoted by different letters(A-D) show statistically significant differences depending on concentration (p < 0.05).

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The reducing powers of the control, CH-Alcalase, and CH-Alcalase <3 kDa are shown (Figure 5B). The reducing power of peptides may also serve as a significant indicator of their antioxidant potential [28,29]. The reducing power of CHs ranged from 0.074 to 0.424 in a dose-dependent manner (p<0.05). The control group had the lowest reducing power, which did not change significantly with increasing concentrations of the control. In contrast to ABTS radical-scavenging activity, the reducing power of CH-Alcalase was higher than that of CH-Alcalase<3 kDa. cistanche tubulosa extract Similar observations suggested that crude collagen hydrolysate may be more effective in reducing power than ultrafiltrated collagen peptides [30].

Inhibition of tyrosinase, collagenase, and elastase activity was used to verify theirs in vitro anti-aging effects [20]. The results of the inhibition of tyrosinase, collagenase, and elastase activity of the control, CH-Alcalase, and CH-Alcalase<3 kDa are summarized (Table 3). Tyrosinase, collagenase, and elastase inhibitors have been used as important ingredients of cosmetics for skin whitening, anti-aging, and anti-wrinkling, respectively. Collagenase and elastase, especially, are known to be major enzymes responsible for dehydration and wrinkle formation on the skin surface[31]. The results indicated that the tyrosinase inhibition effect of vitamin C(95.50%,1 mg/mL, positive control) was higher than that of the other treatments(Table 4). Tyrosinase inhibition effects of control, CH-Alcalase, and CH-Alcalase<3 kDa groups were 28.21%, 15.44%, and 30.20%, respectively. Thus, the CHs did not show a better skin whitening effect compared to the control. Collagenase inhibition by the control, CH-Alcalase, and CH-Alcalase<3 kDa groups were 6.45%,54.37%, and 61.90%, respectively. In addition, CH-Alcalase and CH-Alcalase<3 kDa inhibition of collagenase corresponded to that of vitamin Cat 1 mg/mL. Thus, CHs obtained from this study may be effective collagenase inhibitors that may possibly play an important role in anti-aging activities. However, all collagen samples did not display elastase inhibition effects, which may result from a lack of skin elasticity. Overall, vitamin C(1 mg/mL) inhibited various activities of the enzymes in the following order: tyrosinase(95.50%)>collagenase(48.09)> elastase(2/.00%o).CH-Alcalase(5 mg/mL)inhibited various activities of the enzymes in the following order: collagenase (54.37%)> tyrosinase (15.44%)> elastase (no activity). CH-Alcalase<3 kDa (5 mg/mL) inhibited these activities in the following order: collagenase (61.90%)>tyrosinase (30.20%)> elastase (no activity). Similar trends reported in another study indicated that collagen hydrolysates showed a potential to act as anti-aging agents and collagenase inhibitors [20].

3. Materials and Methods

3.1. Porcine Skin Pretreatment

Porcine skin was purchased from a local supplier (Seoul, Korea), and all visible fat and connective tissues of the porcine skin were removed using a razor blade. The porcine skin used in this study was obtained from one porcine in order to minimize biological variation. Trimmed porcine skin was washed in water at 90°C for 1 min four times to remove fat and residual materials. The skin was then cut into 1 cm square sections and pulverized in distilled water for 3 min using a four-wing blade blender(CNHR-26, Bosch, Hong Kong, China). The pulverized porcine skin was homogenized at high-speed (25,000rpm) for 5 min using an Ultra Turrax(T25, IKA Labotechnik, Staufen, Germany). Approximately 100g of the porcine skin mixture (50% final solid contents)was vacuum-packaged and frozen at -20 °C and stored for use within 1 month.

3.2. Commercial Proteases and Reagents

Alcalase, Flavorzyme, Neutrase, and Protamex were purchased from Novozymes(Bagsvaerd, Denmark). Bromelain and Papain were purchased from Daesong Sangsa(Seoul, Korea). All chemicals for antioxidant and anti-aging tests were purchased from the Sigma-Aldrich Chemical Company (St.Louis, MS, USA). All other reagents and solvents used in this study were of analytical grade.

3.3. Enzyme Hydrolysis

Enzymatic hydrolysis was developed for the respective food-grade commercial enzymes used (based on the manufacturer's recommendations; Table 4). The prepared porcine skin mixture was diluted with distilled water to a final solid content of 5%. This concentration was selected to ensure flow behavior because of its low viscosity. The 5% porcine skin mixture was termed the collagen suspension (or control). The collagen suspension was hydrolyzed in reactors using six food-grade commercial enzymes at an enzyme: substrate ratio of 1:100. Sample aliquots (5 mL)were drawn at 1, 3,6, 12, and 24 h of hydrolysis and immediately heated at 100°C for 10 min to inactivate the enzyme, followed by cooling to 0°C using ice water. During the time of sampling, pH was controlled using 1 M NaOH as appropriate. After cooling, samples were centrifuged at 4000×g for 15 min, and the supernatant (collagen hydrolysate; CH) was collected. CH was concentrated further by ultrafiltration, using an AmiconStirred Cells system(Catalog No. UFSC 20001, EMD Millipore Corporation, Burlington, MA, USA)with a 3 kDa molecular weight cut-off (MWCO)(UltracelMembrane, EMD Millipore Corporation, Burlington, MA, USA)at 60 psi nitrogen gas, at 20°C. cistanche tubulosa reviews The prepared CH was freeze-dried and kept in air-tight containers at 20 °C until analysis.

3.4. Determination of pH, Protein Recovery, Solubility Free Amino Group Content, and Production Yield The pH of the samples (control and CHs)was determined using a pH meter (Model S220, Mettler Toledo GmbH, Columbus, OH, USA). The protein recovery or solubility was determined by estimating protein content using bicinchoninic acid (BCA) protein assay, according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MS, USA) with serum albumin as the standard. Free amino group content was determined via a 2,4,6-trinitrobenzene sulfonic acid(TNBSA) assay according to the manufacturer's instructions(Thermo Fisher Scientific, Waltham, MA, USA)using L-leucine as the standard. Both wet and dry weights were measured to calculate the production field.

3.5. Molecular Weight Distribution

3.5.1.Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The SDS-PAGE patterns of samples (control and CHs) were measured according to a previously reported method [10]. Samples were diluted with 8 M urea (final protein concentration,4 mg/mL). Each sample was mixed with one part of KTG 020 sample buffer(10% of glycerol,2% of SDS,0.003%of bromophenol blue,5% of β-mercaptoethanol, and 63 mM Tris-HCl, pH6.8)from KOMA Biotech Inc.， （Seoul， Korea）， and boiled for 2 min. The sample mixture （20 μL） was loaded into EzWayTM PAG 6% acrylamide gels (KOMA Biotech Inc., Seoul, Korea). Following electrophoresis, the gel was fixed, stained, and de-stained. The molecular weights were determined using wide-range molecular weight standards between 10 and 210 kDa.

3.5.2. Gel Permeation Chromatography (GPC)

The molecular weight distribution of samples was determined according to a previously reported method [3]. Gel permeation chromatography(GPC) was performed using a YL9100 high-performance liquid chromatography (HPLC)system(YL9100, Youngling Instrument Co., Ltd, Gyeonggi-do, Korea) equipped with three Ultrahydrogel TM 120 columns(7.8×3000 mm) from Waters(Milford, MA, USA). The mobile phase was distilled/deionized water at a flow rate of 1.0 mL/min, and the molecular weight distributions of the collagen peptides were monitored using a YL 9100 refractive index detector at 40℃. A molecular weight standards kit (106-67,500 Da, Polymer Standards Service, Mainz, Germany)served as the standard.

3.6.Amin0 Acid Composition

The amino acid composition of the samples was analyzed through derivatization with 9-fluorenyl ethoxy carbonyl (FMOC)-chloride and o-phthalic dialdehyde (OPA)on an Ultimate 3000 HPLC system (Dionex, Idstein, Germany)equipped with two detectors (a fluorescence detector and a UV detector） and a VDSpher 100 C18-E （4.6 mm ×150 mm， 3.5 μm particle size， VDS Optilab， Berlin, Germany). The injection volume was 1.0 L, and the mobile phase was composed of two eluents: a 40 mM sodium phosphate dibasic (pH 7) and a 45%(o/v)acetonitrile/45%(v/v) methanol solution. By connecting a UV detector and fluorescence detector, ultraviolet rays were detected at 338 nm, OPA derivative was detected at 450 nm of an emission wavelength and 340 nm of an excitation wavelength, FMOC derivative was detected at 305 nm of an emission wavelength of and 266 nm of an excitation wavelength. An amino acid mix(1.0 nmol mL-I for each amino acid) was used for calibration.

where Atotalamino acid was the content after hydrolysis using 6 M HCl (at 130 °C for 24 h), and Afree amino acid was the content after solubilizing in distilled water.

3.7. Evaluation of Antioxidant Activity

3.7.1.2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)Radical-Scavenging Activity

The ABTS radical-scavenging activity of CH was measured according to a previously reported method [20]. The ABTSradical cation was generated by mixing ABTS stock solution (7.0 mM) with potassium persulfate (2.45 mM) and incubating the resultant mixture in the dark at room temperature overnight. The ABTS radical solution was diluted in5.0 mM phosphate-buffered saline (pH7.4) to an absorbance level of 0.70±0.02 at 734 nm. One mL of the diluted ABTS radical solution was mixed with 1 mL of each sample. Ten minutes later, sample (A sample, with sample)and control(A control without sample) absorbances were measured at 734 nm. The ABTS radical-scavenging activity(%)Was calculated as:

3.7.2.Reducing Power

The reducing power of CH was measured according to a previously reported method [20]. One mL of each sample was mixed with1 mL of 0.2 M phosphate buffer(pH6.6) and 1 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min, and then 1 mL of 10% trichloroacetic acid was added. An aliquot of 2 mL from this incubation mixture was mixed with2 ml of distilled water and 0.4 mL of 0.1% ferric chloride. After 10min, the absorbance of the resulting solution was measured at 700 nm on a spectrophotometer (OPTIZEN, Mecasys Co., Daejeon, Korea)Increased absorbance (at 700 nm) of the reaction mixture was considered to indicate increased reducing power.

3.8. Evaluation of the Anti-Aging Effect

3.8.1. Inhibition of Tyrosinase Activity

Tyrosinase inhibition was determined by a previously described method [20]. A premixture solution containing 70 μL of 0.1 M phosphate buffer （pH6.8），30uLof mushroom tyrosinase（167U/mL; Sigma-Aldrich, USA), and 20 L of the sample was incubated for 5 min at 30°C. Approximately 100μL of 3，4-dihydroxy phenyl-L-alanine （L-DOPA）was then added to initiate the enzymatic reaction. Absorbance at 492 nm was measured for 20 min to monitor L-DOPA formation. Ascorbic acid (1 mg/mL)served as a positive control, which was used for comparison. The inhibition ratio was calculated as follows:

where A was a mixture with tyrosinase without sample; B was a mixture without sample and tyrosinase; C was a mixture with sample and tyrosinase, and D was a mixture with a sample but without tyrosinase.

3.8.2.Inhibition of Collagenase Activity

Collagenase inhibition was determined by a previously described method [20]. Briefly, 50 mM tricine buffer (pH7.5)containing 10 mM calcium chloride and 400 mM sodium chloride was prepared. Then,50mLof a 1.0mM N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala solution and 0.2 mg/ml collagenase (from Clostridium histolyticum, Type IA, 0.5-50 FALGPA U/mg solid; Sigma-Aldrich, USA) were added in the presence and absence of samples. The reaction was stopped by adding citric acid(6%). The reaction mixture was separated by adding ethyl acetate. The absorbance of the supernatant was measured at 345 nm. Ascorbic acid (1 mg/mL) served as the positive control and was used for comparison. The percentage of inhibition was calculated as:

where A was a mixture with collagenase without sample; B was a mixture without sample and collagenase; C was a mixture with sample and collagenase, and D was a mixture with a sample but without collagenase.

3.8.3.Inhibition of Elastase Activity

Elastase inhibition was determined by a previously described method [20]. N-succinyl-Ala-Ala-Ala-p-nitroanilide (Suc-Ala-Ala-Ala-pNA) served as the substrate, and the release of p-nitroaniline was monitored for 20 min at 25°C.A portion(1 ug) of type IV porcine pancreatic elastase (PPE) was dissolved in 1 mL of 0.2 M Tris-HCl buffer(pH 8.0).The reaction mixture contained 0.2 M Tris-HCl buffer(pH8.0), 1 ppm PPE, 0.8 mM Suc-Ala-Ala-Ala-pNA, the sample, and the aforementioned substrate. Absorbance at 214 nm was measured. Ascorbic acid(1 mg/mL)served as the positive control used for comparison. The inhibition ratio was calculated as follows:

where A was a mixture with elastase and without sample; B was a mixture without sample and elastase; C was a mixture with sample and elastase, and D was a mixture with a sample but without elastase.

3.9.Statistical Analysis

Data are presented as the mean±standard deviation (SD). The significance of differences between groups was assessed using multiple comparisons and analysis of variance(ANOVA), followed by the Tukey honest significant difference (HSD) test. Differences with p values of less than 0.05 were considered statistically significant.

4. Conclusions

In this study, collagen hydrolysates, a functional food ingredient, were successfully produced through enzymatic hydrolysis, followed by ultrafiltration and purification. Various commercial proteases were tested for their potential usefulness in manufacturing proper collagen hydrolysates. CHS hydrolyzed by Alcalase was the most effectively hydrolyzed CHs, and ultrafiltration followed by purification was effective in generating active peptides with low molecular weights. Results showed that the CHs displayed excellent antioxidant and collagenase inhibition activities. Therefore, CHs obtained by this study may be used in food, cosmetics, or pharmaceutical industries as a natural additive, possessing anti-oxidative and anti-aging properties. Further studies involving an in vivo evaluation of the aging activities of active peptides in human skin may prove to be useful.

This article is extracted from Molecules 2019, 24, 1104; doi:10.3390/molecules24061104 www.mdpi.com/journal/molecules