Title of Invention

A PROCESS FOR THE PREPARATION OF PROTEIN HYDROLYSATE FROM PROTEINACEOUS CHROME WASTES

Abstract

A process for the preparation of protein hydrolysate from proteinaceous chrome wastes obtained as by product of different industries A process for the preparation of protein hydrolysate from proteinaceous chrome wastes obtained as by product of different industries by growing Alcaligenes, having the characteristics in a chrome waste, supplimented with conventional assimilable protein in a known manner at a pH in the range of 6-10 and above till completion of hydrolysis, optionally under shaking, optionally filtering the broth by known method, treating the filtrate with a conventional chrome precipitant, removing the precipitate by known methods to get clear solution and drying the said clear solution by conventional methods to get protein hydrolysate.

Full Text

The present invention relates to a process for the preparation of protein hydrolysate from proteinaceous chrome wastes. More particularly, the present invention relates to a novel microbial process for the preparation of hydrolysate from proteinaceous chrome wastes for industrial applications. The hydrolysate of the present invention has potential use in the Leather industry as filler as well as retanning agent while manufacturing various types of leather. It may also be used as animal feed supplement. It may even find application as potential ecofriendly biofertilizer. An additional feature of the present invention is that the chrome cake, which is produced as a by-product of this process, may be reused for chrome tanning as well as retanning while manufacturing leather. As reported by Erickson (World leather, 10, 57, 1997), this hydrolysate can be used as an animal food enhancer for hogs and cattle. Unlike the conventional fillers and synthetic tanning agents, protein fillers would not impair, because of their fibrous nature, the physical properties of the resulting leathers. In fact, the proteinous materials are likely to be more compatible to leather and hence can play a major role in maintaining the natural feel of leathers, thereby providing an option to replace the expensive synthetic fillers. With the rapid industrialisation all over the globe, there has been a steady increase in the amount of proteinaceous chrome wastes, which are formed as by-products of different industries. Leather industry has been contributing to the ever increasing load of chrome tanned leather shavings, trimmings and splits, which pose a major disposal problem especially in the wake of increased awareness of environmental considerations and strict regulations. Efforts have been made by the researchers to hydrolyse these wastes chemically to get value added protein based products. Erickson (World leather, 10, 57, 1997) has reported the modification of the batch operation of heating shavings with water under alkaline conditions, by way of a continuous feed system, wherein the shavings are converted mechanically into slurry by treating with lime and then stirred in a reactor at 200°F, followed by filtration and subsequent drying of the hydrolysate. As reported by Taylor et al (Journal of the Society of Leather Technologists and Chemists, 81, 5, 1997), the shavings obtained from chrome leather have been hydrolysed by using sulphuric acid to prepare leather hydrolysate containing chromium and the same has been used not only as retanning agent and surfactants for leather making, but also as leather filler, which was prepared by copolymerizing the hydrolysate with vinyl monomers. It has also been reported by Taylor et al (Journal of American Leather Chemists Association, 88, 358, 1993) that although the acidic and basic hydrolysis of leather shavings and trimmings yield animal feed and fertiliser, they result in low molecular weight protein of low economic returns, rendering the chemical hydrolysis cost-ineffective. Moreover, considering the toxic nature of chromium especially under the newly emerging ecofriendly scenario, it is also necessary that the hydrolysate produced from the proteinaceous wastes should be free from any chromium to ensure that the same may be used as value added product. The above drawbacks have been overcome by adopting enzymatic hydrolysis. In fact, the processes of hydrolysing chrome shavings by the hitherto known techniques have mostly been based mainly on the chemical hydrolysis with or without the application of proteolytic enzymes for extraction of gelatin. As reported by Taylor et al (Journal of American Leather Chemists Association, 88, 358, 1993), the trimmings have been hydrolysed by using an alkaline protease having optimum activity at a pH in the range of 8.3-9 and a temperature of 55-65°C to obtain an almost chrome free protein product and a chromium cake that may be chemically treated and recycled. The process involves the separation of gelable protein by treating the leather shavings with non-ionic surfactant in alkaline condition over a period of 7-8 hrs, followed by protease treatment at 70-72°C over a period of 3.5 hrs. The enzymatic hydrolysis is associated with the following limitations. i) Availability of enzyme is limited. ii) Storage of enzyme requires strict maintenance of specific conditions of temperature and humidity. iii) Use of enzyme in the hydrolysis makes the process costly. iv) Specific reaction condition like temperature in the range of 60-72°C has to be maintained over a period of 3-4 hrs for performing hydrolysis, necessitating for the requirement of substantial amount of energy consumption. The above drawbacks may, to a certain extent, be overcome by using microbial source to hydrolyse the leather wastes, because of the following reasons. i) The strain of the microorganism can be cultured as and when required to ensure that the supply of the enzyme is abundant. ii) Since the enzyme can be extracted from the microorganism as and when required.is not necessary to store the enzyme, thereby avoiding not only the maintenance of critical parameters necessary for different enzymes, but also the dependence on shelf life of the concerned enzyme. iii) Since the strain of an microorganism can be stored for using the same at any time, it is not necessary to procure enzyme, which is per se costly. iv) Contrast to the enzymatic hydrolysis, maintenance of temperature in microbial hydrolysis is lower, thereby suggesting reduction in the cost of investment on thermal energy. It is also very important that the microbial source should be effective in hydrolysing protein in presence of chromium. No prior art is available on the hydrolysis of protein by any hitherto known microbial source in presence of chromium. Since investigations on the application of microbes for hydrolysis of these leather wastes have rather been scanty, there is a wide scope to explore this front to provide better result. Our copending patent application no. 1122/DEL/1999 has disclosed to have isolated a new bacterial strain of Alcaligenes odorans, designated as BL-11 and deposited in the Central Leather Research Institute ( CLRI), Chennai, for producing extracellular alkaline protease. The main objective of the present invention is to provide a process for the preparation of protein hydrolysate from proteinaceous chrome wastes, which obviates the drawbacks stated above. Another objective of the present invention is to provide a process to hydrolyse proteinaceous wastes in the presence of chromium. Yet another objective of the present invention is to provide a process for hydrolysing proteinaceous chrome wastes wherein the reuse of chromium is ensured, so that the same does not pose any environmental problem. Still another objective of the present invention is to use the strain of genus Alcaligenes having characteristics, as mentioned in our copending patent application no.1122/Del/99 for hydrolysing the proteinaceous chrome wastes. Yet another objective of the present invention is to provide a ecofriendly as well as cost effective method to prepare hydrolysate from proteinaceous chrome wastes. Still another objective of the present invention is to utilise the proteinaceous chrome wastes for the benefit of mankind. Accordingly the present invention provides a process for the preparation of protein hydrolysate from proteinaceous chrome wastes obtained as by product of different industries which comprises: i) growing Alcaligenes, having the characteristics, as herein described, in a chrome waste, supplimented with conventional assimilable protein in a known manner at a pH in the range of 6-10 and above till completion of hydrolysis, optionally under shaking, ii) optionally filtering the broth, as formed in step (i), by known method, iii) treating the filtrate, as obtained in step (ii), with a conventional chrome precipitant, iv) removing the precipitate by known methods to get clear solution and drying the said clear solution by conventional methods to get protein hydrolysate. In an embodiment of the present invention, the sources of assimilable protein used may be such as peptone, skimmed milk powder, yeast extract, soyabean meal, either individually or in combination. In another embodiment of the present invention, the amount of assimilable protein may be in the range of 0.1-2% w/v. In yet another embodiment of the present invention, the chrome precipitants used may be calcium oxide, magnesium oxide, sodium carbonate, sodium hydroxide, either individually or in combination. In still another embodiment of the present invention, the amount of chrome precipitant used may be maximum l%w/v. In yet another embodiment of the present invention, pH of the medium may be in the range of 6-10. In still another embodiment of the present invention, proteinaceous chrome wastes used may be such as chrome shavings, chrome leather trimmings, chrome leather buffing dust. In yet another embodiment of the present invention, the amount of the proteinaceous chrome wastes used may be upto 20% w/v. In still another embodiment of the present invention, the cell count of the isolate of genus Alcaligem in the inoculum may be in the range of 104-109 per ml. In yet another embodiment of the present invention, the amount of inoculum added may be in the range of 0.5 to 2% v/v, of the medium. In still another embodiment of the present invention, the amount of chrome precipitant added may be in the range of 0.4 -l%w/v, on the basis of filtrate. In yet another embodiment of the present invention, the shaking may be carried out at 100-320rpm. In still another embodiment of the present invention, the mesh size for the filtering device may be in the range of 0.2-0.3 mm. In yet another embodiment of the present invention, the drying methods may be such as water bath boiling, lyophilisation, sun drying, spray drying, vacuum drying, either individually or in combination. 0.1-2% w/v, of a protein substrate is dissolved in distilled water in the absence or presence of maximum 1% w/v, of a chrome precipitant on the protein medium., while ensuring that the pH of the solution is in the range of 6-10. An amount of maximum of 20%w/v, of proteinaceous chrome wastes is added to the above solution at 28-3 7°C. The resulting suspension is sterilised by conventional method and is then allowed to cool down to a temperature in the range of 28-3 7°C. An inoculum of cell count in the range of 104 to 109per ml is prepared separately from the selected isolate of the new strain of genus Alcaligenes, having characteristics as mentioned in our copending patent application no. NF94/99, deposited in the Central Leather Research Institute, Chennai and available with the accession no. BL-11. The sterilised suspension is then inoculated with. 5 to 2% v/v, of protein medium of this inoculum and the resulting inoculated suspension is incubated at 28-37°C in a shaker running at 100 to 320 rpm. After a period of 2-7 days, the whole mass is filtered by conventional method using a filtering device having mesh size in the range of 0.2-0.3 mm and the filtrate is heated with 0.4-1% wv, of chrome precipitant on the filtrate volume, for 15-45 min at a temperature in the range of 70-100°C. The resulting suspension is again filtered by conventional method using similar filtering mesh and the filtrate is concentrated, as an optional step, at -20 to 100°C by known method. The novelty of the present invention lies in identifying a microbial source capable of hydrolysing proteinous materials even in the presence of chromium and also in providing a process to hydrolyse proteinaceous wastes containing chromium, which would otherwise have created environmental problem, thereby not only solving a major problem of waste disposal, but also suggesting an ecofriendly option of creating wealth out of waste. The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the invention. Example I 2.5 gms of peptone and 10 gms of calcium oxide were taken in a Hofflcins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 7.5. Then 250 gms of dried chrome shavings were added to the mixture while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 109 cells per ml. 12.5 ml of the inoculum containing 1 x 109 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 28°C in a rotary shaker running at 200 rpm. After a period of 5 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of solid calcium oxide was added to it with continuous shaking and the mixture was heated at 70°C for 30 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was concentrated by boiling off the surplus water using a steam jacket vessel for 4 hrs. The resulting product obtained in the form of paste was stored in plastic container at 10°C. Example II 25 gms of peptone and 10 gms of magnesium oxide were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 7. 500gms of dried chrome shavings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRJ, was precultured in peptone broth to prepare an inoculum containing 1 x 107 cells per ml. 25 ml of the inoculum containing 1 x 107 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 37°C in a rotary shaker running at 300 rpm. After a period of 2 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of magnesium oxide was added to it with continuous shaking and the mixture was heated at 100°C for 30 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was lyophilised.The resulting powder of protein hydrolysate was stored in an air tight polythene cover. Example III 20 gms of peptone and 30 gms of skimmed milk powder were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 6. 250gms of dried leather trimmings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 104 cells per ml. 50 ml of the inoculum containing 1 x 104 cells per ml of precultured cells were added to the above sterilised leather trimmings suspension and the flask was incubated at 30°C in a rotary shaker running at 100 rpm. After a period of 7 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 5 gms of each of solid calcium oxide and magnesium oxide were added to it with continuous shaking and the mixture was heated at 70°C for 45 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was concentrated by boiling off the surplus water using a steam jacket vessel for 2hrs. The resulting paste was stored in a polythene container at 10°C. Example IV 5 gms of peptone, 5gms of skimmed milk powder, 5 gms of calcium oxide and 5 gms of magnesium oxide were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 7.3. 100 gms of dried chrome buffings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 107 cells per ml. 25 ml of the inoculum containing 1 x 107 cells per ml of precultured cells were added to the above sterilised leather buffings suspension and the flask was incubated at 30°C in a rotary shaker running at 300 rpm. After a period of 2 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of solid calcium oxide was added to it with continuous shaking and the mixture was heated at 100°C for 15 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was concentrated by boiling off the surplus water using a steam jacket vessel for 2.5 hrs. The resulting paste was spray dried for 1 hour and the powder, formed thereby, was stored in an air tight polythene cover. Example V 5 gms of peptone, 5 gms of soya bean meal and 10 gms of calcium oxide were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 7.5. 250 gms of dried chrome shavings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 109 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 28°C in a rotary shaker running at 320 rpm. After a period of 5 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of sodium carbonate was added to it with continuous shaking and the mixture was heated at 100°C for 30 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was vacuum dried to get protein hudrolysate powder,which was stored in an air-tight polythene cover. Example VI 5 gms of peptone, 5 gms of yeast extract and 5 gms of calcium oxide were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 7. 100 gms of dried chrome shavings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 104 cells per ml. 25 ml of the inoculum containing 1 x 104 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 37°C in a rotary shaker running at 100 rpm. After a period of 5 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of magnesium oxide was added to it with continuous shaking and the mixture was heated at 100°C for 30 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth. The filtrate was then sun dried for 6 hrs to get the chrome free hydrolysate in a paste form, which was stored in a plastic container at 10°C.. Example VII 10 gms of peptone and 10 gms of sodium carbonate were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 6.8. 50 gms of dried chrome shavings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 107 cells per ml. 50 ml of the inoculum containing 1 x 107 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 37°C in a rotary shaker running at 100 rpm. After a period of 7 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of magnesium oxide was added to it with continuous shaking and the mixture was heated at 80°C for 45 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was concentrated by boiling off the surplus water using a steam jacketed vessel for 2hrs. The resulting paste of protein hydrolysate was stored in a plastic container at 10°C. Example VIII 25 gms of peptone and 10 gms of sodium hydroxide were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 7. 125 gms of dried chrome shavings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 106 cells per ml. 12.5 ml of the inoculum containing 1 x 106 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 37°C in a rotary shaker running at 100 rpm. After a period of 7 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of magnesium oxide was added to it with continuous shaking and the mixture was heated at 100°C for 30 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was sun dried. The resulting paste, thus formed, was stored in a plasic container at 10°C. Example IX 5 gms of peptone, 5 gms of yeast extract and 25 gms of calcium oxide were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was found to be to 10. 100 gms of dried chrome shavings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 104 cells per ml. 25 ml of the inoculum containing 1 x 104 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 37°C in a rotary shaker running at 100 rpm. After a period of 5 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of magnesium oxide and 15 gms of calcium oxide were added to it with continuous shaking and the mixture was heated at 100°C for 30 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was concentrated by boiling off the surplus water using a steam jacketed vessel for 2 hrs. The resulting product was stored in a plastic container at 10°C. Example X 25 gms of peptone and 10 gms of calcium oxide were taken in a Hoffkins flask and 2.5 lit of distilled water was added to it with stirring. pH of the solution was noted to be 7.5. 250 gms of dried chrome shavings were added to the bath while shaking the flask continuously. The flask was then autoclaved at 121°C for 15 min. at a pressure of 15 Ibs/inch2 to sterilise the leather shaving suspension. The bacterial culture of the selected isolate of Alcaligenes odorans, designated as BL-11 and available in CLRI, was precultured in peptone broth to prepare an inoculum containing 1 x 107 cells per ml. 25 ml of the inoculum containing 1 x 107 cells per ml of precultured cells were added to the above sterilised leather shaving suspension and the flask was incubated at 30°C in a rotary shaker running at 300 rpm. After a period of 5 days, the contents of the flask were filtered by synthetic cloth of mesh 0.2 to 0.3 mm and the filtrate was collected in another flask. 10 gms of magnesium oxide was added to it with continuous shaking and the mixture was heated at 100°C for 30 min for complete precipitation. The precipitate was separated by filtration using the above mentioned filter cloth and the filtrate was lyophilised. The resulting powder was stored in an air tight polythene cover. The advantages of the present invention are the following 1. The process provides an ecofriendly as well as cost effective method to prepare hydrolysate from proteinaceous chrome wastes. 2. The process provides a simple process to hydrolyse proteinaceous wastes in the presence of chromium. 3. Since this process uses an isolated microbial source, which has been deposited in CLRI, no shortage in supply in respect of the hydrolysing source is anticipated. 4. Chromium present in the proteinaceous wastes can be recovered as a byproduct in the form of chrome cake and the leached chromium thereof can be recycled as a tanning agent for leather processing in the manufacture of chrome tanned leather. 5. No difficult control parameter is involved for carrying out the hydrolysis. WE CLAIM: 1. A process for the preparation of protein hydrolysate from proteinaceous chrome wastes obtained as by product of different industries which comprises i) growing Alcaligenes, having the characteristics, as herein described, wherein the cell count of the isolate of genus Alcaligens in the inoculum is in the range of 104-109 per ml. in a chromewaste, supplimented with conventional assimilable protein in an amount in the range of 0.1 to 2% wt in a known marmer at a pH in the range of 6-10 and above till completion of hydrolysis, optionally under shaking, ii) optionally filtering the broth with a filtering device as formed in step (i), by known method, iii) treating the filtrate, as obtained in step (ii), with a conventional chrome precipitant, such as herein described in an amoimt 20%w/v. iv) removing the precipitate by known methods to get clear solution and drying the said clear solution by conventional methods to get protein hydrolysate. 2. A process, as claimed in claim 1, wherein the sources of assimilable protein used are peptone, skimmed milk powder, yeast extract, soyabean meal, either individually or in combination. 3.process, as claimed in claims 1 to 5, wherein proteinaceous chrome wastes used are chrome shavings, chrome leather trimmings, chrome leather buffing dust. 4.A process, as claimed in claims 1 to 10, wherein the mesh size for the filtering device is in the range of 0.2-0.3 mm. 5. A process for the preparation of protein hydrolysate from proteinaceous chrome wastes, substantially as herein described with reference to the examples.

Documents:

1575-del-1999-abstract.pdf

1575-del-1999-Claims-(24-05-2011).pdf

1575-del-1999-claims.pdf

1575-del-1999-Correspondence Others-(24-05-2011).pdf

1575-del-1999-correspondence-others.pdf

1575-del-1999-correspondence-po.pdf

1575-del-1999-description (complete).pdf

1575-del-1999-form-1.pdf

1575-del-1999-form-19.pdf

1575-del-1999-form-2.pdf

1575-del-1999-Form-5-(24-05-2011).pdf

1575-del-1999-Petition-137-(24-05-2011).pdf