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Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis
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Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis Monjurul Haqa,b , Truc Cong Hoa , Raju Ahmeda , Adane Tilahun Getachewa , Yeon-Jin Choa , Jin-Seok Parka , Byung-Soo Chuna,* a b
Department of Food Science and Technology, Pukyong National University, 45 Yongso-ro, Nam-Gu, Busan 48-513, Republic of Korea Department of Fisheries and Marine Bioscience, Jashore University of Science and Technology, Jashore 7408, Bangladesh
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 July 2019 Received in revised form 1 September 2019 Accepted 15 September 2019 Available online xxx
Bacterial collagenolytic protease-extracted Bigeye tuna skin collagen was subjected to catalysts-assisted subcritical water hydrolysis. The degree of hydrolysis was the highest for hydrolysates obtained using sodium bicarbonate catalysts. The color and pH values of the hydrolysates varied depending of the catalysts used. Four different assays of antioxidant activities varied with respect to the various collagen hydrolysates with hydrolysates obtained using sodium bicarbonate catalysts showing the highest activity. Ten bacterial species were studied to evaluate antibacterial activity among which nine showed positive results. The average molecular size of the peptides in the obtained collagen hydrolysates varied between 300 and 425 Da. The total amount of structural and total amino acids varied between 116.63 and 49.51, and 196.86 and 265.68 mg/100 g hydrolysate, respectively. The collagen hydrolysates obtained showed enormous potential to be used in the food and pharmaceutical industries. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Biofunctional Bacterial collagenolytic protease-extracted collagen Catalyst-assisted Subcritical water hydrolysis
Introduction Collagen is the most abundant structural biopolymer protein comprising approximately of around one-third of the total protein in vertebrates and playing major structural roles in fibrous tissues (ligaments, tendons, and skin) and bones of animals [1]. It is also found in the corneas, gut, blood vessels, intervertebral discs, and the dentin of teeth. Triple helices of amino acids twist together to form the collagen structure. The rigidness of different collagens, such as those in the bone, tendon, or cartilage, depends upon the degree of mineralization. In the past decade, numerous studies have concentrated on the field of collagen-based biomaterials and its applications, such as tissue engineering applications, bone regeneration scaffolds, and crosslinking methods, etc. At present, collagen is widely used in research and medical applications [2]. Its commercial production is derived from bovine and porcine sources, but there are some issues in using this, including the transmission of some diseases along with cultural and religious restrictions [3]. Therefore, collagen originating from fish or other aquatic species is being
* Corresponding author. E-mail address: [email protected] (B.-S. Chun).
considered as popular ingredients in the food and pharmaceutical industries. Extracellular proteins including collagen, are known to contain biofunctional peptides that can be released under certain conditions and can show divergent biological activities from the parent proteins [1]. Many researchers have reported on biofunctional activities of collagen peptides such as antioxidant [4], antiobesity [5], antihypertensive [6,7], and antibacterial [1] activities. Activation of these bonded peptides requires physical alterations of the molecule which can be performed by proteolytic processing [8,9]. Enzymatic hydrolysis is widely used to produce functional peptides from collagen [7]; however, subcritical water hydrolysis of collagen, a cheap and easy-to-adopt technology, is considered advantageous. Subcritical water hydrolysis is a clean and fast biomass reduction process that can be applied as a suitable alternative to acidic, basic, and enzymatic hydrolysis because of its faster reaction period, lower residue production, less corrosion, lower degradation product generation, nontoxic solvent use, and so on [10]. Moreover, subcritical water can be used as a tunable reactant as well as a solvent in the conversion of macromolecules, without the need for acid or alkali, thereby avoiding environmental hazards [11]. Acid/alkaline hydrolysis needs extreme reaction conditions and, avoiding uncontrolled and undesired reaction requires purification steps to separate hazardous chemicals causing environmental pollution from the
https://doi.org/10.1016/j.jiec.2019.09.023 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Haq, et al., Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.09.023
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final products. Moreover, enzyme-catalyzed hydrolysis has a longer reaction time to complete the reaction cycle [12]. Subcritical water hydrolysis combines high temperature and pressure, converting collagen macromolecules into smaller molecules of peptides and amino acids [13]. The peptides are converted into free amino acids and subsequently converted to organic acids under higher treatment temperature and longer hydrolysis time [14]. The suitable temperature and time for subcritical water hydrolysis depend on the substrate source, for example, protein from animal sources need an extended reaction time or higher temperature than that from vegetable sources [15]. In addition, some researchers have reported on the incorporation of catalysts to increase biofunctional materials in the subcritical water hydrolysis process [12,16–19]. Subcritical water hydrolysis of bacterial collagenase extracted fish skin collagen converts collagen macromolecules into bioactive peptides/amino acids, which can ideally be used as bioactive ingredients in food, pharmaceutical, and cosmetic industries as these active compounds delivered to the muscle faster than original collagen. Enhancement of the biofunctional activity of the protein substrate because of subcritical water hydrolysis is proven by many previous researchers using various protein sources such as by-products of animals [18,20], by-products of fish [21,22], plant materials [23] and even pure protein, such as hemoglobin, beta-casein [14,24] and bovine serum albumin. In a previous study, collagen from tuna fish skin was extracted using collagenase isolated from collagenase-producing bacteria [1] to avoid collagen extraction by using porcine-originated pepsin. In another study, pepsin-extracted collagen was subjected to subcritical water hydrolysis, the reaction parameters were optimized, and positive biofunctional activities of collagen peptides were found [25]. Thus far, there is no report on the subcritical water hydrolysis of bacterial collagenase-extracted fish collagen
and its biofunctional activity. In addition, collagen peptide-rich hydrolysates were obtained by catalyst-assisted subcritical water hydrolysis at previously optimized conditions [25] using bacterial collagenase-extracted tuna skin collagen. Therefore, the aims of this study were to hydrolyze bacterial collagenase extracted tuna skin collagen enzyme using catalyst-assisted subcritical water and to evaluate the physiochemical and biofunctional properties of the hydrolysates. Materials and methods Chemicals and reagents Acetic acid and iron (III) chloride 6-hydrate (FeCl36H2O) were purchased from Merck (Darmstadt, Hessen, Germany). DPPH, ABTS, trolox, gallic acid, and Folin–Ciocalteu reagent were purchased from Sigma-Aldrich Co. (St. Louis, MI, USA). Only analytical/HPLC grade reagents and solvents were used in this study. Sample collection, preparation, and collagen extraction The collection of Bigeye tuna skin and extraction of collagen using bacterial collagenase is described in a previous study [1]. Catalyst-assisted subcritical water hydrolysis Different catalysts such as sodium chloride, acetic acid, and sodium bicarbonate were prepared at concentration of 0.6 M, and distilled water was used as a control for comparing the effects of the catalyst solutions in the hydrolysis process. A batch-mode Hastelloy reactor facilitated by a temperature regulator (Fig. 1) was used. The solid-to-liquid ratio (W/V) was maintained at 1:200, and
Fig. 1. Schematic diagram of the laboratory-scale subcritical water apparatus.
Please cite this article in press as: M. Haq, et al., Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.09.023
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150 mL of liquid was loaded in the 200 mL size reactor. After closing the reactor tightly, it was heated using an electric heating system at a temperature of 250 C. The pressure inside the reactor was controlled at 50 bar using N2 gas, and the temperature and pressure were continuously monitored through a temperature and pressure gauge, respectively. The sample was stirred at 150 rpm using a four-blade impeller. It took 45 min to reach the desired reactor temperature and the reaction period was maintained at 3 min. The reactor was cooled immediately by circulating refrigerated water to avoid further degradation. Then, the hydrolysate sample was collected from the reactor, filtered using a filter paper, and stored at 4 C. Determination of the degree of hydrolysis The degree of hydrolysis of the hydrolysates was measured according to the method described by Thiansilakul et al. [26] with some modifications. In brief, 125 mL of the hydrolysate sample was added to the reaction reagent. The reaction reagent was prepared by mixing 2.0 mL of sodium phosphate (0.2125 M, pH 8.2) with 1 mL 2,4,6-trinitrobenzene sulfonic acid (0.01%, pH 8.2). The mixture sample was incubated at 50 C for 30 min in dark. Then, 2.0 mL of 0.1 M sodium sulfite was added to the sample to stop the reaction. Later, the sample solution was cooled at room temperature for 15 min, and the absorbance was measured at 420 nm. A calibration curve was measured using glycine. The degree of hydrolysis was measured using the following equation: Degree of hydrolysis ¼
Lt L0 100 Lmax L0
where Lt is the α-amino acid in the hydrolysate, L0 is the α-amino acid in the original sample homogenate, and Lmax is the α-amino acid in the original sample obtained using acid hydrolysis (6 N HCl,100 C for 24 h).
Chelating activity ð%Þ ¼
3
DPPH scavenging activity The DPPH radical scavenging capacity was measured following the method of Asha et al. [28] with some modifications. In total, 50 mL of the hydrolysate sample (5 mg/mL) was added to 950 mL of the DPPH solution (0.1 mM) and incubated in dark at room temperature for 30 min. Then, the absorbance was measured at 517 nm. The DPPH scavenging activity was expressed as mg VCEAC/g of collagen hydrolysate. A standard curve was prepared using vitamin C. Ferric reducing antioxidant power assay The ferric reducing antioxidant power (FRAP) aasay was conducted according to the method of Asha et al. [28] after some alterations. Fifty microliter of the hydrolysate sample (5 mg/mL) was mixed with 0.95 mL of the FRAP reagent and incubated at 37 C for 30 min in dark. The FRAP solution without the sample was considered as the blank, and the absorbance was measured at 595 nm. FRAP activity was expressed as mg VCEAC/g of collagen hydrolysate. Metal chelating activity The metal chelating activity of the collagen hydrolysates was measured following the procedure described by Klompong et al. [29], with some modifications. The sample (100 mL at 5 mg/mL) was mixed with 870 mL of distilled water. Then, 10 mL of 2 mM FeCl2 and 20 mL of 5 mM ferrozine were added and incubated at room temperature for 10 min. Subsequently, the absorbance was recorded at 562 nm. Distilled water instead of the sample was used as a control. The metal chelating activity was measured using the following equation:
Absorbacne of control at 562 nm Absorbacne of sample at 562 nm 100 Absorbacne of control at 562 nm
Color and pH measurement
Determination of antimicrobial activity of the collagen hydrolysates
The color of the hydrolysates was measured using of a portable reflectance spectrophotometer (Lovibond RT Series, Amesbury, UK). The instrument was standardized with white and black references before each measurement and color parameters L* (lightness/ brightness), a* (redness/greenness), and b*(yellowness/blueness) were recorded. The pH of the hydrolysates was determined using a Mettler Toledo pH meter (EasyPlus, Switzerland) at 25 C. Technical buffer solutions were used to calibrate the pH meter prior to the measurements.
The antimicrobial activity of the collagen hydrolysates for 10 bacterial strains was determined according to the method of Mosquera et al. [30] with some modifications. The tested strains and procedures are described in detail in a previous study [1].
Radical scavenging activity measurement ABTS scavenging activity The ABTS radical scavenging activity of the collagen hydrolysates was determined following the methods of Aleman et al. [27]. The diluted hydrolysate sample (5 mg/mL; 20 mL) was added to 980 mL of the ABTS solution (OD value, 0.70 0.02 at 734 nm), kept in dark for 16 h at room temperature, and the absorbance was measured at 734 nm. A standard curve of vitamin C was prepared (50–500 ppm). The result was expressed as mg vitamin C equivalent antioxidant capacity (VCEAC)/g of hydrolysate.
Analysis of amino acid composition The collagen hydrolysates were analyzed for both structural and free amino acid contents using an amino acid auto analyzer (Models: S433-H and S430-H, SYKAM, Germany) following the methods described in detail in the study by Asaduzzaman and Chun [21]. First, the samples were filtered and injected onto the analyzer facilitated by the cation separation column LCA K06/Na (4.6 150 mm) and LCA K07/Li (4.6 150 mm), with column temperatures of 57–74 C and 37–74 C and buffer pH ranges of 3.45–10.85 and 2.90–7.95 for structural and free amino acid analyses, respectively. As a mobile phase, 5 mM p-toluenesulfonic acid solution was used (0.45 mL/min) for both. As a post-column reagent, a mixture of 5 mM p-toluenesulfonic acid, 100 mM EDTA, and 20 mM bis–tris was used (0.25 mL/min). Excitation and emission wavelengths were maintained at 440 and 570 nm, respectively, for both operating conditions. The values were presented as mg/100 g of liquid collagen hydrolysate.
Please cite this article in press as: M. Haq, et al., Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.09.023
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MALDI-TOF spectroscopy analysis The molecular profile of the obtained hydrolysates was analyzed using the MALDI-TOF Bruker Autoflex III following the method of Meillisa et al. [31]. A small beam laser was facilitated by using an instrument, and the spectrum was obtained with a voltage of 20 kV. Water/acetonitrile (25:75) was used as a solvent, and 2,5-dihydroxybenzoic acid as a matrix. A hydrolysate sample of 0.5 mL was mixed with 0.5 mL of the matrix solution. The matrix solution was prepared from norharmane-acetonitrile and trifluoroacetic acid at a rate of 7:3, where the norharmaneacetonitrile solution was prepared by dissolving 10 mg norharmane in 1 mL acetonitrile. Statistical analysis Values are presented as a mean standard deviation of triple determinations. One-way analysis of variance was conducted using SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). Duncan’s multiple range test was performed to determine significant differences among the means, and p 0.05 was regarded to be significant (Fig. 2). Results and discussion Degree of hydrolysis, color, and pH values The degree of hydrolysis, color, and pH values of the bacterial collagenolytic protease-extracted collagen hydrolysates obtained using subcritical water hydrolysis using different catalytic solutions are shown in Table 1. The degree of hydrolysis varied depending on the type of catalyst used in this study. The highest value of degree of hydrolysis was found in hydrolysates obtained using sodium bicarbonate catalysts (21.81 0.48%), whereas the lowest value was found in those obtained using distilled water (15.28 0.36%). The same degree of hydrolysis was obtained when pepsin-extracted tuna skin collagen was hydrolyzed by the subcritical water hydrolysis process using only water as the solvent [25]. Asaduzzaman et al. [12] hydrolyzed mackerel fish
muscles and reported that the addition of acidic and alkaline catalysts influenced the hydrolysis yield. These catalysts are corrosive on the substrate, and in addition, the polarity of the solvent and dielectric constant are increased, which enhanced depolymerization of the substrate. Fish collagen has very poor solubility in water at ambient temperature because of aggregation by hydrophobic interactions, which can be altered by different catalyst-assisted subcritical water hydrolysis. The degree of whey protein hydrolysate was increased fourfold with sodium bicarbonate catalysts compared with that with only water, and the molecular weight of the peptides was reduced because of the application of sodium bicarbonate catalysts [32]. The degree of hydrolysis is considered significant for determining the molecular weight of peptides and the exposure of the terminal amino groups of the resulting product, thereby influencing different biopotential activities of the hydrolysate [1]. The color values of the collagen hydrolysates varied depending on the catalysts used for hydrolysis (Table 1). The highest L value (lightness) was obtained with the collagen hydrolysate in acetic acid catalysts, whereas the lowest L value was obtained with that using the sodium bicarbonate catalysts. The color of the collagen hydrolysates plays an important role in food ingredients [33]. The color differences in the collagen hydrolysates may be due to the presence of Maillard reaction products [34]. The variation in the pH values of the collagen hydrolysates might be due to the conversion of collagen to acidic compounds. Antioxidant activity ABTS radical scavenging activity ABTS is a popular method determining both hydrophilic as well as lipophilic antioxidants [12]. The highest ABTS radical scavenging activity value of 54.17 mg VCEAC/g of hydrolysate was found in the collagen hydrolysate obtained using sodium bicarbonate catalysts (Fig. 3). The lowest ABTS radical scavenging activity of 35.70 mg VCEAC/g of hydrolysate was found in the collagen hydrolysate obtained using only water. The free radical scavenging activity of the collagen hydrolysate depends on their amino acid composition as well as the sequences of their peptides [35]. Similar ABTS radical
Fig. 2. Summarized scheme of all the steps of the work.
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Table 1 Degree of hydrolysis, color and pH values of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Parameters Degree of hydrolysis (%) Color
Water only d
L a* b*
pH
15.28 0.36 52.90c 0.75 0.48b 0.05 15.61b 0.39 8.25c 0.09
Sodium chloride b
19.64 0.10 54.68b 0.95 0.57c 0.03 6.09c 0.24 9.62b 0.28
Acetic acid c
17.61 0.41 68.37a 1.22 1.62d 0.06 4.61d 0.55 2.37d 0.02
Sodium bicarbonate 21.81a 0.48 35.61d 0.96 4.11a 0.29 23.34a 0.96 12.70a 0.32
Values are presented as means standard deviations of triplicates. Different small letters in the same row of the table indicate significant differences (P < 0.05).
Fig. 3. Antioxidant activity of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions.
scavenging activity was reported by Ahmed et al. [1] when they conducted a study on the hydrolysis of pepsin-extracted collagen using distilled water. The collagen molecules were subjected to degradation based on the different types of catalysts and produced various types/sizes of peptides and amino acids that can have an effect on the ABTS radical scavenging activity. Guillén et al. [36] hydrolyzed tuna gelatin using alcalase enzyme and reported a ABTS radical scavenging activity value of 17.57 1.62 VCEAC/g of protein. The variations in radical scavenging activity of different hydrolysates are due to the substrate nature, solid–liquid ratio, and peptides/amino acid formation extent. Small–size peptides/amino acids in collagen hydrolysates have proton/electron donating and chain breaking ability; thus, they react with free radicals and stabilize them, terminating the free radical chain reaction. Hydrophobic amino acids such as tyrosine, histidine, methionine, tryptophan and lysine are supposed to have radical scavenging ability [37]. The catalyst-assisted collagen hydrolysis in this study showed higher ABTS radical scavenging activity than that reported in previous studies. DPPH radical scavenging activity The DPPH radical scavenging activity of different hydrolysates varied between 17.47 1.53 and 38.81 2.06 mg VCEAC/g hydrolysate. Maximum DPPH radical scavenging activity was found in the collagen hydrolysate obtained using sodium bicarbonate followed by that of the collagen hydrolysate obtained using acetic acid, sodium chloride, and water as solvent (Fig. 3). In the presence of antioxidant compounds, the DPPH radicals are scavenged by the proton-donating antioxidant. In presence of different catalysts during hydrolysis of collagen molecules, various peptides and amino acids were formed, which acted as a proton donor and stabilized the DPPH free radicals. Some antioxidant compounds
display ABTS radical scavenging activity but may not display DPPH radical scavenging activity. This is because ABTS radical scavenging activity is applicable to hydrophilic and hydrophobic antioxidant systems, whereas DPPH scavenging activity is only applicable to hydrophobic systems [38]. The collagen hydrolysates obtained in this study showed both ABTS and DPPH free radical scavenging activities containing both hydrophobic and hydrophilic amino acids that contribute to the antioxidant properties. FRAP assay The collagen hydrolysates showed FRAP activity, and the values varied depending on the catalyst solution used for hydrolysis. Higher FRAP activity of 24.41 1.18 mg VCEAC/g of hydrolysate was found in hydrolysates obtained using sodium bicarbonate solutions followed by 21.10 0.51, 19.70 0.27, and 17.94 0.67 mg VCEAC/g of hydrolysate in hydrolysates obtained using sodium chloride, acetic acid, and only water (Fig. 3). The different peptides obtained from the in different hydrolysates have the ability to provide electrons to the radicals. The principle of the FRAP assay is based on electron transfer instead of hydrogen ion transfer. It determines the ability of peptides and amino acids to reduce Fe3+ to Fe2+. The FRAP reaction is conducted at an acidic pH to maintain iron solubility, which decreases the ionization potential and enhances redox potential. Because of the transformation of Fe3+ to Fe2+, there is a formation of a colored compound (595 nm) in the presence of the FRAP reagent. Ahmed and Chun [25] hydrolyzed pepsin-extracted collagen using water and reported the FRAP activity to be 17.17 mg VCEAC/g of hydrolysate at 250 C, which is similar to that observed in present study, but the FRAP activity was found to be higher when different acidic and alkaline catalysts were used. This might be due to the generation of various bioactive peptides by the various catalytic solutions used
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during the hydrolysis process. The FRAP activity was found to be 11.55 VCEAC/g of protein in enzyme-hydrolyzed tuna gelatin [36]. Metal chelating activity The highest metal chelating activity was found to be 60.76 4.72% and the lowest activity was found to be 48.61 0.45 in the collagen hydrolysates obtained using sodium bicarbonate and only water, respectively (Fig. 3). Peptides and amino acids in the collagen hydrolysated were chelated with Fe2+ and showed metal chelation activity. The mechanisms underlying the process is as follows: ferrozine forms a complex with Fe2+, a colorful solution but metal chelating antioxidant compounds in the collagen hydrolysate inhibit the colorful complex formation resulting in decrease in the color [38]. Ahmed et al. [1] found the metal chelating activity of pepsin-extracted collagen hydrolysate to be 50.54% in hydrolysates obtained at 250 C with only water as a solvent. The use of different catalysts during the hydrolysis process increased the metal chelating activity in the present study. Asaduzzaman and Chun [39] found a maximum metal chelating activity of 54% in mackerel muscle hydrolysate. The collagen hydrolysates showing metal chelating activity can be used as functional food ingredients to improve the bioavailability of
minerals. Nakchum and Kim [40] studied the ferrous chelating ability of squid skin-derived collagen and found positive results. Guo et al. [41] also reported that Alaska Pollock skin-derived collagen peptides showed iron and copper chelating activity and fish collagen hydrolysates showed calcium chelating activity [41,42]. Antimicrobial activity The antimicrobial activity of the collagen hydrolysate is shown in Table 2. Except for Enterococcus faecium, all bacteria studied were found to be sensitive to the collagen hydrolysates. The collagen hydrolysates obtained using catalysts revealed higher activity than those obtained using only water. Antimicrobial activity is closely associated with hydrophobic amino acids in hydrolysates [43]. This hydrophobic nature of the peptides facilitates entrance into the bacterial cell membrane, and the positive charge initiates the peptide interaction with the negatively charged bacterial membrane [44]. The mechanisms of microbe and peptide interactions include formation of disconnected channels in lipid bilayers, resulting in the discretion of lipid bilayers. In addition, specific lipid–peptide interaction results in detergent-like solubilization of the membrane and even the
Table 2 Antimicrobial activity of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Bacterial strains
Water only
Sodium chloride
Acetic acid
Sodium bicarbonate
Bacillus cereus Bacillus subtilis Enterococcus faecium Escherichia coli Klebsiella pneumonia Listeria monocytogenes Pseudomonas putida Pseudomonas aeruginosa Staphylococcus aureus Salmonella typhimurium
+
++ + + + +
++ ++ + +++ ++ +
++ + ++ + ++ +
N.B.: inhibition zones: +++: >1 cm; ++: 0.5 cm, +: 0.25–0.5 cm; : <0.25 cm and : 0.1 cm.
Fig. 4. MALDI-TOF mass spectra chromatogram of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions.
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Table 3 Structural amino acids content (mg/100 g) in bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Amino acids
Water only
Sodium chloride
Acetic acid
Sodium bicarbonate
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Ammonia Histidine Arginine Proline Total
1.28 0.12 0.80 0.04 0.73 0.10 22.08 2.51 29.61 2.15 19.64 2.45 1.54 0.23 4.67 0.54 0.83 0.10 1.80 0.15 5.94 0.34 1.75 0.11 4.23 0.41 6.15 0.23 5.97 0.71 1.21 0.43 2.90 0.25 15.03 1.65 126.16
0.12 0.06 0.12 0.07 0.07 0.02 21.20 2.65 30.81 2.91 20.55 2.95 1.83 0.16 4.27 0.34 1.59 0.07 0.99 0.12 4.79 0.21 0.85 0.15 3.81 0.36 4.81 0.26 4.61 0.32 0.92 0.04 0.89 0.16 14.39 1.57 116.63
0.15 0.02 0.12 0.09 0.15 0.07 20.75 2.48 30.22 3.16 20.96 2.41 2.91 0.21 4.10 0.21 0.99 0.10 1.07 0.10 4.43 0.31 0.80 0.09 3.56 0.12 5.40 0.41 8.68 0.27 0.86 0.12 3.25 0.11 14.11 1.37 122.51
0.10 0.09 0.20 0.06 0.02 0.01 23.28 2.76 41.77 3.76 28.35 3.12 2.98 0.62 6.59 0.32 0.21 0.05 1.64 0.31 7.47 0.12 1.32 0.06 5.15 0.40 10.54 0.63 3.17 0.51 0.53 0.13 0.19 0.04 15.86 0.34 149.51
Values are presented as means standard deviations of triplicates.
peptides interact with the membranes [45,46]. Antimicrobial peptides can destroy the targeted harmful microbes and demonstrate a wide scale of activities, including antimicrobial activity against some antibiotic-resistant microorganisms. Antimicrobial peptides from natural sources can kill microorganisms and modulate inflammatory responses [47]. Therefore, it is feasible to use them widely in food preservation and offer extra health aids.
size of the peptides in a hydrolysate is important for determining its functional activity [49]. Molecular analysis of the hydrolysates by MALDI-ToF spectroscopy revealed, it is seen that many lower molecular weight compounds were obtained because of the hydrolysis treatment.
Molecular weight analysis
The structural and free amino acid contents of the different collagen hydrolysates obtained using different catalysts are shown in Tables 3 and 4, respectively. The total amount of structural amino acids varied between 116.63 and 49.51 mg/100 g hydrolysates, where the highest content was found in hydrolysates obtained using sodium bicarbonate catalysts and the lowest amount was found in those obtained using only water. Among the structural amino acids found in different collagen hydrolysates, glycine, alanine, and proline were the most abundant. Usually, these amino acids are also dominant in raw collagens [50] and
The molecular weight of the collagen hydrolysates is shown in Fig. 4. The molecular profile of the peptides in all the hydrolysates showed that the molecular size of the peptides was <425 Da. The application of subcritical water hydrolysis depolymerized the collagen molecules in the presence of different catalysts and water as well. The average molecular size of the peptides in the obtained hydrolysates was 300–425 Da. Most of the peptides showing biofunctional activity were <1 kDa [36,48]. The average molecular
Analysis of amino acids in the collagen hydrolysates
Table 4 Free amino acids content (mg/100 g) in bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Free amino acids
Water only
Sodium chloride
Acetic acid
Sodium bicarbonate
Phosphoserine Taurine Aspartic acid Threonine Serine Glutamic acid α-Amino adipic acid Glycine Alanine A-amino-n-butyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine β-alanine β-amino isobutyric acid Ethanol amine Lysine Histidine Hydroxy proline Proline Total
8.83 0.43 1.84 0.14 1.14 0.12 1.20 0.12 0.49 0.14 3.46 0.21 ND 90.70 4.56 19.46 2.43 3.62 0.65 19.86 1.56 8.53 0.45 ND 6.73 0.46 12.65 0.29 10.51 0.58 11.15 0.48 4.10 0.65 7.13 0.62 8.58 0.53 2.17 0.56 2.75 0.37 22.56 2.56 247.55
6.99 0.54 ND 0.94 0.07 1.63 0.21 ND 3.37 0.31 ND 91.57 5.34 16.40 1.45 4.46 0.34 24.84 2.27 11.63 0.62 18.50 1.95 5.55 0.41 12.47 0.87 15.22 0.45 ND 3.54 0.37 5.28 0.73 11.62 1.05 2.77 0.38 11.93 0.98 16.91 1.73 265.68
7.40 0.82 ND 0.62 0.14 ND ND 4.06 0.18 ND 62.28 3.65 13.73 1.34 7.05 0.32 16.01 1.93 6.89 0.49 17.81 1.56 6.95 0.73 ND 3.42 0.71 13.76 0.28 9.72 0.62 3.33 0.25 7.29 0.82 2.02 0.27 ND 14.44 1.93 196.86
7.62 0.39 ND 0.70 0.13 1.34 0.13 ND 6.04 0.22 0.94 0.54 55.15 5.34 12.97 1.34 11.24 1.04 23.60 1.36 6.15 0.34 21.58 1.45 6.82 0.24 12.29 0.67 4.83 0.68 8.04 0.91 ND ND 8.57 0.38 2.09 0.36 1.71 0.27 13.47 1.50 205.23
ND: Not detected. Values are presented as means standard deviations of triplicates.
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gelatin hydrolysates [36]. There were differences among the structural amino acid contents of the collagen hydrolysates because of the different catalyzing effects of the catalysts used. Asaduzzaman et al. [12] also reported that the amino acid content of mackerel muscle hydrolysate obtained using sodium bicarbonate was higher that obtained to using only water. Among the different solvents used for hydrolysate preparation, there might be a higher conversion of protein to amino acids in hydrolysates obtained using sodium bicarbonate solution. Sodium bicarbonate and acetic acid solutions showed higher contents of amino acid production than only water, whereas sodium chloride showed lower amino acid production. Amino acid production was found to be significantly higher in whey protein hydrolysates obtained using subcritical water hydrolysis [17]. The free amino acid contents in the collagen hydrolysates varied between 196.86 and 265.68 mg/100 g; the maximum content was found in hydrolysates obtained using sodium chloride catalysts and the minimum content was found in those obtained using acetic acid catalysts. Among the free amino acids found in different collagen hydrolysates, glycine, glutamic acid, alanine, and proline have the highest content. A balanced diet containing essential amino acid is a prerequisite for proper protein synthesis. The collagen hydrolysates obtained using different catalysts contained balanced and important essential amino acids (Tables 3 and 4) suitable for use as a good nutritional supplement. Moreover, differences in the antioxidant and antioxidant activities of the collagen hydrolysates might be due to the composition and sequence of amino acids. Conclusion Bacterial collagenolytic protease-extracted collagen hydrolysates obtained by subcritical water hydrolysis showed significant antioxidant and antimicrobial potential. The activities were higher in the hydrolysates obtained using different catalyst-assisted subcritical water hydrolysis than in those obtained using only water. The functional activities varied depending on the hydrolysates observed using different catalysts, whereas the maximum activities were obtained in hydrolysates obtained using sodium bicarbonate. The molecular profile of peptides showed that the molecular weight was reduced because of catalyst-assisted subcritical water hydrolysis, and the molecular weight of peptides in all the hydrolysates was <425 Da. The collagen hydrolysates obtained by environmentally subcritical water hydrolysis, a very fast and green technique, could be applied as a potential ingredient in functional food, pharmaceutical, and cosmetics industries for human health benefits. Acknowledgments The research was funded by Basic Science Research Program, Nation Research Foundation in Korea (NRF), Ministry of Education (2016R1D1A1B03936128).
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
[37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50]
R. Ahmed, A.T. Getachew, Y.J. Cho, B.S. Chun, LWT Food Sci. Technol. 89 (2018) 44. R. Parenteau-Bareil, R. Gauvin, F. Berthod, Materials 3 (2010) 1863. M. Ahmad, S. Benjakul, Food Chem. 120 (2010) 817. S. Ketnawa, O. Martínez-Alvarez, S. Benjakul, S. Rawdkuen, Food Chem. 192 (2016) 34. M. Saito, C. Kiyose, T. Higuchi, N. Uchida, H. Suzuki, J. Agric. Food Chem. 57 (2009) 10477. Y. Zhuang, L. Sun, B. Li, Food Bioprocess Tech. 5 (2012) 1622. P. Banerjee, C. Shanthi, Process Biochem. 47 (2012) 2335. F.X. Maquart, S. Pasco, L. Ramont, W. Hornebeck, J.C. Monboisse, Cr. Rev. OncolHeem. 49 (2004) 199. S. Schenk, V. Quaranta, Trends Cell Biol. 13 (2003) 366. Y. Zhao, H.T. Wang, W.J. Lu, H. Wan, Chem. Eng. J. 166 (2011) 868. B.M. Pedras, M. Nascimento, I. Sá-Nogueira, P. Simões, A. Paiva, S. Barreiros, J. Ind. Eng. Chem. 72 (2019) 453. A.K.M. Asaduzzaman, M. Haq, B.S. Chun, J. Ind. Eng. Chem. 68 (2018) 301. I. Marcet, C. Álvarez, B. Paredes, M. Díaz, Waste Manage. 49 (2016) 364. T. Rogalinski, S. Herrmann, G. Brunner, J. Supercrit. Fluid. 36 (2005) 49. J.Y. Je, Z.J. Qian, S.H. Lee, H.G. Byun, S.K. Kim, J. Med. Food 11 (2008) 629. K.Y. Kang, B.S. Chun, Korean J. Chem. Eng. 21 (2004) 654. A.D. Espinoza, R.O. Morawicki, J. Agric. Food Chem. 60 (2012) 5250. H. Cheng, X. Zhu, C. Zhu, J. Qian, N. Zhu, L. Zhao, J. Chen, Bioresour. Technol. 99 (2008) 3337. Z.H.U. Xian, Z.H.A.O. Chao, H. Liang, Cheng, Chin. J. Chem. Eng. 16 (2008) 456. M. Esteban, A. García, P. Ramos, M. Márquez, Bioresour. Technol. 101 (2010) 2472. A.K.M. Asaduzzaman, B.-S. Chun, J. Food Sci. Technol. 52 (2015) 793. D.O. Kim, K.W. Lee, H.J. Lee, C.Y. Lee, J. Agric. Food Chem. 50 (2002) 3713. A.D. Espinoza, R.O. Morawicki, T. Hager, J. Food Sci. 77 (2012). T. Powell, S. Bowra, H.J. Cooper, Anal. Chem. 88 (2016) 6425. R. Ahmed, B.S. Chun, J. Supercrit. Fluid. 141 (2018) 88. Y. Thiansilakul, S. Benjakul, F. Shahidi, J. Food Biochem. 31 (2007) 266. A. Alemán, B. Giménez, P. Montero, M.C. Gómez-Guillén, LWT Food Sci. Technol 44 (2011) 407. K.K. Asha, K.R. Kumari, K.A. Kumar, N.S. Chatterjee, R. Anandan, S. Mathew, Int. J. Pept. Res. Ther. 22 (2016) 421. V. Klompong, S. Benjakul, D. Kantachote, F. Shahidi, Food Chem. 102 (2007) 1317. M. Mosquera, B. Giménez, S. Ramos, M.E. López-Caballero, M.D.C. GómezGuillén, P. Montero, J. Aquat. Food Prod. Technol. 25 (2016) 444. A. Meillisa, H.C. Woo, B.S. Chun, Food Chem. 171 (2015) 70. Y. Zhuang, L. Sun, B. Li, Food Bioprocess Tech. 5 (2012) 1622. A.D. Espinoza, R.O. Morawicki, T. Hager, J. Food Sci. 77 (2012) 20. K. Watchararuji, M. Goto, M. Sasaki, A. Shotipruk, Bioresour Technol. 99 (2008) 6207. H.C. Wu, H.M. Chen, C.Y. Shiau, Food Res. Int. 36 (2003) 949. G. Guillén, G.M.E. López Caballero, A. Alemán, A. López de Lacey, B. Giménez, P. Montero García, Antioxidant and antimicrobial peptide fractions from squid and tuna skin gelatin. Sea by-products as a real material: new ways of application, (2010) p. 89. S.Y. Dong, M.Y. Zeng, D.F. Wang, Z.Y. Liu, Y.H. Zhao, H.C. Yang, Food Chem. 107 (2008) 1485. P. Kittiphattanabawon, S. Benjakul, W. Visessanguan, H. Kishimura, F. Shahidi, Food Chem. 119 (2010) 1519. A.K.M. Asaduzzaman, B.S. Chun, Fish. Sci. 80 (2014) 369. L. Nakchum, S.M. Kim, Prep. Biochem. Biotechnol. 46 (2016) 123. L. Guo, P.A. Harnedy, M.B. O’Keeffe, L. Zhang, B. Li, H. Hou, R.J. FitzGerald, Food Chem. 173 (2015) 536. L. Guo, P.A. Harnedy, L. Zhang, B. Li, Z. Zhang, H. Hou, X. Zhao, R.J. FitzGerald, J. Agric. Food Chem. 95 (2015) 1514. M. Mosquera, B. Giménez, S. Ramos, M.E. López-Caballero, M.D.C. GómezGuillén, P. Montero, J. Aquat. Food Prod. Technol. 25 (2016) 444. T. Wieprecht, M. Dathe, R.M. Epand, M. Beyermann, E. Krause, W.L. Maloy, D.L. MacDonald, M. Bienert, Biochemistry 36 (1997) 12869. R.E.W. Hancock, A. Patrzykat, Curr. Drug Targets Infect. Disord. 2 (2002) 79. A. Patrzykat, S.E. Douglas, Protein Pept. Lett. 12 (2005) 19. D.A. Devine, R.E. Hancock, Curr. Pharm. Design 8 (2002) 703. B. Li, F. Chen, X. Wang, B. Ji, Y. Wu, Food Chem. 102 (2007) 1135. P.J. Park, W.K. Jung, K.S.F. Nam, F. Shahidi, S.K. Kim, J. Am. Oil Chem. Soc. 78 (2001) 651. J.W. Woo, S.J. Yu, S.M. Cho, Y.B. Lee, S.B. Kim, Food Hydrocolloid. 22 (2008) 879.
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Journal of Industrial and Engineering Chemistry xxx (2019) xxx–xxx
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Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis Monjurul Haqa,b , Truc Cong Hoa , Raju Ahmeda , Adane Tilahun Getachewa , Yeon-Jin Choa , Jin-Seok Parka , Byung-Soo Chuna,* a b
Department of Food Science and Technology, Pukyong National University, 45 Yongso-ro, Nam-Gu, Busan 48-513, Republic of Korea Department of Fisheries and Marine Bioscience, Jashore University of Science and Technology, Jashore 7408, Bangladesh
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 July 2019 Received in revised form 1 September 2019 Accepted 15 September 2019 Available online xxx
Bacterial collagenolytic protease-extracted Bigeye tuna skin collagen was subjected to catalysts-assisted subcritical water hydrolysis. The degree of hydrolysis was the highest for hydrolysates obtained using sodium bicarbonate catalysts. The color and pH values of the hydrolysates varied depending of the catalysts used. Four different assays of antioxidant activities varied with respect to the various collagen hydrolysates with hydrolysates obtained using sodium bicarbonate catalysts showing the highest activity. Ten bacterial species were studied to evaluate antibacterial activity among which nine showed positive results. The average molecular size of the peptides in the obtained collagen hydrolysates varied between 300 and 425 Da. The total amount of structural and total amino acids varied between 116.63 and 49.51, and 196.86 and 265.68 mg/100 g hydrolysate, respectively. The collagen hydrolysates obtained showed enormous potential to be used in the food and pharmaceutical industries. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Biofunctional Bacterial collagenolytic protease-extracted collagen Catalyst-assisted Subcritical water hydrolysis
Introduction Collagen is the most abundant structural biopolymer protein comprising approximately of around one-third of the total protein in vertebrates and playing major structural roles in fibrous tissues (ligaments, tendons, and skin) and bones of animals [1]. It is also found in the corneas, gut, blood vessels, intervertebral discs, and the dentin of teeth. Triple helices of amino acids twist together to form the collagen structure. The rigidness of different collagens, such as those in the bone, tendon, or cartilage, depends upon the degree of mineralization. In the past decade, numerous studies have concentrated on the field of collagen-based biomaterials and its applications, such as tissue engineering applications, bone regeneration scaffolds, and crosslinking methods, etc. At present, collagen is widely used in research and medical applications [2]. Its commercial production is derived from bovine and porcine sources, but there are some issues in using this, including the transmission of some diseases along with cultural and religious restrictions [3]. Therefore, collagen originating from fish or other aquatic species is being
* Corresponding author. E-mail address: [email protected] (B.-S. Chun).
considered as popular ingredients in the food and pharmaceutical industries. Extracellular proteins including collagen, are known to contain biofunctional peptides that can be released under certain conditions and can show divergent biological activities from the parent proteins [1]. Many researchers have reported on biofunctional activities of collagen peptides such as antioxidant [4], antiobesity [5], antihypertensive [6,7], and antibacterial [1] activities. Activation of these bonded peptides requires physical alterations of the molecule which can be performed by proteolytic processing [8,9]. Enzymatic hydrolysis is widely used to produce functional peptides from collagen [7]; however, subcritical water hydrolysis of collagen, a cheap and easy-to-adopt technology, is considered advantageous. Subcritical water hydrolysis is a clean and fast biomass reduction process that can be applied as a suitable alternative to acidic, basic, and enzymatic hydrolysis because of its faster reaction period, lower residue production, less corrosion, lower degradation product generation, nontoxic solvent use, and so on [10]. Moreover, subcritical water can be used as a tunable reactant as well as a solvent in the conversion of macromolecules, without the need for acid or alkali, thereby avoiding environmental hazards [11]. Acid/alkaline hydrolysis needs extreme reaction conditions and, avoiding uncontrolled and undesired reaction requires purification steps to separate hazardous chemicals causing environmental pollution from the
https://doi.org/10.1016/j.jiec.2019.09.023 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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final products. Moreover, enzyme-catalyzed hydrolysis has a longer reaction time to complete the reaction cycle [12]. Subcritical water hydrolysis combines high temperature and pressure, converting collagen macromolecules into smaller molecules of peptides and amino acids [13]. The peptides are converted into free amino acids and subsequently converted to organic acids under higher treatment temperature and longer hydrolysis time [14]. The suitable temperature and time for subcritical water hydrolysis depend on the substrate source, for example, protein from animal sources need an extended reaction time or higher temperature than that from vegetable sources [15]. In addition, some researchers have reported on the incorporation of catalysts to increase biofunctional materials in the subcritical water hydrolysis process [12,16–19]. Subcritical water hydrolysis of bacterial collagenase extracted fish skin collagen converts collagen macromolecules into bioactive peptides/amino acids, which can ideally be used as bioactive ingredients in food, pharmaceutical, and cosmetic industries as these active compounds delivered to the muscle faster than original collagen. Enhancement of the biofunctional activity of the protein substrate because of subcritical water hydrolysis is proven by many previous researchers using various protein sources such as by-products of animals [18,20], by-products of fish [21,22], plant materials [23] and even pure protein, such as hemoglobin, beta-casein [14,24] and bovine serum albumin. In a previous study, collagen from tuna fish skin was extracted using collagenase isolated from collagenase-producing bacteria [1] to avoid collagen extraction by using porcine-originated pepsin. In another study, pepsin-extracted collagen was subjected to subcritical water hydrolysis, the reaction parameters were optimized, and positive biofunctional activities of collagen peptides were found [25]. Thus far, there is no report on the subcritical water hydrolysis of bacterial collagenase-extracted fish collagen
and its biofunctional activity. In addition, collagen peptide-rich hydrolysates were obtained by catalyst-assisted subcritical water hydrolysis at previously optimized conditions [25] using bacterial collagenase-extracted tuna skin collagen. Therefore, the aims of this study were to hydrolyze bacterial collagenase extracted tuna skin collagen enzyme using catalyst-assisted subcritical water and to evaluate the physiochemical and biofunctional properties of the hydrolysates. Materials and methods Chemicals and reagents Acetic acid and iron (III) chloride 6-hydrate (FeCl36H2O) were purchased from Merck (Darmstadt, Hessen, Germany). DPPH, ABTS, trolox, gallic acid, and Folin–Ciocalteu reagent were purchased from Sigma-Aldrich Co. (St. Louis, MI, USA). Only analytical/HPLC grade reagents and solvents were used in this study. Sample collection, preparation, and collagen extraction The collection of Bigeye tuna skin and extraction of collagen using bacterial collagenase is described in a previous study [1]. Catalyst-assisted subcritical water hydrolysis Different catalysts such as sodium chloride, acetic acid, and sodium bicarbonate were prepared at concentration of 0.6 M, and distilled water was used as a control for comparing the effects of the catalyst solutions in the hydrolysis process. A batch-mode Hastelloy reactor facilitated by a temperature regulator (Fig. 1) was used. The solid-to-liquid ratio (W/V) was maintained at 1:200, and
Fig. 1. Schematic diagram of the laboratory-scale subcritical water apparatus.
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150 mL of liquid was loaded in the 200 mL size reactor. After closing the reactor tightly, it was heated using an electric heating system at a temperature of 250 C. The pressure inside the reactor was controlled at 50 bar using N2 gas, and the temperature and pressure were continuously monitored through a temperature and pressure gauge, respectively. The sample was stirred at 150 rpm using a four-blade impeller. It took 45 min to reach the desired reactor temperature and the reaction period was maintained at 3 min. The reactor was cooled immediately by circulating refrigerated water to avoid further degradation. Then, the hydrolysate sample was collected from the reactor, filtered using a filter paper, and stored at 4 C. Determination of the degree of hydrolysis The degree of hydrolysis of the hydrolysates was measured according to the method described by Thiansilakul et al. [26] with some modifications. In brief, 125 mL of the hydrolysate sample was added to the reaction reagent. The reaction reagent was prepared by mixing 2.0 mL of sodium phosphate (0.2125 M, pH 8.2) with 1 mL 2,4,6-trinitrobenzene sulfonic acid (0.01%, pH 8.2). The mixture sample was incubated at 50 C for 30 min in dark. Then, 2.0 mL of 0.1 M sodium sulfite was added to the sample to stop the reaction. Later, the sample solution was cooled at room temperature for 15 min, and the absorbance was measured at 420 nm. A calibration curve was measured using glycine. The degree of hydrolysis was measured using the following equation: Degree of hydrolysis ¼
Lt L0 100 Lmax L0
where Lt is the α-amino acid in the hydrolysate, L0 is the α-amino acid in the original sample homogenate, and Lmax is the α-amino acid in the original sample obtained using acid hydrolysis (6 N HCl,100 C for 24 h).
Chelating activity ð%Þ ¼
3
DPPH scavenging activity The DPPH radical scavenging capacity was measured following the method of Asha et al. [28] with some modifications. In total, 50 mL of the hydrolysate sample (5 mg/mL) was added to 950 mL of the DPPH solution (0.1 mM) and incubated in dark at room temperature for 30 min. Then, the absorbance was measured at 517 nm. The DPPH scavenging activity was expressed as mg VCEAC/g of collagen hydrolysate. A standard curve was prepared using vitamin C. Ferric reducing antioxidant power assay The ferric reducing antioxidant power (FRAP) aasay was conducted according to the method of Asha et al. [28] after some alterations. Fifty microliter of the hydrolysate sample (5 mg/mL) was mixed with 0.95 mL of the FRAP reagent and incubated at 37 C for 30 min in dark. The FRAP solution without the sample was considered as the blank, and the absorbance was measured at 595 nm. FRAP activity was expressed as mg VCEAC/g of collagen hydrolysate. Metal chelating activity The metal chelating activity of the collagen hydrolysates was measured following the procedure described by Klompong et al. [29], with some modifications. The sample (100 mL at 5 mg/mL) was mixed with 870 mL of distilled water. Then, 10 mL of 2 mM FeCl2 and 20 mL of 5 mM ferrozine were added and incubated at room temperature for 10 min. Subsequently, the absorbance was recorded at 562 nm. Distilled water instead of the sample was used as a control. The metal chelating activity was measured using the following equation:
Absorbacne of control at 562 nm Absorbacne of sample at 562 nm 100 Absorbacne of control at 562 nm
Color and pH measurement
Determination of antimicrobial activity of the collagen hydrolysates
The color of the hydrolysates was measured using of a portable reflectance spectrophotometer (Lovibond RT Series, Amesbury, UK). The instrument was standardized with white and black references before each measurement and color parameters L* (lightness/ brightness), a* (redness/greenness), and b*(yellowness/blueness) were recorded. The pH of the hydrolysates was determined using a Mettler Toledo pH meter (EasyPlus, Switzerland) at 25 C. Technical buffer solutions were used to calibrate the pH meter prior to the measurements.
The antimicrobial activity of the collagen hydrolysates for 10 bacterial strains was determined according to the method of Mosquera et al. [30] with some modifications. The tested strains and procedures are described in detail in a previous study [1].
Radical scavenging activity measurement ABTS scavenging activity The ABTS radical scavenging activity of the collagen hydrolysates was determined following the methods of Aleman et al. [27]. The diluted hydrolysate sample (5 mg/mL; 20 mL) was added to 980 mL of the ABTS solution (OD value, 0.70 0.02 at 734 nm), kept in dark for 16 h at room temperature, and the absorbance was measured at 734 nm. A standard curve of vitamin C was prepared (50–500 ppm). The result was expressed as mg vitamin C equivalent antioxidant capacity (VCEAC)/g of hydrolysate.
Analysis of amino acid composition The collagen hydrolysates were analyzed for both structural and free amino acid contents using an amino acid auto analyzer (Models: S433-H and S430-H, SYKAM, Germany) following the methods described in detail in the study by Asaduzzaman and Chun [21]. First, the samples were filtered and injected onto the analyzer facilitated by the cation separation column LCA K06/Na (4.6 150 mm) and LCA K07/Li (4.6 150 mm), with column temperatures of 57–74 C and 37–74 C and buffer pH ranges of 3.45–10.85 and 2.90–7.95 for structural and free amino acid analyses, respectively. As a mobile phase, 5 mM p-toluenesulfonic acid solution was used (0.45 mL/min) for both. As a post-column reagent, a mixture of 5 mM p-toluenesulfonic acid, 100 mM EDTA, and 20 mM bis–tris was used (0.25 mL/min). Excitation and emission wavelengths were maintained at 440 and 570 nm, respectively, for both operating conditions. The values were presented as mg/100 g of liquid collagen hydrolysate.
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MALDI-TOF spectroscopy analysis The molecular profile of the obtained hydrolysates was analyzed using the MALDI-TOF Bruker Autoflex III following the method of Meillisa et al. [31]. A small beam laser was facilitated by using an instrument, and the spectrum was obtained with a voltage of 20 kV. Water/acetonitrile (25:75) was used as a solvent, and 2,5-dihydroxybenzoic acid as a matrix. A hydrolysate sample of 0.5 mL was mixed with 0.5 mL of the matrix solution. The matrix solution was prepared from norharmane-acetonitrile and trifluoroacetic acid at a rate of 7:3, where the norharmaneacetonitrile solution was prepared by dissolving 10 mg norharmane in 1 mL acetonitrile. Statistical analysis Values are presented as a mean standard deviation of triple determinations. One-way analysis of variance was conducted using SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). Duncan’s multiple range test was performed to determine significant differences among the means, and p 0.05 was regarded to be significant (Fig. 2). Results and discussion Degree of hydrolysis, color, and pH values The degree of hydrolysis, color, and pH values of the bacterial collagenolytic protease-extracted collagen hydrolysates obtained using subcritical water hydrolysis using different catalytic solutions are shown in Table 1. The degree of hydrolysis varied depending on the type of catalyst used in this study. The highest value of degree of hydrolysis was found in hydrolysates obtained using sodium bicarbonate catalysts (21.81 0.48%), whereas the lowest value was found in those obtained using distilled water (15.28 0.36%). The same degree of hydrolysis was obtained when pepsin-extracted tuna skin collagen was hydrolyzed by the subcritical water hydrolysis process using only water as the solvent [25]. Asaduzzaman et al. [12] hydrolyzed mackerel fish
muscles and reported that the addition of acidic and alkaline catalysts influenced the hydrolysis yield. These catalysts are corrosive on the substrate, and in addition, the polarity of the solvent and dielectric constant are increased, which enhanced depolymerization of the substrate. Fish collagen has very poor solubility in water at ambient temperature because of aggregation by hydrophobic interactions, which can be altered by different catalyst-assisted subcritical water hydrolysis. The degree of whey protein hydrolysate was increased fourfold with sodium bicarbonate catalysts compared with that with only water, and the molecular weight of the peptides was reduced because of the application of sodium bicarbonate catalysts [32]. The degree of hydrolysis is considered significant for determining the molecular weight of peptides and the exposure of the terminal amino groups of the resulting product, thereby influencing different biopotential activities of the hydrolysate [1]. The color values of the collagen hydrolysates varied depending on the catalysts used for hydrolysis (Table 1). The highest L value (lightness) was obtained with the collagen hydrolysate in acetic acid catalysts, whereas the lowest L value was obtained with that using the sodium bicarbonate catalysts. The color of the collagen hydrolysates plays an important role in food ingredients [33]. The color differences in the collagen hydrolysates may be due to the presence of Maillard reaction products [34]. The variation in the pH values of the collagen hydrolysates might be due to the conversion of collagen to acidic compounds. Antioxidant activity ABTS radical scavenging activity ABTS is a popular method determining both hydrophilic as well as lipophilic antioxidants [12]. The highest ABTS radical scavenging activity value of 54.17 mg VCEAC/g of hydrolysate was found in the collagen hydrolysate obtained using sodium bicarbonate catalysts (Fig. 3). The lowest ABTS radical scavenging activity of 35.70 mg VCEAC/g of hydrolysate was found in the collagen hydrolysate obtained using only water. The free radical scavenging activity of the collagen hydrolysate depends on their amino acid composition as well as the sequences of their peptides [35]. Similar ABTS radical
Fig. 2. Summarized scheme of all the steps of the work.
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Table 1 Degree of hydrolysis, color and pH values of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Parameters Degree of hydrolysis (%) Color
Water only d
L a* b*
pH
15.28 0.36 52.90c 0.75 0.48b 0.05 15.61b 0.39 8.25c 0.09
Sodium chloride b
19.64 0.10 54.68b 0.95 0.57c 0.03 6.09c 0.24 9.62b 0.28
Acetic acid c
17.61 0.41 68.37a 1.22 1.62d 0.06 4.61d 0.55 2.37d 0.02
Sodium bicarbonate 21.81a 0.48 35.61d 0.96 4.11a 0.29 23.34a 0.96 12.70a 0.32
Values are presented as means standard deviations of triplicates. Different small letters in the same row of the table indicate significant differences (P < 0.05).
Fig. 3. Antioxidant activity of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions.
scavenging activity was reported by Ahmed et al. [1] when they conducted a study on the hydrolysis of pepsin-extracted collagen using distilled water. The collagen molecules were subjected to degradation based on the different types of catalysts and produced various types/sizes of peptides and amino acids that can have an effect on the ABTS radical scavenging activity. Guillén et al. [36] hydrolyzed tuna gelatin using alcalase enzyme and reported a ABTS radical scavenging activity value of 17.57 1.62 VCEAC/g of protein. The variations in radical scavenging activity of different hydrolysates are due to the substrate nature, solid–liquid ratio, and peptides/amino acid formation extent. Small–size peptides/amino acids in collagen hydrolysates have proton/electron donating and chain breaking ability; thus, they react with free radicals and stabilize them, terminating the free radical chain reaction. Hydrophobic amino acids such as tyrosine, histidine, methionine, tryptophan and lysine are supposed to have radical scavenging ability [37]. The catalyst-assisted collagen hydrolysis in this study showed higher ABTS radical scavenging activity than that reported in previous studies. DPPH radical scavenging activity The DPPH radical scavenging activity of different hydrolysates varied between 17.47 1.53 and 38.81 2.06 mg VCEAC/g hydrolysate. Maximum DPPH radical scavenging activity was found in the collagen hydrolysate obtained using sodium bicarbonate followed by that of the collagen hydrolysate obtained using acetic acid, sodium chloride, and water as solvent (Fig. 3). In the presence of antioxidant compounds, the DPPH radicals are scavenged by the proton-donating antioxidant. In presence of different catalysts during hydrolysis of collagen molecules, various peptides and amino acids were formed, which acted as a proton donor and stabilized the DPPH free radicals. Some antioxidant compounds
display ABTS radical scavenging activity but may not display DPPH radical scavenging activity. This is because ABTS radical scavenging activity is applicable to hydrophilic and hydrophobic antioxidant systems, whereas DPPH scavenging activity is only applicable to hydrophobic systems [38]. The collagen hydrolysates obtained in this study showed both ABTS and DPPH free radical scavenging activities containing both hydrophobic and hydrophilic amino acids that contribute to the antioxidant properties. FRAP assay The collagen hydrolysates showed FRAP activity, and the values varied depending on the catalyst solution used for hydrolysis. Higher FRAP activity of 24.41 1.18 mg VCEAC/g of hydrolysate was found in hydrolysates obtained using sodium bicarbonate solutions followed by 21.10 0.51, 19.70 0.27, and 17.94 0.67 mg VCEAC/g of hydrolysate in hydrolysates obtained using sodium chloride, acetic acid, and only water (Fig. 3). The different peptides obtained from the in different hydrolysates have the ability to provide electrons to the radicals. The principle of the FRAP assay is based on electron transfer instead of hydrogen ion transfer. It determines the ability of peptides and amino acids to reduce Fe3+ to Fe2+. The FRAP reaction is conducted at an acidic pH to maintain iron solubility, which decreases the ionization potential and enhances redox potential. Because of the transformation of Fe3+ to Fe2+, there is a formation of a colored compound (595 nm) in the presence of the FRAP reagent. Ahmed and Chun [25] hydrolyzed pepsin-extracted collagen using water and reported the FRAP activity to be 17.17 mg VCEAC/g of hydrolysate at 250 C, which is similar to that observed in present study, but the FRAP activity was found to be higher when different acidic and alkaline catalysts were used. This might be due to the generation of various bioactive peptides by the various catalytic solutions used
Please cite this article in press as: M. Haq, et al., Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.09.023
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during the hydrolysis process. The FRAP activity was found to be 11.55 VCEAC/g of protein in enzyme-hydrolyzed tuna gelatin [36]. Metal chelating activity The highest metal chelating activity was found to be 60.76 4.72% and the lowest activity was found to be 48.61 0.45 in the collagen hydrolysates obtained using sodium bicarbonate and only water, respectively (Fig. 3). Peptides and amino acids in the collagen hydrolysated were chelated with Fe2+ and showed metal chelation activity. The mechanisms underlying the process is as follows: ferrozine forms a complex with Fe2+, a colorful solution but metal chelating antioxidant compounds in the collagen hydrolysate inhibit the colorful complex formation resulting in decrease in the color [38]. Ahmed et al. [1] found the metal chelating activity of pepsin-extracted collagen hydrolysate to be 50.54% in hydrolysates obtained at 250 C with only water as a solvent. The use of different catalysts during the hydrolysis process increased the metal chelating activity in the present study. Asaduzzaman and Chun [39] found a maximum metal chelating activity of 54% in mackerel muscle hydrolysate. The collagen hydrolysates showing metal chelating activity can be used as functional food ingredients to improve the bioavailability of
minerals. Nakchum and Kim [40] studied the ferrous chelating ability of squid skin-derived collagen and found positive results. Guo et al. [41] also reported that Alaska Pollock skin-derived collagen peptides showed iron and copper chelating activity and fish collagen hydrolysates showed calcium chelating activity [41,42]. Antimicrobial activity The antimicrobial activity of the collagen hydrolysate is shown in Table 2. Except for Enterococcus faecium, all bacteria studied were found to be sensitive to the collagen hydrolysates. The collagen hydrolysates obtained using catalysts revealed higher activity than those obtained using only water. Antimicrobial activity is closely associated with hydrophobic amino acids in hydrolysates [43]. This hydrophobic nature of the peptides facilitates entrance into the bacterial cell membrane, and the positive charge initiates the peptide interaction with the negatively charged bacterial membrane [44]. The mechanisms of microbe and peptide interactions include formation of disconnected channels in lipid bilayers, resulting in the discretion of lipid bilayers. In addition, specific lipid–peptide interaction results in detergent-like solubilization of the membrane and even the
Table 2 Antimicrobial activity of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Bacterial strains
Water only
Sodium chloride
Acetic acid
Sodium bicarbonate
Bacillus cereus Bacillus subtilis Enterococcus faecium Escherichia coli Klebsiella pneumonia Listeria monocytogenes Pseudomonas putida Pseudomonas aeruginosa Staphylococcus aureus Salmonella typhimurium
+
++ + + + +
++ ++ + +++ ++ +
++ + ++ + ++ +
N.B.: inhibition zones: +++: >1 cm; ++: 0.5 cm, +: 0.25–0.5 cm; : <0.25 cm and : 0.1 cm.
Fig. 4. MALDI-TOF mass spectra chromatogram of bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions.
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Table 3 Structural amino acids content (mg/100 g) in bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Amino acids
Water only
Sodium chloride
Acetic acid
Sodium bicarbonate
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Ammonia Histidine Arginine Proline Total
1.28 0.12 0.80 0.04 0.73 0.10 22.08 2.51 29.61 2.15 19.64 2.45 1.54 0.23 4.67 0.54 0.83 0.10 1.80 0.15 5.94 0.34 1.75 0.11 4.23 0.41 6.15 0.23 5.97 0.71 1.21 0.43 2.90 0.25 15.03 1.65 126.16
0.12 0.06 0.12 0.07 0.07 0.02 21.20 2.65 30.81 2.91 20.55 2.95 1.83 0.16 4.27 0.34 1.59 0.07 0.99 0.12 4.79 0.21 0.85 0.15 3.81 0.36 4.81 0.26 4.61 0.32 0.92 0.04 0.89 0.16 14.39 1.57 116.63
0.15 0.02 0.12 0.09 0.15 0.07 20.75 2.48 30.22 3.16 20.96 2.41 2.91 0.21 4.10 0.21 0.99 0.10 1.07 0.10 4.43 0.31 0.80 0.09 3.56 0.12 5.40 0.41 8.68 0.27 0.86 0.12 3.25 0.11 14.11 1.37 122.51
0.10 0.09 0.20 0.06 0.02 0.01 23.28 2.76 41.77 3.76 28.35 3.12 2.98 0.62 6.59 0.32 0.21 0.05 1.64 0.31 7.47 0.12 1.32 0.06 5.15 0.40 10.54 0.63 3.17 0.51 0.53 0.13 0.19 0.04 15.86 0.34 149.51
Values are presented as means standard deviations of triplicates.
peptides interact with the membranes [45,46]. Antimicrobial peptides can destroy the targeted harmful microbes and demonstrate a wide scale of activities, including antimicrobial activity against some antibiotic-resistant microorganisms. Antimicrobial peptides from natural sources can kill microorganisms and modulate inflammatory responses [47]. Therefore, it is feasible to use them widely in food preservation and offer extra health aids.
size of the peptides in a hydrolysate is important for determining its functional activity [49]. Molecular analysis of the hydrolysates by MALDI-ToF spectroscopy revealed, it is seen that many lower molecular weight compounds were obtained because of the hydrolysis treatment.
Molecular weight analysis
The structural and free amino acid contents of the different collagen hydrolysates obtained using different catalysts are shown in Tables 3 and 4, respectively. The total amount of structural amino acids varied between 116.63 and 49.51 mg/100 g hydrolysates, where the highest content was found in hydrolysates obtained using sodium bicarbonate catalysts and the lowest amount was found in those obtained using only water. Among the structural amino acids found in different collagen hydrolysates, glycine, alanine, and proline were the most abundant. Usually, these amino acids are also dominant in raw collagens [50] and
The molecular weight of the collagen hydrolysates is shown in Fig. 4. The molecular profile of the peptides in all the hydrolysates showed that the molecular size of the peptides was <425 Da. The application of subcritical water hydrolysis depolymerized the collagen molecules in the presence of different catalysts and water as well. The average molecular size of the peptides in the obtained hydrolysates was 300–425 Da. Most of the peptides showing biofunctional activity were <1 kDa [36,48]. The average molecular
Analysis of amino acids in the collagen hydrolysates
Table 4 Free amino acids content (mg/100 g) in bacterial protease extracted collagen hydrolysates obtained by subcritical water treatment using different catalytic solutions. Free amino acids
Water only
Sodium chloride
Acetic acid
Sodium bicarbonate
Phosphoserine Taurine Aspartic acid Threonine Serine Glutamic acid α-Amino adipic acid Glycine Alanine A-amino-n-butyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine β-alanine β-amino isobutyric acid Ethanol amine Lysine Histidine Hydroxy proline Proline Total
8.83 0.43 1.84 0.14 1.14 0.12 1.20 0.12 0.49 0.14 3.46 0.21 ND 90.70 4.56 19.46 2.43 3.62 0.65 19.86 1.56 8.53 0.45 ND 6.73 0.46 12.65 0.29 10.51 0.58 11.15 0.48 4.10 0.65 7.13 0.62 8.58 0.53 2.17 0.56 2.75 0.37 22.56 2.56 247.55
6.99 0.54 ND 0.94 0.07 1.63 0.21 ND 3.37 0.31 ND 91.57 5.34 16.40 1.45 4.46 0.34 24.84 2.27 11.63 0.62 18.50 1.95 5.55 0.41 12.47 0.87 15.22 0.45 ND 3.54 0.37 5.28 0.73 11.62 1.05 2.77 0.38 11.93 0.98 16.91 1.73 265.68
7.40 0.82 ND 0.62 0.14 ND ND 4.06 0.18 ND 62.28 3.65 13.73 1.34 7.05 0.32 16.01 1.93 6.89 0.49 17.81 1.56 6.95 0.73 ND 3.42 0.71 13.76 0.28 9.72 0.62 3.33 0.25 7.29 0.82 2.02 0.27 ND 14.44 1.93 196.86
7.62 0.39 ND 0.70 0.13 1.34 0.13 ND 6.04 0.22 0.94 0.54 55.15 5.34 12.97 1.34 11.24 1.04 23.60 1.36 6.15 0.34 21.58 1.45 6.82 0.24 12.29 0.67 4.83 0.68 8.04 0.91 ND ND 8.57 0.38 2.09 0.36 1.71 0.27 13.47 1.50 205.23
ND: Not detected. Values are presented as means standard deviations of triplicates.
Please cite this article in press as: M. Haq, et al., Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.09.023
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gelatin hydrolysates [36]. There were differences among the structural amino acid contents of the collagen hydrolysates because of the different catalyzing effects of the catalysts used. Asaduzzaman et al. [12] also reported that the amino acid content of mackerel muscle hydrolysate obtained using sodium bicarbonate was higher that obtained to using only water. Among the different solvents used for hydrolysate preparation, there might be a higher conversion of protein to amino acids in hydrolysates obtained using sodium bicarbonate solution. Sodium bicarbonate and acetic acid solutions showed higher contents of amino acid production than only water, whereas sodium chloride showed lower amino acid production. Amino acid production was found to be significantly higher in whey protein hydrolysates obtained using subcritical water hydrolysis [17]. The free amino acid contents in the collagen hydrolysates varied between 196.86 and 265.68 mg/100 g; the maximum content was found in hydrolysates obtained using sodium chloride catalysts and the minimum content was found in those obtained using acetic acid catalysts. Among the free amino acids found in different collagen hydrolysates, glycine, glutamic acid, alanine, and proline have the highest content. A balanced diet containing essential amino acid is a prerequisite for proper protein synthesis. The collagen hydrolysates obtained using different catalysts contained balanced and important essential amino acids (Tables 3 and 4) suitable for use as a good nutritional supplement. Moreover, differences in the antioxidant and antioxidant activities of the collagen hydrolysates might be due to the composition and sequence of amino acids. Conclusion Bacterial collagenolytic protease-extracted collagen hydrolysates obtained by subcritical water hydrolysis showed significant antioxidant and antimicrobial potential. The activities were higher in the hydrolysates obtained using different catalyst-assisted subcritical water hydrolysis than in those obtained using only water. The functional activities varied depending on the hydrolysates observed using different catalysts, whereas the maximum activities were obtained in hydrolysates obtained using sodium bicarbonate. The molecular profile of peptides showed that the molecular weight was reduced because of catalyst-assisted subcritical water hydrolysis, and the molecular weight of peptides in all the hydrolysates was <425 Da. The collagen hydrolysates obtained by environmentally subcritical water hydrolysis, a very fast and green technique, could be applied as a potential ingredient in functional food, pharmaceutical, and cosmetics industries for human health benefits. Acknowledgments The research was funded by Basic Science Research Program, Nation Research Foundation in Korea (NRF), Ministry of Education (2016R1D1A1B03936128).
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
[37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50]
R. Ahmed, A.T. Getachew, Y.J. Cho, B.S. Chun, LWT Food Sci. Technol. 89 (2018) 44. R. Parenteau-Bareil, R. Gauvin, F. Berthod, Materials 3 (2010) 1863. M. Ahmad, S. Benjakul, Food Chem. 120 (2010) 817. S. Ketnawa, O. Martínez-Alvarez, S. Benjakul, S. Rawdkuen, Food Chem. 192 (2016) 34. M. Saito, C. Kiyose, T. Higuchi, N. Uchida, H. Suzuki, J. Agric. Food Chem. 57 (2009) 10477. Y. Zhuang, L. Sun, B. Li, Food Bioprocess Tech. 5 (2012) 1622. P. Banerjee, C. Shanthi, Process Biochem. 47 (2012) 2335. F.X. Maquart, S. Pasco, L. Ramont, W. Hornebeck, J.C. Monboisse, Cr. Rev. OncolHeem. 49 (2004) 199. S. Schenk, V. Quaranta, Trends Cell Biol. 13 (2003) 366. Y. Zhao, H.T. Wang, W.J. Lu, H. Wan, Chem. Eng. J. 166 (2011) 868. B.M. Pedras, M. Nascimento, I. Sá-Nogueira, P. Simões, A. Paiva, S. Barreiros, J. Ind. Eng. Chem. 72 (2019) 453. A.K.M. Asaduzzaman, M. Haq, B.S. Chun, J. Ind. Eng. Chem. 68 (2018) 301. I. Marcet, C. Álvarez, B. Paredes, M. Díaz, Waste Manage. 49 (2016) 364. T. Rogalinski, S. Herrmann, G. Brunner, J. Supercrit. Fluid. 36 (2005) 49. J.Y. Je, Z.J. Qian, S.H. Lee, H.G. Byun, S.K. Kim, J. Med. Food 11 (2008) 629. K.Y. Kang, B.S. Chun, Korean J. Chem. Eng. 21 (2004) 654. A.D. Espinoza, R.O. Morawicki, J. Agric. Food Chem. 60 (2012) 5250. H. Cheng, X. Zhu, C. Zhu, J. Qian, N. Zhu, L. Zhao, J. Chen, Bioresour. Technol. 99 (2008) 3337. Z.H.U. Xian, Z.H.A.O. Chao, H. Liang, Cheng, Chin. J. Chem. Eng. 16 (2008) 456. M. Esteban, A. García, P. Ramos, M. Márquez, Bioresour. Technol. 101 (2010) 2472. A.K.M. Asaduzzaman, B.-S. Chun, J. Food Sci. Technol. 52 (2015) 793. D.O. Kim, K.W. Lee, H.J. Lee, C.Y. Lee, J. Agric. Food Chem. 50 (2002) 3713. A.D. Espinoza, R.O. Morawicki, T. Hager, J. Food Sci. 77 (2012). T. Powell, S. Bowra, H.J. Cooper, Anal. Chem. 88 (2016) 6425. R. Ahmed, B.S. Chun, J. Supercrit. Fluid. 141 (2018) 88. Y. Thiansilakul, S. Benjakul, F. Shahidi, J. Food Biochem. 31 (2007) 266. A. Alemán, B. Giménez, P. Montero, M.C. Gómez-Guillén, LWT Food Sci. Technol 44 (2011) 407. K.K. Asha, K.R. Kumari, K.A. Kumar, N.S. Chatterjee, R. Anandan, S. Mathew, Int. J. Pept. Res. Ther. 22 (2016) 421. V. Klompong, S. Benjakul, D. Kantachote, F. Shahidi, Food Chem. 102 (2007) 1317. M. Mosquera, B. Giménez, S. Ramos, M.E. López-Caballero, M.D.C. GómezGuillén, P. Montero, J. Aquat. Food Prod. Technol. 25 (2016) 444. A. Meillisa, H.C. Woo, B.S. Chun, Food Chem. 171 (2015) 70. Y. Zhuang, L. Sun, B. Li, Food Bioprocess Tech. 5 (2012) 1622. A.D. Espinoza, R.O. Morawicki, T. Hager, J. Food Sci. 77 (2012) 20. K. Watchararuji, M. Goto, M. Sasaki, A. Shotipruk, Bioresour Technol. 99 (2008) 6207. H.C. Wu, H.M. Chen, C.Y. Shiau, Food Res. Int. 36 (2003) 949. G. Guillén, G.M.E. López Caballero, A. Alemán, A. López de Lacey, B. Giménez, P. Montero García, Antioxidant and antimicrobial peptide fractions from squid and tuna skin gelatin. Sea by-products as a real material: new ways of application, (2010) p. 89. S.Y. Dong, M.Y. Zeng, D.F. Wang, Z.Y. Liu, Y.H. Zhao, H.C. Yang, Food Chem. 107 (2008) 1485. P. Kittiphattanabawon, S. Benjakul, W. Visessanguan, H. Kishimura, F. Shahidi, Food Chem. 119 (2010) 1519. A.K.M. Asaduzzaman, B.S. Chun, Fish. Sci. 80 (2014) 369. L. Nakchum, S.M. Kim, Prep. Biochem. Biotechnol. 46 (2016) 123. L. Guo, P.A. Harnedy, M.B. O’Keeffe, L. Zhang, B. Li, H. Hou, R.J. FitzGerald, Food Chem. 173 (2015) 536. L. Guo, P.A. Harnedy, L. Zhang, B. Li, Z. Zhang, H. Hou, X. Zhao, R.J. FitzGerald, J. Agric. Food Chem. 95 (2015) 1514. M. Mosquera, B. Giménez, S. Ramos, M.E. López-Caballero, M.D.C. GómezGuillén, P. Montero, J. Aquat. Food Prod. Technol. 25 (2016) 444. T. Wieprecht, M. Dathe, R.M. Epand, M. Beyermann, E. Krause, W.L. Maloy, D.L. MacDonald, M. Bienert, Biochemistry 36 (1997) 12869. R.E.W. Hancock, A. Patrzykat, Curr. Drug Targets Infect. Disord. 2 (2002) 79. A. Patrzykat, S.E. Douglas, Protein Pept. Lett. 12 (2005) 19. D.A. Devine, R.E. Hancock, Curr. Pharm. Design 8 (2002) 703. B. Li, F. Chen, X. Wang, B. Ji, Y. Wu, Food Chem. 102 (2007) 1135. P.J. Park, W.K. Jung, K.S.F. Nam, F. Shahidi, S.K. Kim, J. Am. Oil Chem. Soc. 78 (2001) 651. J.W. Woo, S.J. Yu, S.M. Cho, Y.B. Lee, S.B. Kim, Food Hydrocolloid. 22 (2008) 879.
Please cite this article in press as: M. Haq, et al., Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalysts-assisted subcritical water hydrolysis, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.09.023