Metal-Leather Protein Hydrolysate Chelates
Because leather protein hydrolysate obtained by CaO hydrolysis consists mainly of free amino acids (Jacob et al. 2016). It has been used in order to prepare metal-leather protein hydrolysate chelates (M-LPHCs). 2 mole of appropriate metal salt (CuSO4·5H2O, ZnSO4·7H2O or FeSO4·7H2O) was separately added to one mole of total free amino acids (the most appropriate ratio). One mole of total free amino acids to 2 moles of metal were chosen because this ratio was preferred (the most appropriate ratio). In this respect, a mixing of 10.0 ml of LPH with each of 1.46 g of (CuSO4·5H2O), 1.72 g of (ZnSO4·7H2O) or 0.912 g of (FeSO4·7H2O) yielded 0.86 g of Cu2+-LPHC, 0.70 g of Zn2+-LPHC or 0.90 g of Fe2+-LPHC, respectively. In this part of the present study, three different M-LPHCs were used: copper(II)-leather protein hydrolysate chelates (Cu2+-LPHC), zinc-leather protein hydrolysate chelates (Zn2+-LPHC) and iron(II)-leather protein hydrolysate chelates (Fe2+-LPHC). The characterization and biological activities of these M-LPHCs were investigated.
Characterization of Metal-Leather Protein Hydrolysate Chelates
Analytical Data and Physical Properties
Some of analytical data, including moisture% of M-LPHCs, ion% chelated with LPHs as a ligand, M-ion% in M-LPHCs and yield% of M-LPHCs, and some physical properties, including the color of M-LPHCs (whether in solid state or solution), melting point (M.P, °C) and UV-vis λ-max (nm) are summarized and presented in Table 1. Regarding to the analytical data of M-LPHCs and the yield of each chelate it can be said that moisture% of M-LPHCs was ranged between about 19 and 26%, about 1/5 or ¼ of M-LPHC. Concerning the reaction of metal salts with ligands (LPH) in order to prepare M-LPHCs, data also revealed that 23.68% of total Cu ions reacted with ligands (LPH) and formed Cu2+-LPHC, 17.59% of total Zn ions reacted with LPH and formed Zn2+-LPHC, and 22.91% of total Fe ions reacted with LPH and formed Fe2+-LPHC. In other word, 23.68%, 17.59% and 22.91% of total Cu, Zn and Fe ions were incorporated in complex ions (Cu2+-, Zn2+ and Fe2+-LPHC, respectively).
Table 1
Some of analytical data and physical properties of metal-leather protein hydrolysate chelates (M-LPHCs) prepared from LPH obtained by CaO hydrolysis (LPH/CaO).
Compound | Moisture (%) | Ion chelated (%) | M-ion (%) | Yield (%) | Color | M.P (°C) | UV-vis λ-max (nm) |
| Solid | Solid | Solid | Solid | Solid | Sol. | Solid | Sol. |
LPH/CaO | | - | - | - | Light-yellow | Light-yellow | - | 314 |
Cu2+-LPHC | 22.03 | 23.68 | 10.3 | 57.72 | Blue | Blue | 138.95 | 234 |
Zn2+-LPHC | 19.05 | 17.59 | 9.8 | 40.70 | Orange | Pale-orange | 135.50 | 214 |
Fe2+-LPHC | 26.48 | 22.91 | 8.4 | 98.68 | Pale-yellow | Light- green | 130.44 | 214 |
The chemical analysis of M-LPHCs revealed that the solid form of M-LPHCs contained a different percentage of metal ion. The percentages of metal ions in the complexes reached 10.3% in Cu-LPHC, 9.8% in Zn2-LPHC and 8.4% in Fe2-LPHC. The yield of complexation reaction percentage (dry weight of M-LPHC/salt weight × 100) was calculated and presented in Table 1. They recorded 57.72% for Cu2-LPHC, 40.70% for Zn2-LPHC and 98.68% for Fe2-LPHC. The obtained results showed that each M-LPHC has a particular color distinct for the chelate. The blue and Pale-yellow colors of Cu2-LPHC and Fe2-LPHC of course are attributed to the ions of copper(II) and ferrous or iron(II), respectively. It is not expected that the Zn2-LPHC have orange color because zinc ion is colorless. The orange color of Zn2-LPHC, whether in the form of solid state or solution, is probably due to the presence of chromium (VI) ion, where LPH contained a considerable amount of chromium ion. Chromium compounds in oxidation state (VI) has a beautifully orange color (Lennartson 2014). The melting points of M-LPHCs were almost close to each other and higher than 130°C (138.95, 135.50 and 130.44°C for Cu2-LPHC, Zn2-LPHC and Fe2-LPHC, respectively). This may be due to that the ligands in all chelates were the same (LPH).
In respect to UV-visible spectroscopy, the symmetry around the metallic ions was determined comparing the LPH and the saturated solution of metallic complexes UV-visible spectra. The electronic spectra of the complexes were recorded in water and their assignments (λ-max, wavelength of maximum absorbance in nm) were given in Table 1. One representative ligand field spectra of M-LPHCs are shown in Fig. 1 and band position is presented in Table 1. The λ-max (wavelength of maximum absorbance) of M-LPHCs was in UV-region (234 nm for Cu-LPHC, 214 nm for each Zn-LPHC and Fe-LPHC). As it's known, the peptide bond absorbs light in the range of 180 to 230 nm (which is called the "far-UV" range). The aromatic residues, tyrosine, tryptophan, and phenylalanine, also absorb light in this region and, in addition, show bands near 260 to 280 nm (in the "near-UV"). Therefore, the λ-max of metal-leather protein hydrolysate chelates may be due to the presence of aromatic amino acids and peptides in all-chelate preparations. Tripathi and Kamal (2015) mentioned the characteristic π − π* transitions are observed in the spectrum of complexes at 257, 288, and 364 nm. The electronic spectrum also exhibits a broad band at 815 nm attributable to d-d transitions, which strongly distorted the octahedral geometry around the Cu(II) ion. The absorption bands of the complexes corresponded to the n→σ*, n→π* and π→π* transitions of -NH2 and –COO. Shifts in these bands and the observed d-d transitions of the complexes indicated coordination. The UV-visible spectra of the complexes show absorption bands assigned to a large band around 634 nm. The presence of the later band mentions an octahedral stereochemistry for these complexes.
The UV-visible spectra of the complexes show absorption bands assigned to a large band around 634 nm. The presence of the later band mentions an octahedral stereochemistry for these complexes.
FTIR spectroscopy
The amino acids are existing as zwitterions in the crystalline state and predominant vibrations for free amino acid ligands are associated with υst(–NH2), υst(C = O), υs(–COO¯), υst(O–H) and υst(C–N). In their complexes, the amino acids (AAs) are generally act as bidentate ligands with respect to pH, it binds with the metal by one oxygen and one nitrogen atom. Also, the noncoordinating groups (C = O) are hydrogen-bonded with the adjacent complex or with lattice water, as well as, it forms weakly bonded with the metal containing neighboring complex. Thus, υ(–COO¯) of AA complexes are affected by each of coordination and intermolecular interactions. The major factor in determining the frequency order in AA complexes is the coordination effect. Our data indicate that, the order of the metal–oxygen interaction increased because of the more asymmetrical of –COO¯ group and the metal–oxygen interaction becomes stronger. The selected vibrations and assignments of LPH/CaO, Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC are described in Table 2.
Table 2
Some important IR bands (cm₋1) of leather protein hydrolysate chelates (LPHC) obtained by CaO hydrolysis (LPH/CaO) and its metal complexes.
Compound | υst(NH2) | υst(C = O) | υs(COO−) | υst(O–H) | υst(C–N) |
LPH/CaO | 3367.10 | 1642.09 | 1412.60 | 3075.90 | 1111.76 |
Cu2+-LPHC | 3406.64 | 1623.77 | 1451.17 | 2514.72 | 1114.65 |
Zn2+-LPHC | 3543.56 | 1624.73 | 1447.31 | 3408.57 | 1132.01 |
Fe2+-LPHC | 3406.64 | 1625.70 | 1434.78 | 2514.72 | 1116.58 |
υ: vibration, st: stretching, s: symmetric. |
The FTIR spectra (Fig. 2) of three metal leather protein hydrolysate chelates showed an absorption pattern in the 4,000–400 cm₋1 region which similar to region of AA. The predominant vibrations of the Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC are associated with υst(–NH2), υst(C = O), υs(–COO¯), υst(O–H) and υst(C–N). The vibration bands for –OH and –NH from 3300–3500 cm₋1 (peak no. 1 in Fig. 2A, B, C, D and peak no. 2 in Fig. 2C) and the possibility of this vibration due to intermolecular hydrogen bond in polypeptides, histidine and arginine. The vibration band of –NH from 3130 − 3030 cm₋1 and represented as peak No. 2 and 3, indicate the presence of free amino acids liberated from LPH (Fig. 2A). The stretching band of –NH in amino acid from 1660 − 1610 cm₋1, peak No. 4 (Fig. 2A) and peak No. 6 (Figure B, C, D). The vibration of C = O from 1550–1610 cm₋1, peak No. 5 (Fig. 2A) related to aspartic and glutamic acid. δCH2 from (1470 − 1430 cm₋1) in proline, peak No. 6 (Fig. 2A) and No. 7 (Fig. 2B, C, D). νC-OH (phenol) from 1230 − 1140 cm₋1 in tyrosine (peak no. 8 in Fig. 2C), and in secondary alcohol (theronine) from 1120 − 1100 cm₋1, peak no. 8 (Fig. 2A, B, D). δCH in glycine from 500–1000 cm₋1, peaks No. 9, 10 and 11 (Fig. 2A, B, C, D) and No. 12 (Fig. 2A, B, C) and No. 13,14 and 15 (Fig. 2A).
The observed vibrational bands of –NH2 groups around 3367.10–3543.56 cm₋1 were very sensitive to the intermolecular interaction effect in the solid state and these bands sometimes appear to be wide broad. Moreover, the Cu(II)–NH2– or Zn(II)–NH2– or Fe(II)–NH2–bond from the υ(–NH2) are strong. In comparison with the free AA, the vibration of N–H bands appears like to be shifted in direction of higher frequency of the three metal-leather protein hydrolysate chelates (Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC), with the involvement of the amine group in the complex formation. The carboxylate ion of AA coordinates to Cu(II) or Zn(II) or Fe(II) as a unidentate mode. The C = O groups of Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC have approximately the same frequency around 1623.77–1642.09 cm₋1 and the υ(CO) is metal-sensitive (Nakamato 2009).
Biological Activities of Metal-Leather Protein Hydrolysate Chelate
Antibacterial Activity
The antibacterial activities of metal-leather protein hydrolysate chelate (Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC) prepared from leather protein hydrolysate obtained by CaO hydrolysis (LPH/CaO) were investigated against isolated gram-positive strains (Bacillus cereus and Micrococcus spp.) and one standard gram-negative bacteria (Escherichia coli). According to the results given in Table 3, all metal-leather protein hydrolysate chelates had potential of antibacterial activity against bacteria species tested. The M-LPHCs were mainly active against gram-positive strains. The data indicated that the volumes of M-LPHCs tested (30, 60 and 90 µl/disc) exhibited varying levels of antibacterial activity as compared with Flumoquine (positive reference standard). Some of M-LPHCs have no antibacterial activity at low level (30 µl/disc). For example, at 30 µl/disc, Zn2+- and Fe2+-LPHC had no activities against E. coli whilst Cu2+-LPHC had no activity against Micrococcus spp. Generally, it was clear that there was a direct correlation between the volume (concentration) of M-LPHC and its inhibitory activity against bacteria species tested. The most active chelate was Zn2+-LPHC followed by Fe2+-LPHC then Cu2+-LPHC. The antibacterial activity of Flumoquine (positive reference standard) and metal-leather protein hydrolysate chelates at a higher volume (90 µl/disc) for Escherichia coli was in the following order: Flumoquine (3.54 mm) > Zn-LPHC (2.70 mm) > Fe-LPHC (1.70 mm) > Cu-LPHC (1.40 mm), while for Bacillus cereus the order was: Fe2+-LPHC (3.75 mm) > Zn2+-LPHC (3.10 mm) > Flumoquine (3.01 mm) > Cu2+-LPHC (1.75 mm) and for Micrococcus spp. the order was: Zn2+-LPHC (5.05 mm) > Cu2+-LPHC (3.50 mm) > Fe2+-LPHC (2.55 mm) > Flumoquine (1.46 mm).
Table 3
Inhibition zone (mm) of metal-leather protein hydrolysate chelates (Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC) prepared from leather protein hydrolysate obtained by CaO hydrolysis against test strains.
Hydrolysate complex | Volume (µl/disc) | Diameter of inhibition zone (mm) |
| E. coli | B. cereus | Micrococcus spp. |
Cu2+-LPHC | 30 | 1.32 | 1.00 | 0.00 |
60 | 1.40 | 1.42 | 2.15 |
90 | 1.60 | 1.75 | 3.50 |
Zn2+-LPHC | 30 | 0.00 | 1.47 | 2.65 |
60 | 1.10 | 2.10 | 3.65 |
90 | 2.70 | 3.10 | 5.05 |
Fe2+-LPHC | 30 | 0.00 | 0.40 | 0.00 |
60 | 0.85 | 1.75 | 0.50 |
90 | 1.70 | 3.75 | 2.55 |
Flumoquine (St., 30 µg/disc) | - | 3.54 | 3.01 | 1.46 |
The previous studies reported that the antibacterial activity of the amino acid complex are affected by its stability, where, the lower stability, the greater antibacterial activity. This may be due to the presence of high free ions in the solution, which enhance the interaction between the ligands and the metal ions (Stanila et al. 2007; Marcu et al. 2008). Moreover, the activity of the complex as antibacterial may be referred to presence of partially sharing between the positive charge of the metal and the ligands donor atoms which increases the lipophilic properties of the metal chelate and facilitation its movement through the phospholipid bilayers of the bacterial cell membranes. In addition to, other factors such as conductivity, solubility and dipole moment may also be the possible reasons of the antibacterial activity increasing (Chohan and Mushtaq 2000).
Application of Metal-Leather Protein Hydrolysate Chelates as Plant Growth Promoters in Hydroponic Nutrient Solution
It was suggested that the complexation of mineral ions, such as Cu, Zn and Fe, with any ligand, such as EDTA and amino acids, might improve the uptake of minerals by the plant. Accordingly, the present study was interested and designed to study the effect of metal-leather protein hydrolysate chelates (Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC) prepared from leather protein hydrolysate obtained by CaO hydrolysis (LPH/CaO) on the spinach grown in hydroponic nutrient solution. The salts of mineral ions (CuSO4, ZnSO4 and FeSO4) in the hydroponic nutrient solution were individually substituted with M-LPHCs (Cu2+-, Zn2+- or Fe2+-LPHC). The growth characteristics of the grown spinach and the content of each meaning mineral in plants (Cu, Fe and Zn) at the end of experiment (18 days) were determined.
Plant Growth Characteristics
The data on the effect of metal-leather protein hydrolysate chelates under investigation on the growth characteristics, including leaf numbers, leaf length (cm), shoot length (cm), shoot weight (g), root numbers, root length (cm) and roots weight (g), of spinach grown in hydroponic nutrient solution were summarized and illustrated in Table 4. Generally, application of M-LPHCs as sources of micronutrients (Cu, Zn and Fe) instated of mineral salts significantly increased growth characteristics of spinach plants relative to control. Application of Cu2+-LPHC and Zn2+-LPHC caused the greatest increase in growth characteristics of spinach plants compared with Fe2+-LPHC. The Zn2+-LPHC was the best to improve the most growth characteristics of spinach plants whilst the Fe-LPHC was the lowest one to improve growth characteristics. From these results it can be say that M-LPHCs had stimulating effects on spinach plants grown in hydroponic nutrient solution. The stimulating effects of M-LPHCs on the growth of plant are depending on the plant cultivar and the type of ligand (amino acid).
Minerals Content of Plants
Table 5 shows the effect of Cu2+-, Zn2+- and Fe2+-LPHC on the plant contents of copper, zinc and iron (ppm) of spinach grown in hydroponic nutrient solution. Our study results confirmed the greater efficacy of Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC in supplying Cu or Zn or Fe to spinach plants compared with that of control (CuSO4, ZnSO4 and FeSO4). As shown in Table, copper and zinc contents of plants grown in hydroponic nutrient solution supplied with Cu-LPHC or Zn-LPHC reached about 200%, or more for Zn, relative to control supplied with mineral salts (CuSO4 or ZnSO4), i.e. the uptake of Cu or Zn by plants was doubly that of control. The same was observed with Fe-LPHC where the ratio reached 126.07% relative to control. This means that LPHs as a ligand increase the uptake of micronutrients by plants. The results indicated that using of metal-leather protein hydrolysate chelates in the plant nutrition (nutrient solution) could improve growth characteristics of spinach plants (stimulating effect) and also supply sufficient amounts of minerals for plant uptake.
Table 4
Effect of metal-leather protein hydrolysate chelates (Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC) prepared from leather protein hydrolysate obtained by CaO hydrolysis on growth characteristics of spinach grown in hydroponic nutrient solution.
Compound | Leaf No. | Leaf length (cm) | Shoot length (cm) | Shoot weight (g) | Root No. | Root length (cm) | Roots weight (g) |
Mineral salts (control) | 3.0a ± 0.87 | 2.75ab ± 0.64 | 4.8a ± 0.45 | 46.6b ± 7.69 | 6.3c ± 1.041 | 6.7a ± 0.88 | 1.3b ± 0.06 |
Cu2+-LPHC | 4.7a ± 0.29 | 3.60a ± 0.21 | 5.7a ± 0.06 | 79.3a ± 6.11 | 17.0b ± 1.32 | 10.7a ± 1.46 | 2.4b ± 0.17 |
Zn2+-LPHC | 4.3a ± 0.29 | 3.63a ± 0.06 | 5.7a ± 0.50 | 82.7a ± 7.59 | 22.0a ± 1.50 | 11.2a ± 1.72 | 3.9a ± 0.22 |
Fe2+-LPHC | 3.3a ± 0.58 | 1.92b ± 0.20 | 4.5a ± 0.46 | 43.4b ± 4.22 | 16.7b ± 1.26 | 8.1a ± 0.40 | 4.1a ± 0.72 |
L.S.D | 2.105 | 1.324 | 1.544 | 24.6871 | 4.861 | 4.6426 | 1.459 |
-Values are means of three replicates ± SE. Numbers in the same column followed by the same letter are not significantly different at P < 0.05.
Table 5
Effect of copper-, zinc- and iron-leather protein hydrolysate chelates (Cu2+-LPHC, Zn2+-LPHC and Fe2+-LPHC) prepared from leather protein hydrolysate obtained by CaO hydrolysis on copper, zinc and iron contents (ppm) of spinach plant grown in hydroponic nutrient solution.
Concentration | Copper | Zinc | Iron |
CuSO4 | Cu-LPHC | ZnSO4 | Zn-LPHC | FeSO4 | Fe-LPHC |
ppm | 0.26 | 0.52 | 0.32 | 0.68 | 26.12 | 32.93 |
% | 100 | 200 | 100 | 212.5 | 100 | 126.07 |
The stimulating effect of metal-leather protein hydrolysate chelates on spinach growth could be due to the amino acids role, which exist in abundance in LPH, in improving the rate of plant growth, cell division, and/or cell development (Abdul-Qados 2009). Pervious study performed by Nassar et al. (2003) who found that the positive effect of Arg on both of bean root and shoot growth was accompanied with elevation of certain plant growth regulators levels. Our study indicated, the growth-stimulating effect of Cu2+-LPHC and Zn2+-LPHC was greater than that of Fe2+-LPHC, this may have revealed to the variation of mineral concentration used. Previous studies reported the effects of various amino acids on plant growth (Rashad et al. 2003; Svennerstam et al. 2007).
The great ability of amino acids to forming complexes with Cu or Zn or Fe increase the bioavailability of these metals for plant uptake (Zhou et al. 2007). Furthermore, stimulated plant growth by amino acids, i.e. LPH, may result in a greater ability for Cu or Zn or Fe uptake in roots. The pervious study performed by Zhang et al. (2009) reported that, the addition of amino acids to the nutrient solutions increase the uptake and translocation of Zn from root to shoot in tomato. Another study observed, the positive effect of amino acids on nutrient uptake and growth in marigold plants (Eid et al. 2011). Regarding to our obtained results, the effect of Zn2+-LPHC on shoot and root Zn accumulation varied with amino acid type. The chelators used in nutrient solutions may be transported into the plant tissue by an undeveloped casparian band at the root tip. A high concentration of chelates can remove calcium (Ca2+) from the cell membrane and impairs root membrane integrity (Vadas et al. 2007).
Uptake of Copper by In Vitro Everted Gut Sacs of Broilers
The effect of Cu2+-LPHC prepared from leather protein hydrolysate obtained by CaO hydrolysis as a source of copper, in comparison to Cu ion (CuSO4 as control) and on the absorption (bioavailability) of Cu was studied. Absorption was measured as the uptake of Cu by everted sacs. This system has been shown to be rapid and useful in predicting the trend of the absorptive response in intact animals. The absorption, in general, can take place by passive transport, involving simple diffusion, provided there is a high concentration of the nutrient outside the cell and a low concentration inside. In this system (in vitro everted gut sacs), the absorption of a nutrient (Cu) from the lumen of the intestine can take place from outside of everted gut sac with a high concentration of nutrient to inside of everted gut sac with a low nutrient concentration.
The observed Cu concentrations (ppb) inside and outside the everted ileam and absorption percentage of Cu2+-LPHC relative to control (CuSO4) by ileum sacs incubated for 80 min are reported in Table 6. The uptake percentage of Cu in the form of chelate by everted ileum sac was about 3 times that in the ionic form. From these results it can be say that LPH chelates (leather protein hydrolysate chelates) increase the bioavailability (absorption) of mineral ions, including Cu, by ileum.
Table 6
Absorption of copper-leather protein hydrolysate chelate (Cu-LPHC) prepared from leather protein hydrolysate obtained by CaO hydrolysis and copper sulphate (CuSO4) in the everted sacs of ileum after incubation time (80 min).
Compound | Cu concentration (ppb) |
Inside the everted ileum sac | Outside the everted ileum sac | Absorption% |
CuSO4 (control) | 7002.8 | 50341.3 | 12.21 |
Cu-LPHC | 20983.0 | 36361.1 | 36.59 |
From the data it could be concluded that LPH as a ligand was effective in facilitating Cu absorption. Organic Cu was more efficiently absorbed than inorganic Cu (CuSO4) under the conditions of this study. An enhancement of Cu absorption by organic ligand has also been reported (Khaled et al. 2020). The absorption of Cu from protein chelate in the separated intestinal segments of mice was 4 times that of CuSO4. These findings support the results of the current study whereas it has been proven that the uptake percentage of Cu-LPHC was about 3 times greater than in the absence of ligand. While there are many different forms of mineral supplements available, it has been demonstrated that amino acid chelates are superior in many respects. Mineral glycine chelates, for example, have been shown to be stable and bioavailable. The unique bonding characteristics of these organic minerals set the amino acid chelate in a class of its own.
Finally, the novelty of our study is, the LPH obtained by CaO (LPH/CaO) hydrolysis is considered a source of free amino acids with low cost. LPH/CaO has potential to be used as complexing agent (ligand) with metal ions (metal-LPH chelates, i.e. Cu2+, Zn2+ & Fe2+-LPHCs). Good results are obtained with M-LPHCs as a source of metal ions in plant nutrition where the synergistic effect was evident. The obtained results in the present research strongly proved that Cu2+, Zn2+ & Fe2+-LPHCs are recommended to increase the plant growth promoters of spinach plants grown under hydroponic nutrient condition. On the other hand, we claim that the cost of 100 grams of M-LPHC crystals in average of 2.5 $ while the cost of imported one liter of metal-amino acid chelates in average of 12.5 $. In conclusion, this research can help the leather industry in solving the difficult CCLW disposal problem and obtain economic benefits.