Stem Cells For Regenerative Medicine And Anti-aging Part 1

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Abstract: To investigate methods for improving the processing of porcine waste, porcine skin was hydrolyzed using different commercially available proteases (Alcalase, Flavorzyme, Neutrase, Bromelain, Protamex, and Papain) under several optimal conditions. Following enzymatic hydrolysis, the collagen hydrolysates(CHs) were fractionated by molecular weight (3 kDa) via membrane ultrafiltration. The CHS were analyzed for physical properties (pH, protein recovery, free amino group content, molecular weight distribution, and amino composition) as well as for functional properties (antioxidant activities and anti-aging activities). Among the CHs, CHs hydrolyzed by Alcalase (CH-Alcalase) exhibited the highest degree of hydrolysis compared to other CHs. Both"CH-Alcalase" and"CH-Alcalase<3 kDa" fractions showed a considerably high antioxidant activity and collagenase inhibition activity. Therefore, resulting bioactive have the potential for development as antioxidants and anti-aging ingredients in the food, cosmetics, and pharmaceuticals, from animal by-products.

Keywords: hydrolysis; commercial proteases; collagen hydrolysate; antioxidant; anti-aging

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1. Introduction

With increasing life expectancy, skin anti-aging procedures are receiving much attention. Collagen, used in skincare products as a cosmeceutical ingredient for anti-aging, is widely used in the cosmetic market. Collagen is a high molecular weight, fibrous protein of animal origin, found abundantly in connective tissue, skin, tendons, cartilage, ligaments, teeth, nails, and hair of humans and animals. Livestock remains the main source of industrial collagen[1]. Annually, large quantities of livestock by-products are discarded worldwide, as waste, by the food and meat processing industries. However, these may be utilized as important protein resources for novel food materials or converted to value-added products via hydrolysis, which is widely applied to improve and upgrade the functional and nutritional properties of proteins [2,3]. Collagen is also increasingly being considered a source of bioactive peptides, which have shown promise as beneficial compounds for use in nutritional or pharmaceutical applications. cistanche benefits, Therefore, hydrolysis of collagen may play an important role in improving its bioavailability and suitability for use in various commercial processes.

Physiological activities of bioactive peptides, which usually contain 2-20 amino acid residues (<6000 Da), are based on the composition and sequence of their amino acids [2,4]. Due to their structural properties such as molecular weight, bioactive peptides may possess specific characteristics that affect numerous physiological processes related to antimicrobial, antioxidant, emulsifying, antihypertensive, antidiabetic, and immunomodulatory functions in organisms[1,5,6]. Generally, low molecular weight hydrolysates display lower viscosity, better dispersion, higher hydrophobicity, and smaller particle size [7]. cistanche cholesterol The degree of hydrolysis directly affects the molecular weight and amino acid composition of the hydrolysates [8]. Therefore, the hydrolysis process may be necessary for producing novel bioactive peptides.

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Protein hydrolysis, leading to the cleavage of peptide bonds, may be carried out via enzymatic or chemical processes [9]. Chemical processes, including alkaline or acid hydrolysis using solvent extractions, are harmful not only to the environment but also to humans who consume the resulting products [4,10]. Chemical processes, which are difficult to control, yield products containing modified amino acids [9]. In contrast, enzymatic hydrolysis may be performed under mild conditions, thus avoiding extreme environments that are required by chemical treatments. Moreover, the processes neither produce side reactions nor decrease the nutritional value of the protein source [8]. Several proteases, including Alcalase, pepsin, Protamex, trypsin, and Neutrase, are commonly used to hydrolyze proteins, resulting in various types of protein hydrolysates and peptides with a variety of bio-functional activities. One study reported producing collagen hydrolysates with antioxidant activity from porcine skin gelatin using enzymatic hydrolysis by pepsin and pancreatin [11]. cistanche deserticola side effects Salmon by-products were hydrolyzed to produce peptic hydrolysates using various proteases, where the final peptides showed excellent antioxidant and anti-inflammatory properties [12].

The objective of the research was:(a)to identify the most active collagen hydrolysates produced by enzymatic hydrolysis using different commercially available proteases (Alcalase, Flavorzyme, Neutrase, Protamex, Bromelain, and Papain);(b) to confirm the fractionation effect of collagen hydrolysates obtained from enzymatic hydrolysis, and (c) to determine theirs in vitro antioxidant and anti-aging activities.

2. Results and Discussion

2.1.Enzymatic Hydrolysis Effect on Collagen Hydrolysates

2.1.1.pH

The functional properties of proteins may be enhanced through hydrolysis by certain proteases. In this study, six different proteases (Alcalase, Flavorzyme, Neutrase, Bromelain, Protamex, and Papain) were used to hydrolyze collagen from porcine skin in order to obtain active collagen hydrolysates with low molecular weights. cistanche dosage reddit The change of pH in collagen hydrolysates (CH) is shown in Figure 1. The pH of the collagen suspension (5% porcine skin mixture)was initially6.39-6.55 (at 0h). CH-Alcalase and CH-Flavorzyme reached optimal pH (pH 8.0) at 12 h of incubation. CH- Protamex, CH-Bromelain, and CH-Papain reached optimal pH(pH7.0) at 6h of incubation, whereas CH-Flavorzyme continued to decrease from pH6.39 to pH5.69 for 24 h incubation(Figure 1). The pH of a protein hydrolysate is an important factor that regulates enzymatic hydrolysis reactions[13]. Therefore, the generation of collagen hydrolysates under conditions of differing pH may be due to pH-dependent changes in enzyme conformation, which may impact their bio-activation and physicochemical properties.

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

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of CHs treated with various proteases for 24 h are shown (Figure2). The peptide profile of the collagen suspension (at 0 h)showed a visible band in the low-to-high molecular weight range. Results indicated rapid hydrolysis in CH-Alcalase, CH-Neutrase, CH-Bronneline, and CH-Protamex, as there were no bands after 3 h incubation. The high molecular weight range bands of CH-Alcalase, CH-Neutrase, and CH-Protamex decreased in a time-dependent incubation manner. However, CH-Bromeline did not vary with incubation time because it has beer completely hydrolyzed after just 1 h incubation. Interestingly, the CHs completely disappeared when the sample reacted with Alcalase or Neutrase for 6h, indicating that collagen acted as a suitable substrate for some of the other enzymes tested. cistanche extract benefits, However, bands of CH-Flavorzyme and CH-Papain appeared in the low-to-high molecular weight range after 24 h. Proteins can be partly hydrolyzed into peptides or amino acids by enzymatic hydrolysis, depending on protease type, incubation time, and the protease-to-substrate ratio (protein amount)[14]. The enzymatic hydrolysis process is mainly determined by whether the protease type involved is an endopeptidase or an exopeptidase [15,16]. Since Alcalase, Neutrase, and Protamex are endopeptidases, their hydrolysis processes may be expected to break peptide bonds from non-terminal amino acids randomly, thereby facilitating further hydrolysis of proteins. On the other hand, Flavorzyme is both an endo- and exopeptidase, which breaks the non- or N-terminal of peptide chains. In this study, collagen protein contained binding sites, which were more susceptible to exopeptidase activity than to endopeptidase activity of Flavorzyme. Thus, due to exopeptidase activity, hydrolysis of collagen proteins by Flavorzyme involved the step-by-step breaking of peptide bonds from the N-terminal of amino acids, which decelerated protein hydrolysis.

2.1.3. Protein Recovery and Free Amino Group Content

Protein recovery of the CHs obtained via different commercial proteases is shown (Figure 3A). The control (collagen suspension) had a protein recovery content of 1.19 mg/mL. Protein recovery is a parameter of the efficiency of enzymatic hydrolysis. The low protein recovery content of CH is indicative of increased protein breakdown. Furthermore, protein recovery correlates well with protein solubility. Higher hydrolytic activity may be caused by an increase in exposed hydrophobic side chains, leading to increased hydrophobic interactions between proteins and/or peptides. These hydrophobic side chains may lead to decreased protein solubility, thus resulting in increased protein recovery of hydrolysates. In our study, protein recovery content was well-correlated with changes in the SDS-PAGE patterns. The lowest and highest protein recovery content levels were recorded for Alcalase and Flavorzyme hydrolysis, respectively. For hydrolysis efficiency related to incubation time, CH-Alcalase, CH-Bromelain, and CH-Protamex drastically decreased within one hour of incubation compared to the control. The decrease in protein recovery content resulting from protein hydrolysis showed an inverse relationship with the amino group content. Furthermore, most CHs, except for CH-Neutrase, exhibited low protein content between3 h and 12 h. After 24hincubation, their protein content increased again in CH-Alcalase, CH-Flavorzyme, CH-Neutrase, CH-Bromeline, CH-Protamex, and CH-Papain as follows:0.83 mg/mL,1.29 mg/mL, 0.75 mg/mL, 0.97 mg/mL, 0.77 mg/ml, and 1.11 mg/mL, respectively. Some reports indicate that the optimum hydrolysis time depends on several factors such as the species selected, the raw material, the enzyme used, the raw material/enzyme rate, the concentration of the enzyme, and the degree of hydrolysis pursued, etc. [16]. Some reports indicate that extended enzymatic hydrolysis(over 24 h) may lead to a reduction in protease activity; thus, enzymatic hydrolysis should be carried out within 24 h of enzymatic hydrolysis [17,18].

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The free amino group content of CH, which is an indicator of enzymatic hydrolysis efficiency, was estimated(Figure 3B). The expected outcome of protein breakdown is that protein may be hydrolyzed into shorter peptide products. The free amino group content of the control was initially 0.14 mg/mL. The free amino group content of all CHs dramatically increased with increasing incubation time. After 24 h incubation, the free amino group content by descending order was: CH-Alcalase (1.00 mg/mL)>CH-Protamex(0.70 mg/mL)> CH-Neutrase(0.68 mg/mL)>CH-Bromeline (0.67 mg/mL)>CH-Flavorzyme (0.53 mg/mL)>CH-Papain(0.40 mg/mL).Common differences in total protein or free amino group content of enzymatically hydrolyzed samples may be related to enzyme specificity. It is dependent on the properties of an enzyme during the enzymatic hydrolysis process [16,17]. For example, alkaline proteases (such as Alcalase)exhibit higher hydrolytic activities compared to acidic or neutral proteases.

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<3kDa was the lowest field (12.42%)observed. Therefore, although ultrafiltration is useful in separating CHs with 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 95mg/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. These amino acids have been reported to enhance antioxidant activity because of their increased solubilities in lipids or via free radical reactions [1,24].