Enzyme-Accelerated Acid Hydrolysis of Untanned Proteinaceous Wastes from Tanning Industry

Enzyme-Accelerated Acid Hydrolysis of Untanned Proteinaceous Wastes from Tanning Industry. ABSTRACT Traditionally, tanning industry has been producing considerable amounts of solid wastes, which raises serious concerns on account of their environmental impact. Out of these, untanned raw trimmings account for about 5-7% of the total quantity of raw materials processed. This waste could be a value-added cheap source of collagen, which has numerous industrial applications if properly and scientifically utilized. This research work deals with the utilization of raw trimmings of solid waste from tanneries in the process of enzymatic hydrolysis, performed by using acetic acid and protease, in order to obtain protein hydrolysate. The hydrolysis was carried out with varying acid concentrations, acid solutions, temperatures and times. The maximum obtained protein hydrolysate was about 88% at 1.5 M acid concentration, 4% enzyme ratio, and 60 ° C.


INTRODUCTION
Tanning raw hides and skins is one of the oldest processes of our ancestors. The search for methods of conserving hides and skins started in the early Stone Age, around 8,000 BCE. Around five thousand years later, the people of Egypt and Mesopotamia are said to have invented plant-based tanning, using the bark or gum of various trees [1]. The modern tanning process involves several steps in converting raw hides and skins into imputrescible substance and also generates large quantities of solid and liquid wastes. On average, the processing of one metric ton of rawhide produces 200 kg of tanned leather, 250 kg of non-tanned waste, 200 kg of tanned waste leather, and 50,000 kg of liquid waste (Table 1). About 50% of leather mass is lost during the tanning process [2]. Table 1. Environmental input of leather processing, adapted from [3] Raw hide ( The tannery sector of Bangladesh is considered as the most polluting of industries, which is well established, and ranked fourth in terms of earning foreign exchange. The first tannery in Bangladesh was established at Narayanganj by R.P. Shaha in 1940. After that, the tannery industry was shifted to Hazaribagh, Dhaka [4]. At present all tanneries of Hazaribagh have been relocated to Savar Tannery Estate in order to protect the environment. As mentioned earlier, tanning processes generate huge amounts of liquid and solid wastes and if these wastes are not properly treated it will degrade the surrounding environment. Although tanneries have been moved to a new location, solid waste management is not properly maintained. Raw trimmings of hides and skins Solid waste in a nearby tannery's yard Raw hides and skins are by-products of meat industry. Before pre-tanning operations, the hides and skins are trimmed (for the convenience of machine operations), which generates huge amounts of un-tanned proteinaceous solid wastes. These trimmings account for about 5-7% of the total quantity of raw materials processed. If these proteinaceous materials are not utilized appropriately, that will create hazardous pollution to the environment [5]. Leather solid wastes can be processed into valuable products such as glue, gelatin, artificial fibrous leathers, and collagen for various industrial uses. A number of authors have reported about various way of turning these wastes into valuable products. Enzymes, such as papain and neutrases [6], acids, such as phosphoric acid [7], sulphuric acid [8], propionic acid [9] and hydrochloric acid [10], and alkali, such as sodium hydroxide [11], magnesium oxide, calcium oxide [8] etc. are used as hydrolyzing agents in the treatment of solid waste generated by the tanning industry. Eco-friendly ultrasound technology can also be used to accelerate the hydrolyzing process [12]. In this research work, acetic acid (CH 3 COOH) and protease enzyme are used as hydrolyzing agents in the process of extracting the protein hydrolysate from untanned raw trimmings from tanneries in Bangladesh.

Material preparation
Raw trimmings from tanneries were collected from the Savar tannery estate, Savar, Dhaka. These were first washed with an excess amount of tap water in order to remove salt, dirt, dung, blood and other impurities. Hairs were removed by the liming process, using calcium oxide (CaO), and finally unhaired trimmings were delimed by washing them several times with water. After that, they were air dried in the open. Then, the dried trimmings were cut into small pieces for hydrolysis. Acetic acid (CH 3 COOH), lime and protease enzyme were purchased from the local market. Glassware (pipettes, beakers, conical flasks, measuring cylinders, test tubes etc.) that was used was the product of Borosil/Ranken. A magnetic hotplate, a stirrer, Kjeldahl apparatus etc. were used. Acetic acid is commonly used as a souring agent in the process of making vinegar, pickled vegetables and sauce, and as a raw material for spices, in diluted concentrations (4 to 8% by mass). For this reason, acetic acid was chosen as a hydrolysis agent in this study.

Experimental procedure
The study was performed in a batch process in a series of beakers equipped with stirrers by stirring dried raw trimmings and using acetic acid in varying concentrations and at varying temperatures. The detailed experimental methodology is given in Figure 2. Hairs were removed by the liming process, using calcium oxide (CaO), and finally unhaired trimmings were delimed by washing them several times with water. After that, they were air dried in the open.
Then, the dried trimmings were cut into small pieces for hydrolysis. Acetic acid (CH 3 COOH), lime and protease enzyme were purchased from the local market. Glassware (pipettes, beakers, conical flasks, measuring cylinders, test tubes etc.) that was used was the product of Borosil/Ranken. A magnetic hotplate, a stirrer, Kjeldahl apparatus etc. were used. Acetic acid is commonly used as a souring agent in the process of making vinegar, pickled vegetables and sauce, and as a raw material for spices, in diluted concentrations (4 to 8% by mass). For this reason, acetic acid was chosen as a hydrolysis agent in this study.

Experimental procedure
The study was performed in a batch process in a series of beakers equipped with stirrers by stirring dried raw trimmings and using acetic acid in varying concentrations and at varying temperatures. The detailed experimental methodology is given in Figure 2.

Collecting raw material
Trimmings are collected from various tanning plants

Washing and dehairing
Washed with water several times and dehaired with lime

Deliming and cutting
Dehaired trimmings are delimed and cut into small fragments

Hydrolysis
Acid/enzyme hydrolysis

Effect of acid concentration:
The effect of acid concentration on the protein hydrolysis can be examined by varying the concentration of acid in the fixed acid solution at a fixed temperature. 50 gm of cleaned and dried raw trimmings was dissolved in 5 ml of 0.25M, 0.5M, 1.0M and 1.5M acetic acid solution. An extra 400 ml of distilled water was added to each and the temperature was set to 40 °C.
Effect of acid solution: 50 gm of cleaned and dried raw trimmings was put in the beaker with the optimal acid concentration and at a fixed temperature of 40 °C.

Effect of temperature:
The examination of the effect of temperature on the protein hydrolysis was carried out with the optimal acid concentration and its solution.

Effect of hydrolysis time:
The examination of the effect of hydrolysis time (hour) was carried out with the optimal acid concentration, its solution and temperature.

Enzymatic hydrolysis:
The examination of the effect of enzyme concentration was carried out with best the optimal concentration, its solution, time and temperature.

Analysis
The protein of the extracted hydrolysate was identified by the Biuret test and the protein concentration was determined by the Kjeldahl method in a Gerhardt digester (Germany).

a) Biuret test
The Biuret test is based on the ability of Cu(II) ions to form a violet-colored chelate complex with peptide bonds (-CONH-groups) in alkaline conditions. This test confirms the presence of proteins in the sample. In this test, 2 ml of extracted hydrolysate solution was taken in a dry test tube. 3 drops of 10% NaOH and 3-6 drops of 0.5% CuSO 4 were added to the sample test tube [13].

b) Kjeldahl method
The Kjeldahl method is used to determine the nitrogen content in organic and inorganic substances. For over a hundred years the Kjeldahl method has been used for the determination of nitrogen in a wide range of samples such as foods and drinks, meat, feeds, cereals and forages. It is also used for nitrogen determination in wastewaters, soils and other samples. The Kjeldahl method has three main steps ( Figure 3): digestion, distillation, and titration.  The higher the temperature, the faster the digestion can be obtained.
The speed of the digestion can be greatly enhanced by the addition of salt and catalysts. Potassium sulfate (K 2 SO 4 ) is added in order to increase the boiling point of sulfuric acid, and catalysts (e.g. CuSO 4 ) are added in order to increase the speed and the efficiency of the digestion procedure [14].
Organic nitrogen is converted into NH 4 + NH 3 is distilled Nitrogen is determined Digestion Distillation Titration

Digestion
The aim of the digestion procedure is to break all nitrogen bonds in the sample and convert all of the organically bonded nitrogen into ammonium ions (NH 4 + ), carbon dioxide and water (equation 1). During digestion, the organic material carbonizes, which can be visualized by the transformation of the sample into black foam. After that, the foam decomposes and finally a clear liquid indicates the completion of the chemical reaction. For this purpose, the sample is mixed with sulfuric acid at temperatures between 350 and 380 °C. The higher the temperature, the faster the digestion can be obtained.
The speed of the digestion can be greatly enhanced by the addition of salt and catalysts. Potassium sulfate (K 2 SO 4 ) is added in order to increase the boiling point of sulfuric acid, and catalysts (e.g. CuSO 4 ) are added in order to increase the speed and the efficiency of the digestion procedure [14].

Distillation
In the distillation step, the ammonium ions (NH 4 + ) are converted into ammonia (NH 3 ) by introducing alkali (NaOH), as showed in equation (ii).
The ammonia (NH 3 ) is captured in absorbing solution like boric acid, sulfuric acid or hydrochloric acid into the receiver vessel by means of steam distillation. Boric acid (H 3 BO 3 ) of 2-4 % concentration is commonly used for capturing the ammonia, forming solvated ammonium ion (equation iii).

Titration
The concentration of captured ammonium ions is determined by either direct titration or back titration.
Both methods indicate the ammonia is present in the distillate with a color change and allow for calculation of unknown protein concentrations. In direct titration, boric acid is used for capturing ammonia by forming ammonia-borate (NH 4 + :H 2 BO 3 -) complex, which is neutralized by the addition of sulfuric acid, producing a change in color. In back titration, sulfuric acid is used for capturing ammonia. The residual sulfuric acid (the excess that did not react with NH 3 ) is titrated with sodium hydroxide standard solution and the amount of ammonia is calculated by difference.

Working procedure
In this work, 0.5 g of hydrolysate sample was taken in a Kjeldahl flask and digested with 15 ml of concentrated sulfuric acid in the presence of a mixture of K 2 SO 4 and CuSO 4 in the ratio of 5:1 and distilled into a 4% boric acid solution. The formed borate anions were titrated with 0.05M H 2 SO 4 by a direct titration procedure which was converted to nitrogen in the sample [13]. The nitrogen content and the crude protein of the raw trimmings were determined by the following formulae [15]. Calculation of nitrogen content (w n ): (4) where W n is the nitrogen content, in grams per kilogram, of the test sample; V 1 is the volume, in milliliters, of the sulfuric acid required for the determination; V 0 is the volume, in milliliters, of the sulfuric acid required for the blank test; c is the concentration, in moles per liter, of the sulfuric acid used for the titrations; M is the molar mass, in grams per mole, of nitrogen (M 14 g/mol); m is the mass, in grams, of the test portion. Calculation of crude protein (w p ):

NUR-E-ALAM M, et al. Enzyme-Accelerated Acid Hydrolysis of Untanne
The concentration of captured ammonium ions is determined by either dir methods indicate the ammonia is present in the distillate with a color c unknown protein concentrations.
In direct titration, boric acid is used for capturing ammonia by form complex, which is neutralized by the addition of sulfuric acid, producing a ch In back titration, sulfuric acid is used for capturing ammonia. The residual s react with NH 3 ) is titrated with sodium hydroxide standard solution and th by difference.

Working procedure
In this work, 0.5 g of hydrolysate sample was taken in a Kjeldahl flask and d sulfuric acid in the presence of a mixture of K 2 SO 4 and CuSO 4 in the ratio acid solution. The formed borate anions were titrated with 0.05M H 2 SO 4 by was converted to nitrogen in the sample [13]. The nitrogen content a trimmings were determined by the following formulae [15].
Calculation of nitrogen content (w n ): where W n is the nitrogen content, in grams per kilogram, of the test sampl the sulfuric acid required for the determination; V 0 is the volume, in millilit the blank test; c is the concentration, in moles per liter, of the sulfuric ac molar mass, in grams per mole, of nitrogen (M 14 g/mol); m is the mass, in g Calculation of crude protein (w p ): or

NUR-E-ALAM M, et al. Enzyme-Accelerated Acid Hydrolysis of Untanned
The concentration of captured ammonium ions is determined by either dire methods indicate the ammonia is present in the distillate with a color ch unknown protein concentrations.
In direct titration, boric acid is used for capturing ammonia by formin complex, which is neutralized by the addition of sulfuric acid, producing a cha In back titration, sulfuric acid is used for capturing ammonia. The residual su react with NH 3 ) is titrated with sodium hydroxide standard solution and the by difference.

Working procedure
In this work, 0.5 g of hydrolysate sample was taken in a Kjeldahl flask and dig sulfuric acid in the presence of a mixture of K 2 SO 4 and CuSO 4 in the ratio o acid solution. The formed borate anions were titrated with 0.05M H 2 SO 4 by was converted to nitrogen in the sample [13]. The nitrogen content an trimmings were determined by the following formulae [15].
Calculation of nitrogen content (w n ): where W n is the nitrogen content, in grams per kilogram, of the test sample the sulfuric acid required for the determination; V 0 is the volume, in milliliter the blank test; c is the concentration, in moles per liter, of the sulfuric acid molar mass, in grams per mole, of nitrogen (M 14 g/mol); m is the mass, in gr Calculation of crude protein (w p ): where W p is the crude protein content, expressed in grams per kilogram or in or (6) where W p is the crude protein content, expressed in grams per kilogram or in percentage.

Yield
The percentage of yield can be derived from the following equation [16]: where actual yield is the amount of product obtained from hydrolysis and the theoretical yield is 68% [2].

FTIR analysis
The FTIR spectrum of the protein hydrolysate obtained from untanned raw trimmings of solid waste from tanneries in the region 500-4000 cm -1 is shown in Figure 4. Wavelength is in the 3600-3200 cm -1 region, resulting from superimposed OH and NH 3+ stretching bands. Signals at 1645 cm -1 and 1541 cm -1 correspond to the carbonyl group (C=O) and N-H, which is the evidence of protein structure [17].
Calculation of crude protein (w p ): where W p is the crude protein content, expressed in grams per kilogram or in percentage

Yield
The percentage of yield can be derived from the following equation [16]: where W p is the crude protein content, expressed in grams per kilogram or i

Yield
The percentage of yield can be derived from the following equation [16]: Protein hydrolysis of raw trimmings from tanneries by acetic acid was performed with the parameters of acid concentration, acid concentration solution, temperature, and hydrolysis time. Protein concentrations (crude protein) of the extracted hydrolysates were determined by the Kjeldahl method.

Effect of acetic acid concentration on protein hydrolysate
The effect of acetic acid concentration (M) on protein hydrolysis of raw trimmings from tanneries was carried out by hydrolyzing 50 gm of raw trimmings with various acid concentrations (M) with fixed acid solution (5ml) and at a fixed temperature (40 °C). It is shown in Figure 6 that the percentage of protein was increasing with the increase in the concentration of acetic acid from 0.25 to 1.5 molar (M). Since acetic acid is a weak acid and used as a souring agent in vinegar, pickled vegetables etc. in diluted concentrations, it is taken as a hydrolyzing agent in this study. As analytical grade acetic was used in this research, the concentration of acid was not more than 1.5M. The maximum of about 32% protein hydrolysate was extracted from 1.5M acid concentration and the yield was about 23%.

Effect of concentration solution on protein hydrolysate
The effect of acetic acid concentration solution (ml) on protein hydrolysis of raw trimmings from tanneries is shown in Figure 7. Solution of 5 ml, 10 ml, 15 ml and 20 ml of 1.5M acetic acid were taken for each 50 gm of raw trimmings. From Figure 7, it can be observed that the percentage of yield was increasing with the increase in acid solution and the maximum crude protein percentage was found with 20 ml solution of 1.5M acetic acid and its yield was about 34%. Effect of temperature on protein hydrolysate Figure 8 shows the effect of temperature on hydrolysis of tannery raw trimmings using best acid concentration (1.5M) and concentration solution (20ml) with temperature variation from 40 to 110 0 C. On heating, trimmings, mainly composed of collagen, are disintegrated and dissolved very quickly. The maximum percentage of protein hydrolysate was obtained about 58 at temperature 80 0 C and yield was about 43%. Temperature above this, the denaturation of protein was occurred and hence yield declined. As raw trimmings are biological materials, the temperature should not increase to high due to avoiding denature of protein.

Effect of enzyme concentration (%) on hydrolysis
10 ml solution of 1 to 6% enzyme on the basis of sample weight was prepared for hydrolysis at 1.5 M acetic acid concentration as shown in Figure 10. Other operating parameters, temperature and hydrolysis time, were 80 °C and 4 hours respectively. Among the various enzyme ratios, about 55% protein was extracted from 4% ratio of enzyme and the yield was about 41%. After that, the percentages were negligible.  Figure 11 shows the effect of enzyme solution on hydrolysis. After the addition of 20 ml enzyme solution, the percentage of protein, which was about 63%, was not increased significantly and the yield was about 46%.

Effect of heating time on enzymatic hydrolysis
Heating time in enzymatic hydrolysis significantly increases the protein extraction, as shown in Figure 12.
The hydrolysis was conducted with the temperature not above 50-60 °C due to enzyme activity in the 1 st stage and the final hour used for solidification of the digested sample at 80 °C in the 2 nd stage. About 22% of protein hydrolysate was obtained by increasing hydrolysis duration from 4 hours to 6 hours with 1.5 M acetic acid concentration and 20 ml of its solution. The maximum of 88% of protein hydrolysate was obtained after 12 hours of hydrolysis and the yield was about 65%.   Some pictures of protein determination by Kjeldhal method are shown in Figure 13 and 14.

APPLICATIONS OF RAW TRIMMIMGS
The collagen / gelatin extracted from untanned raw trimmings of hides and skins has huge application potential in the field of packaging, biomedicine and cosmetics according to recent studies. The gelatin extracted by acetic acid from raw trimmings was blended with polyvinyl alcohol (PVA) and used to prepare a kind of biodegradable packing material. Here, PVA was used to reduce high water-solubility of gelatin. Collagen burn-healing membrane was prepared from the collagen extracted from pig skin by enzymatic hydrolysis. Hybrid films can be prepared by using collagen extracted from the trimmed skin by acetic acid and then blending with starch/soy protein [18]. Collagen can bind large fat quantities as an emulsifier in meat products. Gelatin is playing a major role in stabilizing ice-cream and other frozen foods. The most common use of gelatin in pharmaceutical industry is covering the outer layer of capsules [19]. Tissue adhesive, vascular grafts, aortic heart valves, drug delivery matrices, wound dressing, and tissue engineering scaffold can be made from collagenous materials derived from leather trimmings [3].

CONCLUSION
Solid wastes generated by the tanning industry pose a great threat to the environment if not properly managed. Untanned raw trimmings do not get any treatment in the tanning process and they can be used as a cheap source of collagen, which has various industrial applications. In this study, the addition of enzyme to the process of hydrolysis with acetic acid accelerated the extraction of protein hydrolysate when compared to hydrolysis with acetic acid alone. About 88% of protein hydrolysate was obtained from enzyme-assisted acid hydrolysis of raw trimmings from tanneries.