Harmandeep
Kaur
a,
Manpreet
Singh
a,
Navdeep
Kaur
b,
Pratap Kumar
Pati
b,
Monika
Rani
c and
Tejwant Singh
Kang
*a
aDepartment of Chemistry, UGC Centre for Advanced Studies-II, Guru Nanak Dev University, Amritsar-143005, India. E-mail: tejwantsinghkang@gmail.com; Tel: +91-183-2258802-Ext-3291
bDepartment of Biotechnology, Guru Nanak Dev University, Amritsar-143005, India
cDepartment of Food Science and Technology, Guru Nanak Dev University, Amritsar-143005, India
First published on 24th June 2024
Collagen is a protein that is hard to dissolve in water and many other solvents, which limits its applications. Herein, deep eutectic solvents (DESs), i.e. choline chloride:
lactic acid (ChCl
:
LA) = 1
:
1 and ethylene glycol
:
zinc chloride (EG
:
ZnCl2) = 4
:
1, are effectively used to dissolve type I collagen under different conditions. Type I collagen is readily soluble at a concentration of 9.5–22.5 w/v% in DESs, and the solubility is governed by the nature of the DES, temperature (45 °C, 70 °C and 90 °C) and the absence or presence of HCl(aq.) (5 × 10−5 M). The dissolved material is regenerated by employing ethanol as an anti-solvent at 4 °C and investigated for alteration in the polymeric structure using Fourier-transform infrared spectroscopy (FTIR), circular dichroism (CD), UV-vis spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), and SDS-PAGE techniques. The increase in temperature and the presence of dilute HCl(aq.) result in a relatively greater disruption of the H-bonded structure of collagen, causing the unwinding of its triple-helical structure coupled with reduction in the helical content of polyproline type-II helices, which exposed vital amino acid residues in the regenerated material. Such an unwinding is accompanied by the formation of low molecular weight polypeptides, which are readily soluble in water and show antimicrobial activity comparable to or more than that exhibited by a model antibiotic Kanamycin towards both Gram-negative and Gram-positive bacteria. DESs are reused for at least 3 cycles for collagen solubilization without alteration in their inherent structure and collagen solubilizing ability, whereas the material regenerated from reused DESs shows properties similar to that shown by the material regenerated from virgin DESs. In this manner, a new sustainable strategy for solubilizing collagen and the direct preparation of essential and active low molecular weight collagen peptides directly from collagen in a single step is established. An inventive approach to using collagen is made possible by the observation that lower molecular weight peptides formed from the sustainable dissolution of collagen with exposed aromatic amino acid residues can demonstrate antibacterial activity.
Sustainable spotlightCollagen is one of the most important and abundant proteins in mammals, and has many important applications. The presence of H, ionic, and hydrophobic bonds as well as electrostatic interactions makes the triple-helical structure of collagen stable; therefore, collagen is considered a hard-to-dissolve polymer that limits its applications. Collagen can otherwise be dissolved in organic solvents or highly acidic media, which is not an environment-friendly option. Therefore, new environment-friendly and cheaper solvents should be used to dissolve collagen and transform it into value-added materials. Herein, the solvents used for the dissolution of collagen, i.e. deep eutectic solvents (DESs), are green, cost-effective, biodegradable, easy to prepare, and environment-friendly compared to other conventional solvents. The material regenerated from DESs after the dissolution of collagen at a relatively lower temperature (45 °C) shows properties similar to native collagen. Moreover, the material regenerated from DESs after the dissolution of collagen at 70 °C and 90 °C exhibits properties akin to gelatin and smaller molecular weight polypeptides. The obtained polypeptides exhibited enhanced antimicrobial activity against both Gram-positive and Gram-negative bacteria, which was even higher than Kanamycin (standard antibiotic). With further research, such materials may be used as biological antimicrobial agents. Further, no harmful organic solvent was used during the synthesis of DESs and thus does not pose any risk to the environment. DESs are recycled and reused, adding to the sustainability of the process. Considering the above premises, the present work certainly emphasizes the following UN sustainable development goals: good health and well-being (SDG3); affordable and clean energy (SDG7); industry, innovation, and infrastructure (SDG9); responsible consumption and production (SDG12), and establish and organize climate action (SDG13). |
Type I collagen exhibits low antigenic and high direct cell adhesion properties and has been extensively used as a biomaterial for the development of tissue engineering constructs and wound dressing systems.9,10 Partial hydrolysis of collagen yields gelatin,11–13 a valuable water-soluble protein that has received considerable attention due to its mineral binding capacity, lipid-lowering effect, immunomodulatory13 and antihypertensive response along with antioxidant13,14 and antimicrobial properties,13 and other applications that are associated with the presence of low molecular weight peptides. The various applications of collagen and its hydrolysed product, gelatin, have made collagen an interesting material to be explored further. Type I collagen is soluble in inorganic (sodium hypochlorite)15 and acts as organic solvents/acids16 but at a relatively higher temperature. The toxic nature of such solvents and the high-temperature conditions of solubilization renders the solubilization non-sustainable and limits the applications of collagen. However, it is non-soluble in water owing to its ordered structure supported by inter- and intra-molecular H-bonds,17 hydrophobic interactions, ionic interactions and van der Waal's forces.18,19 Therefore, new sustainable methods of dissolution, modification or preparation/extraction of collagen peptides/collagen hydrosylates, and subsequent regeneration need to be devised to widen the application of collagen. A detailed comparison of the method in this study with previously reported methods for dissolving collagen and preparing collagen peptides is provided in Annexure S1 (ESI),† revealing that the present method considers all the goals of “Green chemistry”.
To advance in this area, ionic liquids (ILs), which comprise only ions20–22 and are accepted as relatively greener solvents, have been tested as media for solubilizing collagen23,24 considering their ability to dissolve many hard-to-dissolve materials.25,26 The solutions of imidazolium-based ILs in acetate buffer have been found to exert stabilizing or destabilizing effects on the helical structure of type I collagen governed by the nature of ions comprising ILs.24 The lyophilized collagen (type I from calf skin) has also been reported to be soluble in concentrated aqueous solutions of imidazolium-based ILs, [C2mim][BF4] and [C2mim][Ac]; however, the maximum yield of dissolution was quite low (3.57 mg ml−1).27 There are few examples of ILs that can dissolve collagen except for 1-butyl-3-methylimidazolium chloride, [C4mim][Cl], which was found to dissolve collagen in good yield by Meng et al.19 The high chloride concentration is effectual in breaking the H-bonds and the ionic bonds in the collagen and thus leads to its dissolution similar to that observed in the case of dissolution of wood,28 cork,29 cellulose,25 wool keratin,30 and silk fibre31 in ILs. Therefore, few advancements in the dissolution of collagen utilizing ILs have been reported. More importantly, the triple helical structure of native collagen and the effectiveness of essential amino acids are noticeably destroyed during the dissolution processes.
The quest to develop new benign solvents has led to another class of widely accepted green solvents called Deep Eutectic Solvents (DESs). DESs are the eutectic mixtures of two or three molecular components (ionic or uncharged), H-bond donor (HBD), and H-bond acceptor (HBA), and they have melting points far below that of the individual components.32–34 The lowering of melting point is ascribed to the charge delocalisation via H-bond interactions of complexing agent (typically H-bond donor) with the halide anion, which in turn reduces the anion's interaction with its parent cation results in the formation of complex anionic species.35 DESs exhibit properties akin to ILs such as low volatility, recyclability, non-flammability, and excellent thermal stability32,36 but are biocompatible, easy to prepare without the use of any organic solvent in a single-step reaction, and cost-effective. This, along with the possibility of offering multiple H-bonding interactions by DESs to polymers/biopolymers,37,38 renders DESs as green solvents of choice for the dissolution and simultaneous preparation of collagen peptides from collagen under optimal conditions. A range of processing methods have been applied to produce collagen peptides or hydrolyzed collagen. However, the collagen structure is severely degraded to the molecular level during the dissolution/extraction processes, leading to a weakened performance of the obtained collagen-based materials with a low yield. What basically hinders the direct extraction of active collagen peptides is the complicated hierarchical structure, non-collagenous proteins and nucleic acid present along with the raw material. This gap drives us to directly obtain lower molecular weight collagen peptides with exposed active amino acids from natural collagen to reserve nanostructures. Although the extraction of collagen peptides from collagen has been reported in DESs,39,40 to the best of our knowledge, there is no report concerned with thorough investigations on the use of DESs for the dissolution and regeneration of collagen along with the formation of gelatin/collagen peptide to be used as antimicrobial agents.
Herein, DESs comprising (i) choline chloride (ChCl) and LA (ChCl:
LA = 1
:
1) and (ii) ethylene glycol (EG) and ZnCl2 (EG
:
ZnCl2 = 4
:
1) have been successfully established as dissolution media for type I collagen. The choice of the components of DESs is because the presence of Cl− and OH-along with the Lewis acidity of ZnCl2 has been found to aid in the dissolution of hard-to-dissolve polymers, such as cellulose,25 keratin,37,41 PET,42,43 polythene44 and wheat straw,45 when present as one of the components in ILs25 or DESs.37,41–45 LA-based DESs have also been used as a solvent for the exfoliation of biopolymers.46,47 ZnCl2 as Lewis acid is expected to activate the –C
O group of amino acids and –OH groups of EG in the presence of light. Further, Cl− of ZnCl2 could form H-bonds with collagen and is thus supposed to break the amide bonds in collagen. In the case of ChCl
:
LA, the –OH and –C
O groups of LA and Cl− of ChCl undergo the H-bonding interactions required to dissolve and degrade collagen.
Various conditions of temperature (45 °C, 70 °C and 90 °C) and the presence of 5 × 10−5 M of dilute HCl(aq.) have been tested for the dissolution and processing of collagen in DESs. A temperature increase is expected to enhance the solubilization of collagen in DESs. Besides, the presence of 5 × 10−5 M concentration of HCl(aq.) in DESs could result in the protonation of the –NH2 group of amino acid residues of collagen, which favour the dissolution of collagen by offering electrostatic repulsion between similarly charged groups. Collagen dissolved in DESs is regenerated using ethanol as an antisolvent at 4 °C. The structure and properties of the regenerated material are explored and compared with those of native collagen using various state-of-the-art techniques. Based on the conditions, the regenerated material is found to be (i) collagen and (ii) low molecular weight collagen peptides. Regenerated collagen and collagen peptides are known to exhibit remarkable antimicrobial activity against Gram-positive and Gram-negative bacteria, which is almost or even better in some cases than that shown by the antibiotic Kanamycin. Compared to ILs and other processing techniques, DESs can disassemble the original triple helical structure and preserve the nanofibrous structure with active vital aromatic amino acids. In a way, a new sustainable method for the dissolution of hard-to-dissolve biopolymer, collagen, in DESs and its transformation to low molecular weight peptides in a single step, which exhibits remarkable antimicrobial activity, is established. The current work is anticipated to offer a new platform not only for the sustainable dissolution and preparation of collagen peptides from collagen but also for the dissolution and processing of many other biologically important polymers for diverse applications.
DESs | 45 °C | 70 °C | 90 °C | ||||||
---|---|---|---|---|---|---|---|---|---|
Time (h) | Solubility (mg ml−1) | Yield (mg) | Time (h) | Solubility (mg ml−1) | Yield (mg) | Time (h) | Solubility (mg ml−1) | Yield (mg) | |
ChCl![]() ![]() |
4.0 | 95 | 55 | 3.0 | 120 | 90 | 2.0 | 150 | 100 |
EG![]() ![]() |
2.5 | 110 | 70 | 2.0 | 185 | 160 | 1.0 | 210 | 170 |
ChCl![]() ![]() |
3.0 | 120 | 90 | 2.0 | 130 | 95 | 1.0 | 195 | 157 |
EG : ZnCl2 + HCl(aq.) | 2.0 | 166 | 145 | 1.0 | 170 | 155 | 0.5 | 225 | 195 |
It is deduced that although collagen mainly preserves its structure, some disorders occur in the triple-helical structure of collagen. With an increase in dissolution temperature, the amide-A band (3287 cm−1) is broadened, where the broadening follows the order R1-(45) < R1-(70) < R1-(90) and is more in the case of R2 compared to R1 at all of the investigated temperatures (Fig. S3, ESI†). This suggests an increasing extent of disruption of the H-bonding network of collagen at higher temperatures, which is more in the case of R2 compared to R1. The ratio of the amide-III to amide-I band decreases as temperature increases (Fig. 1B) following the order R1-(45) < R1-(70) < R1-(90), which falls even below that shown by gelatin, a linear polypeptide and hydrolysis product of collagen in the case of R1-(90) and R2-(90).
This indicates the complete loss of 2°-structure and unfolding of collagen at higher temperatures, leading to the formation of random coils and other structures. The heterogeneity in the carbonyl groups and their coupling in stretching modes results in complexity in the amide-I region; thus, it is important to focus on this area to understand the different structural components of collagen. Therefore, deconvolution of the amide-I region is performed to compare the 2°-structure of native collagen and regenerated materials.55 The different structural components in native collagen, such as β-sheets, random coils and triple-helical structures, are centred around 1612 cm−1, 1630 cm−1 and 1664 cm−1, respectively, in the amide-I band.55 The change in band area and position in regenerated material represents the changes in the relative content of 2° structural components under different conditions (Fig. 1C and Table S1, ESI†). In comparison with collagen, R1-(45) shows a ∼15% increase in the content of random coils at the cost of a decrease in the content of β-sheets to a similar extent, which is relatively more in the case of R2-(45), where the content of triple-helical structures remains approximately the same. Relatively more loss in the content of β-sheets is observed in the case of R1-(70) and R2-(70). However, a drastic decrease in the content of the triple-helical structure of collagen from 75% to 40% and 27%, and an increase in the content of random coils from 15% in collagen to 45% and 55% in the case of R1-(90) and R2-(90), respectively, suggests complete disruption of the triple-helical structure of collagen at elevated temperatures. The observance of low molecular weight peptides along with the partially unfolded collagen in the case of R1-(70) and R2-(70) and the peptides only at R1-(90) and R2-(90) supports the above inference (discussed later). At different temperatures, the impact of HCl(aq.) in DESs on the properties of regenerated material is also investigated. No significant change in the characteristic properties of R1-(45)-A and R2-(45)-A is observed, as inferred from FTIR investigations (Fig. S4A, ESI†). Moreover, R1-(70)-A and R2-(70)-A are found to be partially soluble in water, whereas R1-(90)-A and R2-(90)-A are completely soluble in water similar to that observed in the case of R1-(90) and R2-(90).
FTIR spectra of water-insoluble material (from R1-(70)-A and R2-(70)-A) (Fig. S4B, ESI†) resemble those of collagen (Fig. S3A, ESI†). Furthermore, water-soluble material from R1-(70)-A and R2-(70)-A (Fig. S4B, ESI†) and (R1-(90)-A and R2-(90)-A) (Fig. S4C, ESI†), expected to be low molecular weight peptides, exhibits a relatively broadened amide-A band (3290 cm−1), red-shifted amide-III (1650 cm−1) and amide II (1550 cm−1) bands mimicking the bands shown by gelatin (Fig. S3B, ESI†). This suggests the formation of water-soluble polypeptides structurally similar to that of gelatin devoid of any 2°-structure, where the complete loss of triple-helical content is supported by the absence of three bands in the amide-III region (1200–1237 cm−1). No significant difference in the 2°-structural components between R1-(45) and R1-(45)-A is observed from the deconvolution of the amide-I band (Fig. 1D). R1-(70)-A exhibits marginal loss in the triple-helical content at the cost of a similar increase in the content of random coils compared to R1-(70), whereas a significant increase in the content of random coils and loss in the triple-helical structure is observed in the case of R2-(70)-A compared to R2-(70) (Fig. 1D). The presence of HCl(aq.) results in the unwinding of the triple-helical structure of collagen, the effect of which is more at higher temperatures, as suggested by relative values of amide III/amide I ratio (Fig. S4D, ESI†), which is in line with the observations made from deconvolution of the amide-I band (Fig. 1D).
![]() | ||
Fig. 2 (A–D) CD spectra of native collagen and the material regenerated from collagen dissolved in DESs under different conditions. |
The ellipticity of the positive band decreases with an increase in the temperature of dissolution, which almost disappears in the case of R1-(90) and R2-(90) without any discrimination towards DES used (Fig. 2A and B). This indicates the complete disruption of the “polyproline-II” helical structure via the breakage of H-bonds between proline/hydroxy-proline amino acid residues.
A single negative band peculiar to the triple-helical structure in collagen transforms into two narrowly separated bands located at ∼205 and ∼210 nm in the case of R1-(70) and R2-(70), respectively, which further shifts red accompanied by a decrease in ellipticity in the case of R1-(90) and R2-(90) (Fig. 2A and B). The band at ∼205 nm closely resembles that shown by polyproline-II56 with a helical structure, the content of which decreases with an increase in temperature as also suggested by the disappearance of the positive band ∼222 nm at higher temperatures. The appearance of the band around 210 nm, which shifts red at higher temperatures, is assigned to the formation of the β-sheet structure, which may be present in the random coils.57 Further, a red shift suggests the breaking of peptide bonds, which could result in the formation of low molecular weight polypeptides with a structure similar to random coils or unordered peptides. Many unordered polypeptides with different compositions stabilized via hydration show such features in the CD spectra.57,58 The presence of HCl(aq.) does not affect the line shape of both CD bands in the case of R1-(45)-A and R2-(45)-A to an appreciable extent compared to that of collagen (Fig. 2C and D) in line with the observations made from FTIR-spectroscopy. Water insoluble R1-(70)-A retains the shape of a negative CD band, and it shifts towards red, whereas a change is observed in the shape of the CD band in the case of water-insoluble R2-(70)-A, water-soluble R1-(70)-A and R2-(70)-A. This along with the complete loss of a positive CD band in the case of water-soluble and water-insoluble R1-(70)-A and R2-(70)-A (Fig. 2C and D) suggests that the triple-helical structure of collagen is appreciably unfolded at higher dissolution temperatures in the presence of HCl(aq.), resulting in its partial transformation to water soluble low molecular weight polypeptides displaying no characteristic band of “polyproline-II” type helices. The appearance of a symmetric negative band at 215 nm and the complete loss of a positive band at 222 nm specific to the “polyproline-II” helical structure in the case of R1-(90)-A and R2-(90)-A (Fig. 2C and D) reveals the complete disruption of the triple-helical structure of collagen and the formation of β-sheets and random coils. On comparing, it is observed that although there is disruption of the H-bonded network of amino-acid residues in both the “polyproline-II” helices and triple-helical structure in the absence of HCl(aq.). However, the presence of HCl(aq.) completely transforms the collagen into polypeptides with random coils and β-sheet structure.
![]() | ||
Fig. 3 UV-vis spectra of native collagen and the material regenerated after dissolution of collagen in (A) ChCl![]() ![]() ![]() ![]() |
Generally, the hydrolysis of collagen occurs via the breakdown of amide bonds in polypeptide chains, which results in the appearance of free –NH2 groups on the side chains of peptides, leading to a red shift in the absorption spectra. Similarly, the retention of the absorption bands in the range 240–300 nm suggests no alteration in the molecular structure of aromatic amino acid residues, whereas a red shift indicates the change in the molecular environment of these amino acid residues in regenerated material caused by disruption of the H-bonded network of collagen.
An increase in temperature results in red shift from ∼215 nm to 230 and 240 nm in the material regenerated from ChCl:
LA and EG
:
ZnCl2, respectively. Besides, an increase in hyperchromicity of the bands (∼215 nm and ∼280 nm) with an increase in temperature for both of the investigated DESs is observed. This depicts the formation of random coil structure and exposure of free –NH2 groups along with unmasking of aromatic amino acid residues (phenylalanine, tyrosine and tryptophan) upon hydrolysis, the extent of which is more in the case of R2 compared to R1.62 This observation is in line with the results obtained from FTIR studies. Further, the absorption band observed at ∼230 nm in the case of material regenerated in the presence of HCl(aq.) after dissolution at 70 °C and 90 °C resembles well the absorbance spectra of gelatin and peptides owing to prominent π–π* transitions (Fig. S5, ESI†). From the observations made from FTIR spectroscopy, CD spectroscopy and UV-vis absorption spectroscopy, it is inferred that there is a negligible effect of HCl(aq.) on the inherent structure and properties of material regenerated after dissolving collagen at 45 °C, whereas the presence of HCl(aq.) and high-temperature synergistically enhances the dissolution of collagen accompanied by the relatively greater unwinding of the triple-helical structure and “polyproline-II” helices, resulting in the formation of water-soluble polypeptides.
![]() | ||
Fig. 4 (A) X-ray diffraction pattern and (B) TGA profiles of native collagen and the material regenerated from ChCl![]() ![]() ![]() ![]() |
Thermogravimetric analysis (TGA) profiles of the native collagen and R1-(45) and R2-(45) (Fig. 4B) show the relatively lower thermal stability of regenerated material with degradation temperature, Td ∼230 °C, compared to native collagen with Td ∼250 °C. The presence of a relatively strong and greater number of H-bonds and ionic bonds in native collagen compared to those present in regenerated material offers higher thermal stability. The weight loss of native and regenerated collagen as a function of temperature is also examined. A small weight loss of ∼100 °C represents the evaporation of physiosorbed and bound water in native collagen and regenerated material (Fig. 4B). Another weight loss in the temperature ranging from 215 to 420 °C is ascribed to the degradation of side-chain groups of amino acids and thermal decomposition of polypeptide chains or higher molecular weight fractions.63 At a temperature above 420 °C, another slight weight loss occurs in the case of native collagen and R1-(45). This loss ensues from the breakdown of the residual organic components such as derivatives of carboxy-terminal crosslinked telopeptides, helical peptides, or some residues with carbonaceous or nitrogenous content, which are the degradation products of collagen and are non-volatile.64–66 A weight loss of ∼20% in the temperature ranging from 420 °C to 600 °C in the case of R2-(45) is assigned to the decomposition of highly unordered polypeptide chains originated by the partial unfolding of triple-helical structure as suggested by FTIR results. Few changes in the thermal stability of the material regenerated at higher temperatures in the presence and absence of HCl(aq.) (Fig. S8 and S9, ESI†) are observed.
![]() | ||
Fig. 5 SDS-PAGE displaying the molecular weight of the marker used, native collagen and the material regenerated after dissolution of collagen at 45 °C and 70 °C in ChCl![]() ![]() ![]() ![]() |
However, in the case of the regenerated material obtained from collagen dissolved at 90 °C, band positions at lower molecular weight with weak intensity represent the intense fragmentation of collagen molecules with the complete disruption of the triple-helical structure both in the absence and presence of HCl(aq.), leading to the formation of polypeptides in both the employed DESs.
The thermal energy offered by high temperatures naturally lowers the activation energy of dissolution via the greater breakage of H-bonds between collagen fibrils. This results in the unfolding of a triple-helical structure as suggested by FTIR, CD and UV-vis absorption measurements and consequently offers more sites for components of DESs to engage in H-bonding with collagen in a synergistic manner and thus enhances the solubility. The solubility of collagen is also observed to increase under mild acidic conditions (Table 1). HCl(aq.) is expected to aid in the breaking of amide bonds in the polypeptides comprising collagen, leading to the appearance of low molecular weight polypeptides. Excess H+ interacts with the imino of proline and hydroxyproline of collagen, resulting in the formation of ammonium salt, which further attracts more of Cl− of DESs as well as of HCl, thus weakening inter- and intra-molecular H-bonding of collagen. An increase in temperature results in greater ionization of HCl in DESs; consequently, relatively more H+ and Cl− interacts with amino acid residues of collagen, leading to the breaking of H-bonds.16 The protonation of the –NH2 group of amino acid residues of collagen favours dissolution by offering electrostatic repulsion between similarly charged groups and facilitating the breakage of the H-bonding network in collagen at the cost of establishing H-bonding with Cl− present in DESs. Comparing the effects of HCl(aq.) and temperature, it is observed that HCl(aq.) does not affect the extent of solubilization of collagen and properties of R1-(45)-A, R2-(45)-A and R1-(70)-A, whereas the effect is more noticeable in the case of R2-(70)-A. Moreover, the structural alterations caused by collagen during dissolution are found to be comparable in the case of material regenerated at 90 °C in both DESs in the presence of HCl(aq.). To ascertain the formation of low molecular weight peptides, especially at higher temperatures and in the presence of HCl(aq.) as suggested by SDS-PAGE measurements, the solubility of the recovered material was examined. It is observed that the R1-(45), R2-(45), R1-(45)-A and R2-(45)-A are not water-soluble, whereas a part of the R1-(70)-A and R2-(70)-A is found to be water soluble. However, the material regenerated after dissolution at 90 °C is spontaneously dissolved in water in both the presence and absence of HCl(aq.). This led to the conclusion that the presence of HCl(aq.) as well as a high dissolution temperature enhances the solubilization of collagen and breaks the collagen into low molecular weight peptides. This happens via the enhanced ionization of HCl(aq.), which in turn hydrolyse the collagen into water-soluble peptides with ionized amino acid residues available to be hydrated by water. During the regeneration process, DESs are washed away, and the H-bonding can be partially restored between the amino acids of collagen chains. Nevertheless, the regenerated material does not have the same amount and location of H-bonds as the native collagen. This results in variations in the structure and properties of regenerated material in comparison to native collagen.
Further, the material regenerated after dissolution of collagen in recycled DESs displays bands similar to those observed in the case of materials regenerated from virgin DESs (Fig. 6B and S13, ESI†). A similar shift in amide bands in comparison to native collagen is observed in samples obtained from recycled DESs. Furthermore, the UV-vis absorbance spectra of regenerated material from recycled DESs (Fig. 6C) are observed to have similar bands at ∼210–230 nm and ∼270–300 nm as those observed for the material regenerated from the original DESs. A similar trend is observed in recycled DESs in the presence of HCl(aq.) (Fig. S14, ESI†). A comparable bathochromic and hyperchromic shift is also observed. This demonstrates the recyclable nature of DESs, which contributes to the sustainability of the dissolution process.
Sample | Escherichia coli | Pseudomonas syringae | Bacillus subtilis | Staphylococcus aureus |
---|---|---|---|---|
a Data represent the mean ± SE of three independent biological replicates. Different letters (a–n) within the column represent values that were significantly different among different samples (Fisher LSD; p ≤ 0.05). | ||||
Buffer | 2.69 ± 0.13g | 10.41 ± 0.54i | 6.16 ± 0.28j | 4.88 ± 0.62n |
Kanamycin | 86.02 ± 1.30a | 90.58 ± 1.73a | 83.21 ± 0.56b | 91.95 ± 0.26a |
Native | 40.30 ± 0.39f | 12.54 ± 0.42h | 27.95 ± 1.03i | 13.53 ± 0.92m |
R1-(45) | 73.41 ± 0.79d | 81.80 ± 0.36c | 71.27 ± 0.58f | 85.39 ± 0.83c |
R2-(45) | 77.33 ± 1.12b | 81.12 ± 0.28c | 62.61 ± 0.21h | 87.35 ± 0.37b |
R1-(45)-A | 75.44 ± 1.13c | 86.46 ± 0.32b | 77.17 ± 0.53d | 54.33 ± 1.31l |
R2-(45)-A | 85.86 ± 1.41a | 59.23 ± 0.28f | 80.03 ± 0.59c | 76.70 ± 1.60g |
R1-(70) | 73.52 ± 1.29d | 70.09 ± 0.34e | 74.88 ± 0.39e | 66.42 ± 0.94i |
R2-(70) | 66.98 ± 1.37e | 80.09 ± 0.37c | 62.57 ± 0.20h | 56.14 ± 0.52k |
R1-(70)-A-water insoluble | 86.66 ± 1.30a | 82.89 ± 0.46c | 65.50 ± 0.50g | 77.22 ± 0.67f |
R2-(70)-A-water insoluble | 86.75 ± 0.92a | 87.51 ± 0.46b | 87.76 ± 0.47a | 91.86 ± 0.58a |
R1-(70)-A-water soluble | 85.94 ± 1.39a | 86.18 ± 0.45b | 82.60 ± 0.38b | 80.84 ± 0.65e |
R2-(70)-A-water soluble | 86.82 ± 1.45a | 86.85 ± 0.61b | 65.66 ± 0.27g | 78.15 ± 0.44f |
R1-(90) | 75.26 ± 1.83c | 71.67 ± 0.73e | 75.32 ± 0.39e | 69.27 ± 0.33h |
R1-(90) | 76.81 ± 1.82c | 50.43 ± 0.74g | 78.60 ± 0.39d | 59.58 ± 0.94j |
R1-(90)-A | 85.79 ± 1.38a | 76.97 ± 1.03d | 76.23 ± 0.51e | 82.00 ± 0.13d |
R2-(90)-A | 78.42 ± 1.07b | 86.67 ± 0.84b | 85.31 ± 0.90a | 86.88 ± 0.42b |
Such positively charged peptides undergo electrostatic interactions with the negatively charged bacterial membranes.13,50 Following this, the aromatic amino acid residues of collagen and collagen peptides are exposed towards solvent upon unfolding during dissolution, especially at high temperatures, and the presence of HCl(aq.) facilitates the anchoring of such hydrophobic amino acid residues to the hydrophobic lipid core of the bacterial membrane.71 According to the barrel-stave model, small cationic peptides adsorb on the surface of bacteria and their hydrophobic amino acid groups are embedded into the membrane, resulting in the formation of pores.72
These pores block bacterial functioning by interacting with their DNAs and RNAs. Further, the efficient antimicrobial activity shown by water soluble R1-(70)-A and R2-(70)-A compared to that of R1-(90), R2-(90), R1-(90)-A and R2-(90)-A can correlate with the decreasing number of peptides in the given fraction with an increase in temperature as evidenced by SDS PAGE as a higher number of collagen peptides are considered to exhibit better antimicrobial properties.13
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4su00122b |
This journal is © The Royal Society of Chemistry 2024 |