A novel Synthesis, Chemical Characterization and Biological Activities of Metal-Leather Protein Hydrolysate Chelates

Leather industries covers a wide chain of production and indirectly contributes to the economic ow. The different stages used in leather processing led to produce huge solid waste volumes. Because of the great effectiveness of amino acids as naturally chelates for minerals, the present study was carried out to recycling leather waste into its protein hydrolysate by CaO hydrolysis. The Leather protein hydrolysates (LPHs) was used to prepare metal-leather protein hydrolysate chelates (Cu 2+ -, Zn 2+ -& Fe 2+ -LPHCs) and some of their physical properties (i.e. λ-max, FTIR spectra, color, melting point) and biochemical properties as its antibacterial activity, as well as using as micronutrient elements for plant were evaluated. Results showed that the Cu 2+ -LPHC gave the highest value of melting point and λ-max than other chelates. All chelates shifted the vibration bands toward a higher frequency than LPH/CaO. Metal-leather protein hydrolysate (M-LPHCs) had antibacterial activities against E. coli, B. cereus and Micrococcus spp. mostly with Zn-LPHC and Fe-LPHC. These complexes also increased the growth characteristics and mineral absorption of spinach plants in hydroponic nutrient solution than that of mineral salts (CuSO 4 , ZnSO 4 and FeSO 4 ). Finally, the study concluded that M-LPHCs can be used as antimicrobial agent, micronutrients for plant and support the minerals bioavailability in animals. or more for Zn, relative to control supplied with mineral salts (CuSO 4 or ZnSO 4 ), 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 sucient amounts of minerals for plant uptake. and iron-leather protein hydrolysate chelates (Cu 2+ -LPHC, Zn 2+ -LPHC and Fe 2+ -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.


Introduction
As ancient and traditional industry, leather processing not only meets the social development demand, but also subsidizes to the global economy all over the world (Koppiahraj et al. 2019). The solid wastes generated from leather processing such as keratin wastes, skin trimmings, chrome shaving waste, eshing wastes and bu ng wastes categorized leather industry to become on a head of polluting industries (Jiang et al. 2016).
Leather manufacturing process are carried out in three sequencing phases; beam house operations, tannery process and nishing process (Dixit et al. 2015). It starts with the animal skin handling with series of chemical treatments named tanning process which causing in release of harmful wastes to the environment (Abdel-Shafy and Mansour 2018; Dwivedi et al. 2019). Tanning operation phase is energetic because of at this stage, the raw material from hide or skin is converted into resistant and durable leather, also it generates e uents and solid wastes. Every year, global production from solid wastes generated by leather industries was recorded as 6 million tons (Rigueto et al. 2020). These solid wastes are classi ed into four types: tanned collagen, untanned collagen, non-collagenous protein waste, non-proteinious waste (Kanagaraj et al. 2006). Tanned collagen is hydrolyzed to a mixture of peptides by different ways of hydrolysis such as alkali, acidic or enzymatic process (Ramalingam et al. 2015;Li et al. 2019). The alkaline treatment is softer than the acid one, and is more appropriate to obtain hydrolysates of industrial application. The enzymatic hydrolysis is highly appreciated because it obtains products with various functional properties (Paul et al. 2012).
Regarding to leather protein hydrolysates production with alkalis, many of previous studies focused on the recycle of the hydrolyzing chromium-containing leather waste (CCLW) to produce of peptides and amino acids for fertilizers and feeds applications. Different methods of treatment of CCLW have been developed based on hydrolysis process used such as using Ca(OH) 2 with steam or NaOH/KOH at elevated temperature and/or pressure, CaO or MgO at moderate temperatures (Haiming et al. 2009; Marcilla et al. 2011). Recently, in our previous study, we used of alkaline hydrolysis (CaO or KOH) and enzymatic hydrolysis (protease or trypsin) to produce leather protein hydrolysates (LPHs) from chromecontaining leather waste (CCLW) and evaluated their antioxidant and metal chelating activities (Jacob et al. 2016).
The amino acids and peptides are considered as natural-chelating agents, they have the ability to coordinate metal ions as Zn via their carboxyl and amino groups (Noam et al. 2020). Therefore, the present study was focused on develop a feasible method for the pollutant eliminating and recycling of the leather solid wastes by synthesis of metal-ion complexes (Cu, Zn and Fe) from leather protein hydrolysate obtained by CaO hydrolysis, as well as, estimate its physical properties and evaluate its biological activities for using as antimicrobial, plant micronutrient and mineral bioavailability supporting in animals.

Materials
Chrome-containing leather waste (CCLW), as animal waste, was obtained from a commercial leather tannery, Al Basatin, Al Maadi, Cairo. Leather was kept at 5°C in refrigerator for further analysis. Spinach (Spinacia oleracea) seeds were obtained from local market, Cairo, Egypt. Escherichia coli 0157, Micrococcus spp. and Bacillus cereus were obtained from Microbiology Laboratory, Faculty of Agriculture, Cairo University, Giza, Egypt. All chemicals used were of analytical reagent grade.

Preparation of Leather Protein Hydrolysate
Alkaline hydrolysis by CaO was applied on leather proteins to produce leather protein hydrolysate (LPH).

Alkaline Hydrolysis
Alkaline hydrolysis of CCLW was carried out according to the method described by Mu et al. (2003) as follows: Fifty grams of CCLW were shaken in 500 ml of distilled water and 5.0 g of CaO were added into digestion ask. The hydrolysis was done at 98°C for 24 h. After cooling at room temperature, clear hydrolysate was obtained by ltration. The ltered solution of protein hydrolysate was completed to 500 ml with distilled water and stored at 4°C for analysis and further studies.

Preparation of Metal Complexes Chelating with LPH as Ligands
After hydrolyzation of leather waste with CaO, 100 ml of LPH were mixed with 300 µl of concentrated phosphoric acid (85%, w/w) then centrifuged at 1968 g for 5-10 min, to remove calcium ion. Clear solution was used to prepare metal-chelate complexes with some individual metal ions (Cu 2+ , Zn 2+ and Fe 2+ ) using the method described by Jie et al. (2008) as follows. In a clean conical ask, 2 mole of appropriate metal salt (CuSO 4 ·5H 2 O, ZnSO 4 ·7H 2 O or FeSO 4 ·7H 2 O) was separately added to one mole of total free amino acids (the most appropriate ratio). The mixture was shaken for one minute and kept at room temperature for 30 minutes. Before mixing in iron chelate complex, the iron salt and 1% of ascorbic acid were added. The obtained product was concentrated by evaporation then washed with ethanol (95%). The washed products were dried in air for 12 h.

Determination of Physical Properties of Metal-ion Complexes General Appearance and Color
The metal-ion complexes obtained from LPH were examined for their appearance and color and noted down. Melting point of each metal-ion complex was measured using a differential thermal analyzer under nitrogen gas (DTA-50, Shimadzu, Japan).

Ultraviolet Visible Absorption Measurements
The Ultraviolet visible absorption of saturated solution of each metal-ion complex was manually recorded at room temperature using a T80 PG UV/VIS Spectrophotometer. For data collection, absorption was begun at 190 nm and stepped at 10 nm intervals to 700 nm. If absorption was over scale, data were recorded at 1 nm interval of wavelength.

IR Spectra
A 0.2 g sample of solid Zn 2+ -, Fe 2+ -or Cu 2+ --chelate was mixed with potassium bromide and pressed into a disc. The structural characteristics of the chelate were determined using infrared spectroscopy (JASCO FTIR 400-4000, Japan).

Assay of Antibacterial Properties
Antibacterial activity of LPH metal-chelate complexes against three different bacteria strains has been tested by disc diffusion method with some modi cations as described by Stanila et al. (2011) as follows: The culture medium was composed (%) from peptone (0.5), beef (0.3), yeast extract, agar (1.5) and sodium chloride (0.5). The pH of medium was adjusted to 6.8 with HCl or NaOH solution at 25°C. Three strains of bacteria (E. coli 0157, Micrococcus spp. and Bacillus cereus) were tested. Antibacterial tests were carried using of suspension containing 10 7 CFU/ml of bacteria, inoculated in nutrient agar. The metal-chelate complexes were sterilized by ltration through 0.45 µm Millipore lters. Sterilized lter paper discs (6 mm) were placed on the inoculated agar. Sterilized lter paper discs were individually saturated with 20, 40 or 60 µl/disc of individually saturated solution of Cu 2+ -LPHC, Zn 2+ -LPHC or Fe 2+ -LPHC with 30, 60 or 90 µl/disc. Sterilized distilled water was used as negative control. Flumequine (30 µg/disc) was used as positive reference standard to determine the sensitivity of each bacterial species tested. The plates were inoculated at 37ºC for 48 h. At the end of the period, antibacterial activity was evaluated by measuring the inhibition zones in millimetres carefully around the paper disc against the test organisms. After farther incubation at 28 ± 2ºC for 30 h, inhibition zones were measured. All inhibitory tests were performed in triplicate. An average of two independent readings for each compound was recorded.
Hydroponic-Culture Experiment Volume) for 15 min then rinsed ve times with distilled water and kept at 25ºC on wet lter paper for germination. Only seedlings with uniform size were transferred to jars under speci c conditions of 10 h light period 25/20ºC day/night temperature. All pots were covered with black polyethylene to prevent passing of light from reaching to solution and roots. Two plants were planted in each pot. All solutions were regularly daily renewed. The lengths, numbers and fresh weights of shoots and roots for each plant were recorded every day. At the experiment of (18 days after seeding), plants were harvested and divided into shoot and roots. Shoot and root dry matter yields were determined for each pot treatment. Plant parts (shoots and roots) were dried and stored for further analysis, determination of metal concentrations.

In Vitro Absorption Experiment
The experiment of everted gut sacs of broilers intestinal segments as described by Ji et al. (2008) was used as follows:

Everted Gut Sacs Procedure
One end of the sac was tied with cotton and 2.0 ml of mineral free Tris-Krebs buffer solution were put into the gut sac and the other end tied. Sacs were incubated for 80 min at 40°C (body temperature of broilers)

Statistical Analysis
Values are presented the means ± standard deviations for 6 replicates. Statistical analysis was performed using the Statically Analysis System for Windows (SAS 2008). One-way analysis of variance was performed to evaluate signi cant differences between sample means, with a signi cance level of p < 0.05. Means were compared by Duncan's test.

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 (CuSO 4 ·5H 2 O, ZnSO 4 ·7H 2 O or FeSO 4 ·7H 2 O) 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 (CuSO 4 ·5H 2 O), 1.72 g of (ZnSO 4 ·7H 2 O) or  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 eld spectra of M-LPHCs are shown in Fig. 1 and band position is presented in Table 1. 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 observed vibrational bands of -NH 2 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.   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 (Cu 2+ -LPHC,

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  could improve growth characteristics of spinach plants (stimulating effect) and also supply su cient amounts of minerals for plant uptake.  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

Uptake of Copper by In Vitro Everted Gut Sacs of Broilers
The effect of Cu 2+ -LPHC prepared from leather protein hydrolysate obtained by CaO hydrolysis as a source of copper, in comparison to Cu ion (CuSO 4 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 Cu 2+ -LPHC relative to control (CuSO 4 ) 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. From the data it could be concluded that LPH as a ligand was effective in facilitating Cu absorption. Organic Cu was more e ciently absorbed than inorganic Cu (CuSO 4 ) 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 CuSO 4 . These ndings 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 Conceptualization, methodology and writing original draft, Adel A fy: Conceptualization, investigation, reviewing and editing, Gamal Gabr: Conceptualization, methodology, investigation, reviewing and editing.
Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.
Competing Interests: The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.