Title of Invention	AN ENZYMATIC COMPOSITION FOR UNHAIRING OF HIDES AND SKIN
Abstract	Abstract An enzymatic composition which obviates the need of lime-sulphide for unhairing of hides and skins comprising enzymes such as Alkaline protease and Pancreatin 8NF along with diluents is disclosed herein. The present invention further discloses a process of unhairing of skins and hides by using the enzymatic unhairing composition. 3 FEB 2005

Full Text

FORM 2 THE PATENT ACT 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule!3) 1. TITLE OF THE INVENTION: "AN ENZYMATIC COMPOSITION FOR UNHAIRING OF HIDES AND SKIN" 2. APPLICANT (a) NAME: ADVANCED BIOCHEMICALS LIMITED (b) NATIONALITY: Indian Company incorporated under the Indian Companies ACT, 1956 (c) ADDRESS: Above Navneet Motors, Gokul Nagar, P.O. Box 182, Thane (W) 400 601 Maharashtra, India 3.PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner in which is to perform. 3-FEB-2005 GRANTED 3-2-2005 Technical field: The present invention relates to an enzymatic composition which obviates the need of lime-sulphide in the composition for unhairing of hides and skins. Further the present invention relates to a process of unhairing skins and hides by using the enzymatic unhairing composition. Background and Prior art: Chemical unhairing based on a mixture of sodium sulphide and lime is an age-old process. This process enjoys a worldwide acceptability due to its user-friendly nature. But the process requires high alkalinity and causes huge pollution. Hence, there is a need for an eco-friendly alternative. Enzymatic unhairing has been highlighted as an alternative to chemical unhairing. This process not only reduces pollution to a large extent, but also increases the area yield. However this process is not as user friendly as lime-sulfide based chemical unhairing as it increases the process time and requires greater control. Chemical unhairing is an age old, well tested process practiced by the tanners all over the world. But the process has following disadvantages • Beam house process generally accounts for 70- 80 % of the total COD of the effluent from all leather making process and out of this the lime and sulphide process alone accounts for over 80 % of the COD and suspended solids and 100 % of sulphide, • The severe alkaline environment is hazardous for the health of the workers. • The hair gets pulped and cannot be recovered and so, it goes into the effluent and significantly increases the BOD value. Further such method results in the loss of valuable protein. • Sulphide is highly toxic, has an obnoxious smell and is also difficult to handle Although it is true that the finished leather obtained from lime - sulphide treated hides/ skins shows good properties, it is well established that this conventional chemical method of dehairing creates enormous pollution problem. In fact, this has become a cause of concern for many of our society. Hence, the need for an alternative to lime-sulphide method is badly felt. WO9012118 describes an enzymatic method for unhairing of hides or skins, which comprises treating the hides or skins with an aqueous float with a pH value of 3.5 to 5, preferably 3.8 to 4.2 and containing an organic acid and a carbohydrase preparation which is able to pass the standard test in the specification as an unhairing carbohydrase. The organic acid is formic and/or acetic acid and/or oxalic acid and/or lactic acid. US3966551 describes process for the preparation of tannable pelts from animal skins or hides wherein concurrent soaking, unhairing, opening up of the hide structure, and bating are effected in a single procedural step by treatment with enzymes in an aqueous bath containing enzymes, the improvement wherein the enzymatic treating bath is free of amines and comprises dissolved therein: a. an effective amount of at least one member selected from the group consisting of a fungal protease whose optimum efficacy towards casein is at a pH above 7.0, trypsin, papain, and a bacterial protease whose optimum efficacy is at a pH between 6 and 9; b. an effective amount of a bacterial protease having an optimum efficacy against hemoglobin at a pH above 9; and c. an effective amount of thioglycolic acid or a salt thereof. The fungal protease (a) is derived from Aspergillus niger or Aspergillus flavus and the bacterial protease (b) is derived from Bacillus subtilis. US4636222 describes an enzymatic method for the unhairing of hides and skins by the use of acid proteases in the alkaline pH range, which comprises treating soaked hides and skins for a period of 12 to 36 hours at a pH range from 9 to 11, with a proteolytic enzyme having an optimum activity in the pH range of from 2 to 7.5 and unhairing, the enzyme being used at a proportion from 2 to 10 m UH6 per gram of salted or dried hides and skins, the enzyme being the sole active unhairing agent. The proteolytic enzyme is a fungal acid protease. The pH value of the liquor is adjusted with alkali, and advantageously with calcined soda, to a value in the range from pH 9 to 11, and preferably to pH 10 ± 0.5. US4457759 discloses an improved hide unhairing and limiting method resulting in an exhaust sewage of reduced hair decomposition products and sulfide ion pollution, which comprises treating hides with a proteolytic enzyme solution to loosen most of the hair present on the hides, removing the loosened hair from the hides without destroying the hair; rinsing the hides of enzyme solution; liming the hides in a sulfide liquor of relatively low strength containing not more than 2.0 percent by weight of the hides of sodium sulfide or sodium hydrogen sulfide, and mixtures thereof; and thereafter adding hydrogen peroxide to the liquor in an amount of 0.25 to 1.0 percent by weight of the hides sufficient to oxidize the sulfide ions present in the liquor. US application 2002182711 describes an enzymatic unhairing agent for use in an unhairing step in tanning for producing leather comprising an alkaline protease as an active component. The pH-adjusting agent used is a combination of calcium hydroxide and sodium hydrogen sulfide, and the pH of the solution is adjusted in the range of 10 to 12. The present invention comprises a combination of enzymes like alkaline protease selected from fungal protease and Pancreatin protease. Summary of the invention: The present invention relates to an enzymatic composition which obviates the need of lime-sulphide for unhairing of hides and skins comprising enzymes such as Alkaline protease and Pancreatin 8NF along with diluents such as Ammonium carbonate; Sodium carbonate; Tris buffer; Barol 08; Calcium chloride; Sodium hydrosulphite powder (sodium sulphhydrate); AOS powder; Dolomite; etc. The composition comprises Alkaline protease (0.4 %); Pancreatin 8NF (0.25 %); Ammonium carbonate (2%); Sodium carbonate (5 %); Tris buffer (1 %); Barol 08 (2 %); Calcium chloride (5 %); Sodium hydro sulphite powder (20 %); AOS powder (2 %); Dolomite (q.s. to make 100 %); etc. Further the present invention relates to a process of unhairing of skins and hides by using the composition. The composition is used in the unhairing process of skins and hides to give wrinkle free material. A wrinkle free material is prepared by the process of unhairing of skins and hides by using the enzymatic unhairing composition The composition reduces the pollution load and gives 5 % to 6% area increment Detailed Description: The present invention discloses an enzymatic composition which obviates the need of lime-sulphide in the composition for unhairing of hides and skins. The present invention discloses a process for unhairing which is a temperature controlled reaction and can be carried out in a drum or paddle. Percentage of feed chemical dosages was based on raw wet salted skins. The skin was continuously washed with plain water for 2 hours and soaked in 0.5 % sodium hydroxide (diluted with water 1:10 ratio) for 50 minutes. This was done in five installments with 10 minutes interval. To this was added soaking agent (SEBsoak developed by Advance biochemical ltd.) 1 % and the skin was soaked for 8 hours. Further the 0.75 % sodium carbonate was added to the mixture and the skin was soaked for 1 hr. Further the skin was left overnight in the drum. Next day the skin was further washed with plain water continuously for 30 minutes. Further a mixture of 200 % v/v water, 3 % sodium chloride, and 1.5 % borax was prepared and the skin was soaked for 30 minutes in this mixture. To this mixture, 2 % unhairing enzymatic composition, SEBlime was added. Further, (0.25 %) preservative was added in three installments of 1 hour to the mixture. The skin was soaked in this mixture for next 24 hours. The treatment was continued for the 24 hours with rotating the drum for 10 minutes in every 1 hour interval. With drum agitation all hair gets separated from the skins. The bath was drained out and skins / hides were taken for fleshing. After fleshing the material was weighed for further operation. Percentage of feed of chemical dosages in the deliming and bating process was based on fleshed weight. The skins/ hide were taken in a drum for washing for 1 hour to remove the dirt of fleshing. Deliming and bating process was carried out by using water, ammonium sulfate and SEBate alkali by conventional technique. The bath was drained out and the skin was washed for 1 hour to remove the short hair. If the hides/ skins contain lot of natural fat then unhairing process comprises degreasing by using SEBdegrease. The skin was washed with continuous water for 1 hour to remove dissolved fat and grease. Further the skin / hides were given an acid treatment (pickling) to lower the pH of the skin/ hides for chrome tanning operation. Further the skins /hides were subjected to chrome tanning for long term preservation. The cross section of the pelt for total penetration of chrome was checked and then sent for basification. The enzymatic composition of the present invention used for unhairing comprises Alkaline protease; Pancreatin 8NF; Ammonium carbonate; Sodium carbonate; Tris buffer; Barol 08; Calcium chloride; Sodium hydrosulphite powder; AOS powder; Dolomite; etc. The preferred unhairing enzymatic composition of the present invention comprises Alkaline protease (0.4 %); Pancreatin 8NF (0.25 %); Ammonium carbonate (2%); Sodium carbonate (5 %); Tris buffer (1 %); Barol 08 (2 %); Calcium chloride (5 %); Sodium hydro sulphite powder (20 %); AOS powder (2 %); Dolomite (q.s. to make 100 %); etc. Alkaline protease used in the present invention is prepared by Advanced Bio Chemicals Ltd and available as Alprolase P (7,50,000 Lvu / gm). Pancreatin 8 NF used in the present invention is prepared by Advanced Bio Chemicals Ltd and available as Pancreatin 8 NF (1,75,000 Lvu / gm). According the present invention, a typical formulation used for enzymatic unhairing is as follows: Comparative study of unhairing was carried out by using only Alkaline protease (Alprolase P) derived from Bacillus sp. and a composition comprising Alkaline protease derived from Bacillus sp. and a animal protease derived from Pancreatin (Pancreatin 8 NF). All the enzymes used in the present study are manufactured by Advanced Bio Chemicals Ltd. The alkaline protease enzyme was produced by the fermentation procedure involving a Bacillus SP. The meaning of fermentation here is growth of the Bacillus sp. on defined nutrient containing liquid medium in a specially designed vessel called as fermenter. The Bacillus sp. organism normally appears in the form of single or double rods, chain formation. The young growing culture is gram positive. Colony on agar becomes opaque with dull smooth surface. The common characteristics of the culture are 1. Growth occurs at 20 °C, 30°C, 40°C and 50°C. 2. Growth positive in presence of 2 to 7% NaCl. 3. Growth positive at pH 5.7 and 6.8. 4. Glucose, Adonitol, Arabinose, Sorbitol fermentation positive. 5. Citrate utilization, Ornithine decarboxylation positive. Lysine decarboxylation and Urease negative. The preservation of culture was done by three methods 1. Cold storage with glycerol at -70 °C. 2. Lyophilization of the culture 3. Subculturing on the slopes of maintenance media. The maintenance media contains the followings in definite proportions Beef extract; Biopeptone; NaCl; Lactose; Agar-Agar powder; etc. The pH of the media was approx. 7.0. After the growth of the bacteria in maintenance media for approximately 2 days, the culture was transferred to the Seed flask. The seed flask comprises sterilized Nitrogen source such as Soya flour, Cotton seed flour, Peptone and a Carbon source such as dextrose, lactose, Maltodextrin etc. After sterilization, the pH of the seed media was approx. 7.0 ±0.3. The culture was cultivated in shake flask at 37 ± 2°C for 40 to 96 hrs and transferred to seed vessels. The composition of the seed vessel media was same as seed flask media, and volume varies from 200 L to 1000 L volume. The initial pH of the seed vessel media was approx 7.0 ± 0.2 and temperature was 30° C. The bacterial growth from seed flask was transferred aseptically to the seed vessels. The seed vessel was rotated from 18 to 30 hrs with required agitation and aeration. After the completion of seed vessel cycle the bacterial growth was transferred to the production media. The production media comprises various growth promoting nutrients and enzyme inducing substrates suspended in water. The nutrients after suspending in the water were sterilized 'in-situ' and after cooling seed were transferred to the production vessel. The production media normally comprises Nitrogen source such as Soya flour, cotton seed flour, Corn Steep Liquor, Yeast Extract, Ammonium sulfate etc; Carbon source as dextrose, malt dextrin, glucose and sucrose; Apart from this some buffer salts, vegetable oil and antifoaming agents. The pH of the growth media was approximately 7.0 After transfer of the seed to the fermentation media the fermenter was rotated at 37 ± 2° C with agitation and aeration. During rotation if required the pH is controlled by adding sterile alkali or acid. The fermentation cycle varies from 2 to 4 days. During fermentation, cell growth, substrate utilization and enzyme activity was monitored at regular intervals. The enzyme activity was measured by Protease assay (PC) method. The activity of the enzyme was calculated by the following formula: PC/g = (Au/As) x (22/30 x W) Wherein 22 = the final volume in ml of the reaction mixture, 30 = the time of the reaction in min, and W = the weight of the original sample taken in gm The enzyme was recovered from the fermentation media as follows: The fermentation broth was filtered through plate and frame filter to separate biomass and unspent media from the enzyme containing liquid. The filtrate thus obtained was concentrated by reverse osmosis process. During concentration procedure, most of the water came out from the membranes whereas the enzyme was retained by the membrane. The concentration process also facilitated removal of other low molecular weight soluble material. The concentrated liquid was stabilized by adding suitable preservatives. The enzyme concentrate was spray dried to convert it into the powder form. The spray drying temperature was adjusted in such a manner that maximum enzyme activity was recovered. The concentrated enzyme was stored at cool and dry place before the use. The pancreatic gland was collected from bovine animal. Further it was crushed and processed to the following flow chart. The determination of activity of pancreatin enzyme was similar to that of alkaline proteases. The activity of pancreatin enzyme is expressed in LVU terms also. LVU ( known as Lohlein - Volhard Method .) LVU method comprises digesting the substrate casein by the proteolytic activity displayed by enzymatic products. During this process carboxylic groups are released which, in turn, increase solution acidity. Non-digested casein can be precipitated with hydrochloric acid and sodium sulfate, and later filtered out. Carboxylic Groups and any hydrochloric acid excess in the filtrate can be titrated with sodium hydroxide 0.1 (N). Alkali consumption during titration is considered a direct measure of enzymatic activity. A Lohlein - Volhard Unit (LV/ g) is defined as follows 1 LV/g = 0.00575 ml NaOH 0.1 (N). The Enzyme quantity in a product was estimated by applying the following formula. N° LVE/g = (V -V;) x f x 0.1 x 300 / weight (in. gr) x 1.725 The activity is expressed in Lohlein- Volhard units per gram: Carbonate of alkali metal helps to maintain a pH in the alkaline zone with the action of spent caustic powder. This operating pH at 9.2 to 9.5 is very much needed for maximum activity of the combination of proteolytic enzymes. This pH kept constant with the help of a buffering action of common salt (NaCl ) and borax which is used at time of processing. Figure I illustrates the effect of pH on activity of enzyme. X-axis (1) indicates the pH of the substrate and Y (2) indicates the enzyme activity. It was found that the enzyme is active till pH was 9.5 to 9.8 but beyond this pH 8 indicated by number 3 enzyme's activity lowered down. Enzyme catalyzed reaction is similar to other reactions in that the rate is increased by increasing temperature up to certain point. Beyond that temperature, the activity of an enzyme declines sharply as shown in figure II. As the temperature increased beyond 45 - 50° C, the rate of activity decreased. At 50° C the activity is maximum then activity decreases with increase in temperature. This decrease in activity is caused by thermal denaturation of the enzyme protein or the inactivation of a thermolabile component in the enzyme system. Figure II illustrates the effect of temperature on activity of enzyme. X-axis (4) indicates the temperature of the substrate and Y (5) indicates the enzyme activity. It was found that the enzyme activity was maximum at 45-50 °C, but it decreases beyond 45-50 °C which was indicated by number (6). It was found that enzyme was very sensitive to temperature. The diluents used in enzymatic composition of unhairing are Ammonium carbonate; Sodium carbonate; Tris buffer; Barol 08; AOS powder; Calcium chloride; Dolomite; Sodium hydro sulphite; etc. The analysis done in different steps of the drain out liquor of the present process as compared with the traditional process is given below: The discharge with the liquor varies greatly depending on processing conditions. A wrinkle free material is prepared by the process of unhairing of skins and hides by using the enzymatic unhairing composition It is evident from the table that the present enzymatic unhairing process decreases the load of pollution. The distinctive features of this enzymatic unhairing are as follows; • It substantially reduces or eliminates the pollution problems associated with sulphide. • It simplifies the pre tanning processes by eliminating the steps of batting, scudding and pickling. • Final leather shows better strength properties, flatter grain and greater surface area. • Hair / wool can be recovered in good strength. • It does not contribute to the increase in COD and BOD of the effluent like conventional method. We claim 1. An enzymatic composition for unhairing of hides and skins to obtain a wrinkle free material comprising Alkaline protease and Pancreatin 8NF along with diluents selected from Ammonium carbonate; Sodium carbonate; Tris buffer; Barol 08; Calcium chloride; Sodium hydrosulphite powder; Alpha olefin sulphonate powder and Dolomite (Calcium Magnesium Carbonate). 2. The composition as claimed in claim 1, wherein said composition comprises Alkaline protease (0.4%); Pancreatin 8NF (0.25%); Ammonium carbonate (2%); Sodium carbonate (5%); Tris buffer (1%); Barol 08 (2%); calcium chloride (5%); Sodium hydro sulphite powder(20%); Alpha olefin sulphonate powder (2%); Dolomite(q.s. to make 100%). 3. The process of unhairing of skins and hides to obtain wrinkle free material using said composition as claimed in claims 1 to 2. Dated this 3rd day of February 2005 Dr. Gopakumar G. Nair Agent for the Applicant

Documents:

110-mum-2005-abstract(03-02-2005).pdf

110-MUM-2005-ABSTRACT(3-2-2005).pdf

110-MUM-2005-ABSTRACT(GRANTED)-(8-7-2006).pdf

110-mum-2005-abstract-(3-2-2005).doc

110-mum-2005-cancelled page(08-06-2005).pdf

110-MUM-2005-CANCELLED PAGES(8-6-2006).pdf

110-mum-2005-claim(granted)-(3-2-2005).doc

110-MUM-2005-CLAIMS(COMPLETE)-(3-2-2005).pdf

110-mum-2005-claims(granted)-(03-02-2005).pdf

110-MUM-2005-CLAIMS(GRANTED)-(8-7-2006).pdf

110-mum-2005-correspondence(23-11-2005).pdf

110-MUM-2005-CORRESPONDENCE(28-3-2006).pdf

110-mum-2005-correspondence(ipo)-(10-7-2006).pdf

110-MUM-2005-CORRESPONDENCE(IPO)-(18-10-2006).pdf

110-MUM-2005-DESCRIPTION(COMPLETE)-(3-2-2005).pdf

110-MUM-2005-DESCRIPTION(GRANTED)-(8-7-2006).pdf

110-MUM-2005-DRAWING(3-2-2005).pdf

110-MUM-2005-DRAWING(GRANTED)-(8-7-2006).pdf

110-mum-2005-drawings(03-02-2005).pdf

110-mum-2005-form 1(03-02-2005).pdf

110-MUM-2005-FORM 1(24-3-2005).pdf

110-mum-2005-form 18(23-12-2005).pdf

110-MUM-2005-FORM 2(COMPLETE)-(3-2-2005).pdf

110-mum-2005-form 2(granted)-(03-2-2005).pdf

110-mum-2005-form 2(granted)-(3-2-2005).doc

110-MUM-2005-FORM 2(GRANTED)-(8-7-2006).pdf

110-MUM-2005-FORM 2(TITLE PAGE)-(COMPLETE)-(3-2-2005).pdf

110-MUM-2005-FORM 2(TITLE PAGE)-(GRANTED)-(8-7-2006).pdf

110-mum-2005-form 26(03-02-2005).pdf

110-mum-2005-form 3(03-02-2005).pdf

110-mum-2005-form 9(18-10-2005).pdf

110-MUM-2005-SPECIFICATION(AMENDED)-(8-6-2006).pdf