Technical Field Statement

The present invention relates to leather made from fish skin, and more particularly Nile Perch (Lates niloticus) skin leather processed as leather by an enzymatic and eco-friendly beam house method. The invention also relates to enzymatic method for loosening hairs and wool on hides or skins using alkaline protease enzyme. Generally this invention is in part a green chemistry and environmentally friendly process of manufacturing leather.

BACKGROUND OF THE INVENTION

Animal hides and skins have hair attached to them, and the hair often is removed in leather preparation processes. Similarly, fish skins have scales attached to them which are also removed in leather processing. Conventional leather processing method is associated with discharges of significant amount of environmental contaminants owing to the use of various toxic chemicals. The conventional dehairing method involves use of high proportions of lime and sulfide, which contributes to 80-90% of the total pollution load in the leather industry and generates noxious gases as well as solid wastes, e.g. hydrogen sulfide and lime (Thanikaivelan et al., 2004). Furthermore, use of lime and sodium sulfide in dehairing result in environmental pollution problems such as increased effluent toxicity, health hazards to the tannery workers and poisonous sludge which blocks sewerage pipes.

Conventional leather processing involves about 14 to 15 steps comprising soaking, liming, deliming, bating, pickling, chrome tanning, basification, rechroming, basification, neutralization, washing, retanning, dyeing, fat-liquoring and fixing. These steps are generally categorized into three main parts i.e., A) Pre-tanning, B) Tanning and C) Post tanning. Prior to pre-tanning, the raw animal skin and hides are preserved by application of salt that restrains microbial attack. Soaking rehydrate the skin and also open up the contracted fibre structure of the skin. Beam house processes (liming and reliming which takes a period of between 12 hours to 6 days) employ lime and sodium sulfide and purifies the skin matrix by the removal of hair, flesh and other unwanted materials. Deliming remove lime, bating split the fibre into fibril, degreasing remove the fat and pickling reduce the pH of the skin from 8 to 2.8-5.0 prepare the skin for subsequent tanning. Tanned skin matrix are further retanned to gain substance, fat liquored to attain required softness and dyed to preferred shades. Tanning renders permanent stability to the skin/hide. The post-tanning operations include retanning, dyeing and fat liquoring.

Pre-tanning operations uses toxic chemicals in large amount which makes leather processing industry one of the world's worst offenders with regard to environment pollution. Generally, liming-reliming process liquors contribute to 50-70% of the total biochemical oxygen demand (BOD) and chemical oxygen demand (COD) load from a tannery waste water and 15— 20% in the case of total solids (TS) load as reported by Aloy et al. 1976 (Tannery and Pollution, Centre Technique Du Cuir: Lyon, France). Natural microbial isolates may be used to produce enzymes, and these natural sources may be obtained from different environments. Extremophile like alkaliphiles bacteria exhibit the ability to grow at the extremely harsh environmental conditions such as high pH and temperature, high levels of salinity or salt, and pressure which critically influence their growth. Enzymes from Extremophiles can be utilized in leather preparation processes to improve leather product quality and reduce pollution load in the industry.

Dehairing of hide and skin is one of the most important steps which define removal of hair, fat, soluble proteins from the raw hide or skins. The traditional beam house processes or wet processing cleans the hides or skins and prepare them for further processing like tanning, retanning, fat liquoring, dyeing and finishing. The beam house process includes the steps of soaking ( dirt removal and re-hydration), dehairing (removal of hair, traditionally part of the liming process), liming (removal of hair and release of fats and proteins as well as swelling of the collagen structure), fleshing (removal of fatty tissue), splitting (horizontal cutting into grain split and flesh split), deliming (releasing lime and reducing pH), bating (removal of proteins, scut removal and fiber opening), and pickling (lowering of pH value to around 3) and tanning (stabilization of the skin or hide matrix). The product of this process is generally known as wet-blue.

Currently, lime blended with sodium sulfide is used to remove wool and hair and dissolve these into a pulp. Additionally, this process opens the fiber structure and plumps the hide due to alkalinity. The duration of the process may vary from 18 hours to 7 days depending upon the method employed. (U.S. Patent No. 20,040,006,825). Similarly, Conventional fish skin processing methods generally include tanning, drying, glazing, etc. The process of the customary pretreatment of the fish skin pelt by preservation, drying, softening in a water bath, descaling and depilating, respectively by lime treatment i.e. decomposing of the keratin by means of calcium hydroxide and sodium sulphide, deliming and scouring as well as pickling in acid and salt containing liquors and tanning in rotating tanning drums containing tanning substances such as chromium salts or tanning substances of plant origin, further washing, fat liquoring, drying, optionally coloring and mechanical treatment (finishing) results usually in soft and limp leather or too rigid leather similar to the leather of reptile skin, but not leather that is simultaneously pliable, soft and of good strength to the touch (US. Pat. No. 4,755,186). In some cases descaling of fish skin is performed in a manual operation by scraping off the scales with the aid of knives or knife-like implements. This is not only a time consuming operation but also frequently causes harm and wounds to the hands of persons scraping.

This process is responsible for the major parts of the COD load from a tannery due to use of lime and sodium sulfide. The water polluted with these chemicals and the solubilized hair leads to an increase in alkalinity, organic nitrogen, high COD (Chemical Oxygen Demand), BOD (Biological Oxygen Demand), TDS (Total Dissolved Solids) loads and the skin is also shrunken and distorted in shape. Moreover, these chemicals results in pollution due to production of hydrogen sulfide and the solid wastes with hair pulp, lime and organic matter forming sludge. The major public concern over tanneries and fish processing industries has traditionally been about odours due to generation of noxious gases, water pollution from untreated discharges and solid waste pollution.

Since the discovery of the enzymatic dehairing process considerable amount of work has been done in an effort to eliminate or reduce environmental pollution in leather processing industry. Examples of enzymatic dehairing are described in US 3,840,433, US 4,636,222, US 13/702,567, and US 5,834,299. However, no enzymes from bacteria originating from Kenyan extremophile environment have been reported so far for dehairing or descaling of skin in the absence of lime and/or lime and sulfide system. Additionally, the enzymatic dehairing and descaling process occurring without pungent smell has also not been reported yet.

Objectives of the Invention

The object of this invention is to provide a simple, rapid, economical, efficient and environmentally friendly method of processing animal hides or skins in Pre-tanning stage which employs crude alkaline protease enzyme and non-toxic chemicals from microbial activities. Another object of the present invention is to provide a method of making fish skin leather having vivid fish skin grains and unique moire patterns. Furthermore, it is the objective of this intervention to eliminate the pungent smell and reduce solid waste load which is the characteristic of many tanneries by recovering hair, wool and scales with less damage, thereby yielding by-products which are in better condition and more valuable. Yet another objective of the present invention is to provide a bio-chemical based beam-house process that provides stronger, softer and smoother leathers.

We can now disclose that the dehairing of animal hides or skins by the use of sodium sulphide or metal sulphide can be eliminated by use of crude alkaline protease enzyme prepared from microbial activities of newly isolate Bacillus Cereus Strain wwcpl. Bacillus Cereus Strain wwcpl Gen Bank accession No.KM201428 isolated from Lake Bogoria Kenya has been found especially more useful in production of crude alkaline protease enzyme which completely and efficiently dehair hide or skin and descale Nile Perch or tilapia skin without any additive.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

FIGURE 1 is a line graph representing an illustrative embodiment of the effect of temperature on dehairing of cowhide using crude alkaline protease enzyme from Bacillus Cereus Strain wwcpl at pH 12.

FIGURE 2 is a line graph of an illustrative embodiment of the thermo stability of protease profile of the crude protease enzyme from Bacillus Cereus Strain wwcpl.

FIGURE 3 is a line graph of an illustrative embodiment of the pH profile of the crude protease enzyme from Bacillus Cereus Strain wwcpl.

DETAILED DESCRIPTION

The present invention provides a new environmentally friendly method for descaling of fish skins and dehairing hides using crude alkaline proteolytic enzyme. This method can advantageously be used for dehairing skins or hides from bovine and also hides or skins from other provenance, e.g. sheep or goat or rabbit or donkeys etc. Moreover, the method can also be effectively used in descale fish skin (e.g. Nile Perch, Tilapia etc.).

Hereinafter, is an enzymatic method of processing animal hide or skin and Fish (Nile Perch Lates niloticus)) skin into leather of the present invention will be described in detail by each step. However, the same description as that of process of manufacturing general leather will not be made in detail.

Soaking

This process is for restoring the protein tissue of original raw skin state to make the hide and Nile Perch skins skin flexible and remove impurities such as dirt, salt contents, blood remains, and soluble proteins on the surface of the fish skin and halt the activities of bacteria and viruses. This also ensures economical use of the crude protease enzymes by reducing unnecessary mobile substrate. The skins are soaked in water of temperature range of between 25 to 35°C and at pH 10 for two hours. The hide and Nile perch skins are then subjected to the main soaking in an alkaline solution maintained at a pH of between pH 11-12 and temperature range of between 25 to 35°C for a period of one to five hours to open the skin fibers through osmotic swelling.

Enzymatic descaling of Nile Perch skin and dehairing of hide or skin

a) Descaling of Nile Perch skin

This process is meant to loosening scales for easy removal, dissolving interfibrillary components such as fats and soluble proteins and open up the fiber structure of the Nile Perch or tilapia skin. The Nile perch skins from process (1) are placed into a paddle or other suitably agitated tank or drum filled with a solution of the crude alkaline protease enzyme at pH 12.0 and a temperature range of between 27°C to 57°C, preferably above 37°C and most preferably at 47°C. The best results are obtained when the Nile perch skins to crude alkaline protease enzyme solution ratio is approximately 1:1 based on the weight. It should be noted that at this optimal pH, the pungent smell which is the characteristic smell in tanneries is greatly reduced or eliminated.

Crude alkaline protease enzyme at this high pH and temperature also aided in the Swelling of the fiber structure of the Nile Perch skin which also enabled the enzyme to hydrolyzing unnecessary water-soluble protein (such as collagen), greasy or fat matter between its outer layer and fibers of the skin. The natural grease and faty acids recovered can be used for commercial value. Agitation or stirring of the enzyme solution with the Nile Perch skin provides for more rapid and uniform descaling process. The loosened scales are removed by exposure to mechanical influence. Small scales under the skin are removed by rubbing the skin on each other in the opposite direction of how the scales are run: from the tail to the head. The descaled Nile Perch skin is then thoroughly washed with clean tap water to remove enzyme remnants, solubilized proteins and fats. The scales are easily recovered by filtering from the enzyme liquor before being washed thoroughly by clean water. Scales recovery from the effluent system greatly helps in reduction of sludge concentration and ultimate pollution load. Moreover, after removal of the scales, the clear enzyme liquor may be re-used alone, or in combination with fresh enzyme solution for the treatment of subsequent batches of Nile Perch skin. This process of enzymatic descaling of fish skin eliminate processes such as liming, deliming and bating which consumes and emit most of toxic chemicals in conventional leather manufacturing processes,

b) Dehairing of hide or skin

This process is meant to loosen hair or wool for easy removal, dissolving interfibrillary components such as fats and soluble proteins and open up the fiber structure of the hide or skin. The hide or skins from process (1) are placed into a paddle or other suitably agitated tank or drum filled with a solution of the crude alkaline protease enzyme at pH 12.0 and a temperature range of between 27°C to 57°C, preferably above 37°C and most preferably at 47°C. The best results are obtained when the hide or skin to enzyme ratio is approximately 1 :1 w/w based on the weight of soaked hides or skins, and crude proteolytic enzyme solution. The time of treatment will generally range from 30 minutes to 12 hours depending both on the operating pH, temperature and type of skin or hide. Crude Protease enzyme destroys the epidermis and loosens the hair or scales which is removed by exposure to mechanical influence such as rubbing the skin on each other in rotating drums. Figure.1. summarizes the effect of temperature on dehairing of hide using crude protease enzyme investigated at pH 12. For maximum activity of alkaline protease enzyme present, the pH should be maintained between 11.0 and 12.5, more preferably in the range of about 12.0 to 12.5. The pH is adjusted by the addition of alkaline substances such as NaOH, Na ₂ C0 ₃ , or other suitable buffers as desired. It should be noted that at this optimal pH, the pungent smell which is the characteristic smell in tanneries is completely eliminated. Table 1 summarizes the effect of pH on dehairing of cowhide using crude alkaline protease enzyme investigated at 37°C

A constant agitation and stirring at frequent intervals ensures uniform temperatures and pH and thus accelerating rate of uniform dehairing. The combined action of the crude alkaline protease enzyme and the agitation or mixing in rotating drums also serves to provide exposure to mechanical influence which rapidly removes the loosened hair or wool from the hide or skin. Still, with less or no agitation, the hair or wool will loosen but may remain attached to the skin. The hair or wool which is removed from the hide or skin during the enzyme treatment is in good condition and readily recovered from the enzyme liquor by decantation, filtration or other suitable separation techniques. The recovered hair or wool is in good condition suitable for further use after washing while the skin or hide is thoroughly washed and ready for pickling and tanning. Recovery of hair and wool from the effluent system greatly helps in reduction of sludge concentration and ultimate pollution load. This process of enzymatic dehairing of hide skin eliminate processes such as liming, deliming and bating which consumes and emit most of toxic chemicals in conventional leather manufacturing processes. The requisite time to carry out this process varies with temperature, pH, enzyme concentration activity and other process variables. It is apparent, however, that in the present procedure, dehairing can be accomplished in less than twelve hours. Significant dehairing can occur within one hour. Enzyme activity is an important and main factor in determining the requisite time.

1. Pickling

This process is a pre-process for tanning meant to lower the pH of the dehaired hide or skin and descaled Nile Perch skin from process (2a and 2b) from pH 10 to pH 2.8 before chrome tanning. This process is particularly important with regard to grain tightness, and improved chrome uptake, thus reducing the input of chrome. It also gives the skin appropriate pH for long term preservation, so that it can be protected from mold fungus and bacteria against deterioration.

2. Tanning

This process is for imparting thermal resistance, corrosion resistance, anti-perishability and flexibility to the hide or skin by transforming the collagen proteins of unstable structure into the property of mineral leather with the use of chrome through a cross-linking action. The tanned hides or skins are tradable intermediate products as wet-blue leather. In the present invention, the fiber structure of the Nile Perch skin and hide is transformed into the property of leather by being subjected to a reaction with chrome. After tanning, leathers are drained, rinsed by hanging up to age and subsequently sammed (brought to a uniformly semi-dry state) to reduce the moisture content before further mechanical action.

3. Retanning This process is performed to improve the wet blue leather characteristics and the re-wetting properties (e.g. the introduction of liquid, such as water, into hides, skins, Nile Perch skin or dried leather) of the hides necessary to facilitate and optimize the subsequent dyeing process. A wide variety of chemicals may be used for the re-tannage of leather, including vegetable tanning extracts, syntans, aldehydes, resins, and mineral tanning agents.

4. Dyeing

This process is for imparting basic color and flexibility to the hide, skin, Nile Perch skin and its color and flexibility are adjusted to an appropriate level and produced depending upon the demand of the markets and their situations. A wide range of dyestuff is available with different characteristics and physico-chemical resistances (e.g. to light, PVC migration, sweat migration, among others) are used. Fat liquoring is the process by which leathers are lubricated to achieve product-specific characteristics and to reestablish the fat content lost in the previous procedures. In the case of present invention, it was necessary to adjust the degree of dyeing in order to exhibit a desired pattern as naturally as possible. If dyed heavily, the moire pattern unique to the Nile Perch skin may not be represented appropriately.

5. Drying

This process is for eliminating water contents completely from the leather which has been subjected to the processes described above, with the method of natural drying or machine drying (such as samming, setting, centrifuging, vacuum drying, toggle drying, paste drying) while optimizing leather quality.

6. Splitting

In general, this process is for eliminating unnecessary tissue of raw hides or for physically adjusting the thickness of raw hides using machine. However, the splitting process that has been used only as a process for adjusting thickness in general leather products is applied as a process for creating unique novel patterns in order to make the characteristics of the fiber structure unique to Nile Perch skin and hide more distinctive in the present invention. If the dyeing dyes are adequately absorbed into the hide or Nile Perch skin during the dying process described above, then the skin is subjected to splitting process to create various and delicate color tones.

7. Finishing

Finishing operations enhance the appearance of the leather and provide the performance characteristics expected in the finished leather with respect to color, gloss, feel, flex, and adhesion as well as other properties including stretch-ability, break, light- and perspiration fastness, water vapor permeability, and water resistance. This process is for coating the hide and Nile Perch skin leather to cover up flaws and scars on the surface thereof, to make the leather suitable for long term preservation, and to make desired

Table.1 Effect of pH on enzymatic dehairing of cowhide at 37°C

8 9 10 11 12 12.5

Complete hair Complete hair Complete Complete Complete Complete removal after removal after 5 hair removal hair hair hair removal 18 hours hours after 5 hours removal removal after 3.0 after 4.5 after 3.5 hours hours hours

Pungent smell Pungent smell none none none none

EXAMPLES

Hereinafter, the constitution and the effects of the present invention will be described in detail with reference to the embodiments and exemplary tests. The present invention provides a novel process for total lime and sulfide free in dehairing hides and descaling of fish skin using crude alkaline protease enzyme from Bacillus Cereus Strain wwcpl. The following embodiments are intended only for the purpose of illustrating the invention and those of ordinary skill in the art will appreciate clearly that the embodiments will not limit the scope disclosed technology and spirit of appended claims of the present invention. That is, changes and modifications to such embodiments of the present invention will be made readily by those skilled in the art, and such changes and modifications are intended to be included within the scope of the invention.

EXAMPLE 1

Protease Isolation:

Crude alkaline protease enzyme was extracted from a bacterial culture of Bacillus Cereus Strain wwcpl and used as part of a leather processing method. Bacillus Cereus Strain wwcpl was obtained from water sample collected from Lake Bogoria, which is an alkaline lake. Location map indicates 00° 15' 00" N and 36° 06' 00" E (Using Google Earth http://earth.google.com/). Biochemical tests of Bacillus Cereus Strain wwcpl showed that it is a gram positive bacterium. The molecular identification of the isolate was based on partial sequence analysis of 16S rDNA.

Genomic deoxyribonucleic acid (DNA) was isolated from the strains by a modified Elisabeth Chachaty and Patrick Saulnier method. DNA samples were amplified and sequenced on an ABI 3730 Genetic Analyser (Applied Biosystems). The sequencing trace files were aligned into contigs with slight modifications of the default conditions using Sequeneher software version 4.6 (Gene Codes Corporation, USA). The sequence was subjected to blast analysis, and also submitted to GenBank where it was accepted under the accession number KM201428. This partial sequence showed an identity of 100% with Bacillus Cereus.

>gi|699208874|gb|KM201428.1| Bacillus cereus strain wwcpl 16S ribosomal RNA gene, partial sequence

GCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTT TGAACCGCATGGTTCG

AAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCT AGTTGGTGAGGTAAC

GGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG GGACTGAGACACGGCC

CAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCG TGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTGAGGGAAGAACAAGTG CTAGTTGAATAAGCTG

GCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCG CGGTAATACGTAGGTG

GCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGT CTGATGTGAAAGCCC ACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGG AAAGTGGAATTCCATG

TGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAAGGCGACT TTCTGGTCTGTAACTG ACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCC ACGCCGTAAACGATGA

GTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCA CTCCGCCTGGGGAGT

ACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTG GAGCATGTGGTTTAATT

CGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAG ATAGGGCTTCTCCTTC

GGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTT GGGTTAAGTCCCGCA ACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGA CTGCCGGTGACAAAC

CGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTAC ACACGTGCTACAATGG

ACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTC TCAGTTCGGATTGTAG

GCTGCAACTCGCCTACAAGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGC CGCGGTGAATACGTTC

CCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAA

The strain was maintained as a glycerol stock at -20°C. 0.5 ml of the cells of the organism Bacillus cereus Strain wwcpl were inoculated into a medium containing (g/1): (Casein, 10; Yeast extract, 1.5; (NH ₄ )S0 ₄ , 1.3; and MgCl ₂ , 0.1 (pH 10.0). The flasks were kept under shaking conditions for 36 h at 30 C. After 36 h the culture filtrate obtained was centrifuged at 13,000 g for 10 minutes to remove the biomass. The enzyme obtained was used for the evaluation of its activity on casein. This crude alkaline protease enzyme preparation was applied in enzymatic dehairing of cow hide and descaling of Nile Perch skin.

Protease Assay: Protease activity was assayed by the modified procedure (Tsuchida et al., 1986) using 1% Casein. The pH was adjusted using the following buffers (0.2 M) of acetate (pH 4.0), phosphate (pH5.0-7.0), Tris-HCl (pH 8.0), and glycine-NaOH (pH 9.0-12.0). The enzyme activity was marked over the entire temperature range of 20° C. to 90° C with maximum activity at 45° C. (Figure. 2). The enzyme depicted more than 70% activity within a range of pH from 9-12, maximum activity being at an alkaline range of 10.5 -11.5 (Figure. 3). The activity of the enzyme at alkaline pH can present an advantage from industrial application point of view since dehairing and descaling best take place in an alkaline environment.

Example 2

Bull hides Leather processing

Fresh bull hide measuring about 2.0 m by 1.8 m (21.75 square feet) with compact thickness range of 0.4 - 0.5 cm and weighing 15 kg (Figure 4) was sourced from a local slaughter house and stored in frozen state at -20 °C.

(1) Soaking

The frozen bull hide was thawed for 5 hours at room temperature to remove the solidified ice and bring it in its natural state. The hide soaked in water at 25°C and at pH 10 to remove impurities such as dirt, salt contents, blood remains, and soluble proteins on the surface of the fish skin and halt the activities of bacteria and viruses. The solution was drained and then hide subjected to the main soaking in an alkaline solution maintained at a pH of between pH 11-12 and temperature of 35°C for three hours with constant agitation and stirring at frequent intervals.

(2) Enzymatic dehairing of bull hide

The hide from process (1) were soaked hours in a bucket filled with a solution of the crude alkaline protease enzyme from Bacillus Cereus Strain wwcpl at pH 12.0 and incubated at temperature of 47°C in at orbital shaker at 150rpm. The hide weight to enzyme ratio was 1:1 based on the basis of mass. The pH was adjusted at regular interval pH to maintain optimum conditions and eliminate the pungent smell. The pH of the enzyme bath was adjusted and maintained at pH12.0 using 2M NaOH. After 4 hours, a good proportion of the hair on the hide had loosened out and could easily be removed manually by gently scraping it with a blunt knife (Figure 5). However, the hide was returned in the enzyme solution after adjusting pH for the next 8 hours to loosen all the hair including the Short hair and scud. The hair free hide (Figure 6) ready for pickling and tanning was thoroughly washed with clean tap water to remove the enzyme solution, hair, solubilized proteins and fats. The removed hair (1.5 kg) in good condition (Figure 7) was recovered by filtering the enzyme liquor before thorough washing and drying on the sun. Enzymatic dehairing eliminated processes such as liming, deliming and bating which consumes and emit most of toxic chemicals in conventional leather manufacturing processes. Table 2 summarizes the physical test results of enzymatically dehaired hide while Table 3 summarize the physical test results of the resultant leather from enzymatic dehairing after post-tanning.

(3) Pickling

Pickling was done to lower the pH of the dehaired hide to pH 2.8 in preparation for chrome tanning. The hide that had been subjected to the process (2) were soaked for 10 minutes in a solution of 25 °C prepared by mixing water and NaCl in the ratio of 150: 4.0 respectively on the basis of mass. Next, the hide was soaked for 20 minutes in a solution at 25 °C prepared by mixing water and sodium chloride, and then formic acid (HCOOH) (85%) in the ratio of 1.0 with respect to 150 of water on the basis of mass was added to the solution. Thereafter, the solution having the hide therein was left as it was for 30 minutes. Sulfuric acid (H ₂ S0 ₄ ) in the ratio of 1.3 with respect to 150 of water on the basis of mass was further added to the solution, and then left for two hours before confirm that pH had reduced to 2.8. The solution was drained.

(4) Tanning

Basic chrome (Cr ₂ 0 ₃ ) in the ratio of 3.0 with respect to 150 of water on the basis of mass were mixed, added to the hide and soaked for 60 minutes. Next, chrome in the ratio of 4.0 with respect to 150 of water on the basis of mass was further added to the solution having hide therein and left for 60 minutes. Thereafter, sodium bicarbonate (NaHC0 ₃ ) in the ratio of 0.7 with respect to 150 of water on the basis of mass was added to the solution, and the solution having the hide therein was added and stirring six times at an interval of 10 hours. Again, sodium bicarbonate in the ratio of 1.0 with respect to 150 of water on the basis of mass was added to the solution, and the solution was stirring for 10 minutes and was left for 50 minutes. Such alternate processes were repeated eight times. While repeating such processes, sodium bicarbonate was added to increase the pH to 4.0-4.3 and the increase, induces cross-linking between the chromium and the collagen. After dehydration, the hide were matured for 5 hours.

(5) Retanning and Dyeing

The hide matured by the process (4) described above was soaked for an hour in a solution of 40 °C prepared by mixing water, basic chrome and sodium formate in the ratio of 200: 4.0: 1.3 on the basis of mass. Next, the hide were left for an hour in a solution of 40 °C prepared by mixing water, sodium formate, and sodium bicarbonate (NaHC0 ₃ ) in the ratio of 150 : 2.0 : 2.5 on the basis of mass respectively. Hot water at 70 °C and synthetic oil in the ratio of 200: 140 respectively on the basis of mass were added into the solution and the solution was left as it was for an hour. Thereafter in the same bath containing hide, 200% of water and 0.2% of Baygenol Black TD 01 added to dissolve and left to run for 30 minutes. 1% granalan Sp (1:4) was dissolved with warm water and then added and left to run for 20 minutes. 3% garoval Ap (liq) was also added, run for 20 minutes before adding 6% dark mimosa powder which run for 40 minutes. The solution was drained then 50% water at 70°C through the axel added, run for 10 minutes before adding Granalan SP 4% which run for an hour. Fixation was done by adding 0.3% formic acid (10%) for 20 mins and the procedure repeated twice before draining and washing the hide with cold water for 10 minutes. The leather was folded with the grain side kept inside and the flesh outside to avoid dirt on the grain side.

(6) Drying

The hide that had been subjected to the processes described in (5) above were put on a hang to dehumidify at 60°C.

(7) Splitting

The hide that had been subjected to the processes described above was split to produce leather with uniform thickness.

(8) Finishing

Finally, hide leather with uniform thickness was completed through general finishing process (Figure 8).

Table 2 summarizes the physical test results of enzymatically dehaired hide while Table. 3 summarize the physical test results of the resultant leather from enzymatic dehairing after post-tanning.

Table. 2. Physical test results of enzymatically dehaired hide

Parameter Unit T/Method KEBS specifications Actual Remarks

Measurement (mean)

Thickness mm L.02-1 0.8-2.0 1.8 pass

Tensile strength at Mpa L.10-1 >10 11.1 pass break

Tear stress N L.13-1 35 35 pass

Elongation at break % L.10-1 10-20 12 pass

Lastometer grain mm L.l l-1 4.0 min 3 fair crack

Lastometer-burst mm L.l l-1 6 .0 min 5 fair

Fat content % 6 max 5 pass

Pressure folds % Physical/ No pressure folds None pass

Visual

Putrefactions marks Physical/ No Putrefactions marks None pass

Visual

Degree of swelling % Physical/ No Swelling None pass

Visual

Stretch /Shrinkage % Physical/ 97 98 pass

Visual

Enlarged flanks /loose Physical/ No Enlarged flanks /loose None pass grain or cracked grain Visual grain or cracked grain

Swelling and Physical/ No Swelling plumping None pass plumping Visual

Staining Physical/ No Staining None pass

Visual Hair removal % Physical/ 97 98 pass

Visual

Short hair and scud % Physical/ 97 85 fair removal Visual

Growth marks and Physical/ No Growth marks and None pass wrinkled grain Visual wrinkled grain

Recommendation: HIDE SUT fABLE FOR TANNING

Table. 3 : Physical test results of leather produced from enzymatic dehairing of hide.

Example 3

Fish skin Leather processing Fresh fish skin of Nile Perch (Lates niloticus) was sourced from a local fish processing industry in Nairobi and stored in frozen state at -20 °C.

(1) Soaking

The frozen Nile perch (Lates niloticus) skins were thawed for 2 hours at room temperature to remove the solidified ice. 8 pieces of Nile Perch skins weighing a total of 6.25 kg were soaked in water at 25 °C and at pH 10 to remove impurities such as dirt, salt contents, blood remains, soluble proteins on the surface of the fish skin and halt the activities of bacteria and viruses. The solution was drained and then Nile Perch skins (Figures 9) subjected to the main soaking in an alkaline solution maintained at a pH of between pH 11-12 and temperature of 35°C for two hours with constant agitation and stirring at frequent intervals.

(2) Enzymatic descaling of Nile Perch skin

The Nile Perch skins from process (1) were soaked for three hours in a bucket filled with a solution of the crude alkaline protease enzyme from Bacillus Cereus Strain wwcpl at pH 12.0 and incubated at a temperature of 47°C in at orbital shaker at 150rpm. The Nile Perch skin weight to enzyme ratio was 1:1 w/w based on the basis of mass. The pH was adjusted at regular interval pH to maintain optimum conditions and eliminate the pungent smell. After 3 hours, the loosened scales were removed by exposure to mechanical influence. Small scales under the skin were removed by rubbing the skin on each other in the opposite direction of how the scales are run: from the tail to the head. The scale free Nile Perch skins (Figures 10) were thoroughly washed with clean tap water to remove the enzyme solution, solubilized proteins and fats. The scales were filtered from the enzyme liquor (Figure 11) and also washed. Enzymatic descaling of fish skin eliminated processes such as liming, deliming and bating which consumes and emit most of toxic chemicals in conventional leather manufacturing processes. (3) Pickling

Pickling was done to lower the pH of the Nile Perch skin to pH 2.8 in preparation for chrome tanning. The Nile perch skins that had been subjected to the process (2) were soaked for 10 minutes in a solution of 25 °C prepared by mixing water and NaCl in the ratio of 150: 4.0 respectively on the basis of mass. Next, the Nile Perch skins were soaked for 20 minutes in a solution at 25 °C prepared by mixing water and sodium chloride, and then formic acid (HCOOH) (85%) in the ratio of 1.0 with respect to 150 of water on the basis of mass was added to the solution. Thereafter, the solution having the Nile Perch skins therein was left as it was for 30 minutes. Sulfuric acid (H ₂ S0 ₄ ) in the ratio of 1.3 with respect to 150 of water on the basis of mass was further added to the solution, and then the Nile Perch skins left two hours before confirm that pH had reduced to 2.8, then the solution was drained to obtain the skins.

(4) Tanning

Basic chrome (Cr ₂ 0 ₃ ) in the ratio of 2.7 with respect to 150 of water on the basis of mass was mixed, added and Nile perch skins soaked, for 60 minutes. Next, chrome in the ratio of 4.0 with respect to 150 of water on the basis of mass was further added to the solution having Nile perch skins therein and left for 60 minutes. Thereafter, sodium bicarbonate (NaHC0 ₃ ) in the ratio of 0.7 with respect to 150 of water on the basis of mass was added to the solution, and the solution having the Nile perch skins therein was added and stirring six times at an interval of 10 hours. Again, sodium bicarbonate in the ratio of 1.0 with respect to 150 of water on the basis of mass was added to the solution, and the solution was stirring for 10 minutes and was left for 50 minutes. Such alternate processes were repeated eight times. While repeating such processes, sodium bicarbonate was added to increase the pH to 4.0-4.3 and increase induces cross-linking between the chromium and the collagen. After dehydration, the Nile perch skins were matured for 5 hours. (5) Retanning and Dyeing

The Nile perch skins matured by the process (4) described above was soaked for an hour in a solution of 40 °C prepared by mixing water, basic chrome and sodium formate in the ratio of 200: 4.0: 1.3 on the basis of mass. Next, the Nile perch skins were left for an hour in a solution of 40 °C prepared by mixing water, sodium formate, and sodium bicarbonate (NaHC0 ₃ ) in the ratio of 150 : 2.0 : 2.5 on the basis of mass respectively. Hot water at 70 °C and synthetic oil in the ratio of 200: 140 respectively on the basis of mass were added into the solution and the solution was left as it was for an hour. Thereafter in the same bath containing Nile perch skins, 200% of water and 0.2% of Baygenol Black TD Oladded to dissolve and left to run for 30 minutes. 1% granalan Sp (1 :4) was dissolved with warm water and then add through the axel left to run for 20 minutes. 3% garoval Ap (liq) was added through the axel, run for 20 minutes before adding 6% dark mimosa powder which run for 40 minutes. The solution was drained then 50% water at 70°C through the axel added, run for 10 minutes before adding Granalan SP 4% which run for an hour. Fixation was done by adding 0.3% formic acid (10%) for 20 minutes. This procedure was repeated twice before draining. The Nile perch skins were washed with cold water for 10 minutes. The leather was folded with the grain side kept inside and the flesh outside to avoid dirt on the grain side.

(6) Drying

The Nile perch skins that had been subjected to the processes described in (5) above were put on a hang to dehumidify at 60°C.

(7) Splitting

The Nile perch skins that had been subjected to the processes described above were split to produce moire patterns unique to the Nile perch skins with uniform thickness.

(8) Finishing Finally, Nile perch skins leather with moire patterns was completed through general finishing process (Fig 12). Table. 4 summarizes Physical test results of leather produced from enzymatically descaled of Nile perch skins

Table. 4 : Physical test results of leather produced from enzymatically descaled Nile perch skins.

Industrial Applicability

The above experiments show that skins or hides can be dehaired in an environmentally friendly way without use of sodium sulfide by means of crude alkaline proteolytic enzyme from Bacillus Cereus Strain wwcpl. The above examples are model experiments. It has been found, that hides or skins dehaired/descaled by means of the method according to the invention are completely dehaired/ descaled without any grain damage whatsoever. Accordingly, it is preferred to be bound not by the specific disclosure herein, but only by the appending claims.

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Our invention can be used for other applications which are currently under investigation and we shall be filling the findings in a later date

Limitations: This invention may not work well if the conditions described in the details of the invention/Innovation are altered.