Title of Invention	METHOD FOR PRODUCING OF ORGANIC CALCIUM CARBONATE FROM CUTTLE FISH BONES
Abstract	ABSTRACT OF THE INVENTION The present invention relates to the extraction of calcium from cuttlefish {Sepia sp.) bones (CFBs). The discarded bones of cuttlefish were used as a source for the extraction of calcium. A methodology was devised to extract calcium by eliminating organic materials associated with the CFB. A gentle alkaline treatment degraded the proteinaceous layer that connects the organic matrix with lamellar matrix of CFB. Thereafter, CFBs were subjected to a controlled heating procedure along with a hydrothermal management at 65°C and 75° which resulted into the peeling off the organic matrix from the calcium carbonate rich lamellar matrix. The lamellar matrices are pulverized into granules of submicrons to 1000 microns of calcium carbonate and the granules are called as organic calcium (OC). The OC was further clarified with alakali-detergent mixture followed by acetone to produce OC deprived of residual glycoprotein complexes. The OC prepared by our method using CFB as a raw material contains 90-95% calcium carbonate in which 40% remain as elemental calcium. The OC is completely free of hazardous contaminants such as arsenic, mercury and lead.

Title of Invention

METHOD FOR PRODUCING OF ORGANIC CALCIUM CARBONATE FROM CUTTLE FISH BONES

Abstract

ABSTRACT OF THE INVENTION The present invention relates to the extraction of calcium from cuttlefish {Sepia sp.) bones (CFBs). The discarded bones of cuttlefish were used as a source for the extraction of calcium. A methodology was devised to extract calcium by eliminating organic materials associated with the CFB. A gentle alkaline treatment degraded the proteinaceous layer that connects the organic matrix with lamellar matrix of CFB. Thereafter, CFBs were subjected to a controlled heating procedure along with a hydrothermal management at 65°C and 75° which resulted into the peeling off the organic matrix from the calcium carbonate rich lamellar matrix. The lamellar matrices are pulverized into granules of submicrons to 1000 microns of calcium carbonate and the granules are called as organic calcium (OC). The OC was further clarified with alakali-detergent mixture followed by acetone to produce OC deprived of residual glycoprotein complexes. The OC prepared by our method using CFB as a raw material contains 90-95% calcium carbonate in which 40% remain as elemental calcium. The OC is completely free of hazardous contaminants such as arsenic, mercury and lead.

Full Text	The raw material used for the extraction of organic calcium is a byproduct of marine food processing industry where these CFBs are discarded as a waste. The CFBs are available all over the world and inexpensive raw material of biological origin. 2. The percent bioavailability of calcium from an organic source is higher than the calcium extracted from limestone and other mineralogical sources. Moreover, the calcium derived from CFBs is devoid of hazardous contaminants like mercury, arsenic and lead. 3. Our method of using CFB as a source of OC could easily replace eco-unfriendly procedures such as exploiting corals, atolls, calcite deposits and sedimentary rocks as a source of OC. 3.2 Background of invention We have focused our research on extraction of OC from a marine organism viz. Cuttlefish (Sepia sp.). Cuttlefish is one of the predominant species that was caught for human consumption all over the world. Cuttlefish has unique physical organization where the bone architecture composed of calcium carbonate that provides buoyancy for the organism to maneuver aound. The uniform perforations found across CFB facilitate seawater to enter into it by means of a capillary action displacing gas found inside the pores of CFB. By this virtue, CFB acts as a 'buoyancy tank' by maintaining an appropriate gas to liquid ratio within the perforated bone. The size, texture and serrated ridges of CFB vary from species to species albeit the general architecture remains same across species. The structural organization of CFB has been described elsewhere in the literature. The dorsal side of CFB is coated with the specialized nacreal cells. In fact, these nacreal cells with the assistance of other scaffolding organic contents present in the cuttlefish skin responsible for the nucleation of aragonite to build CFB. The nacres of cuttlefish produce a hydrothermal solution that flows in a defined orientation with the assistance of an organic matrix and construct the calcium carbonate crystallites to produce CFB. In this mode of physical organization, almost the whole CFB is exposed to absorb seawater into it except the organic matrix which is robustly cemented with the ventral side skin of the cuttlefish. The ventral portion of cuttlefish skin that encapsulates CFB is predominantly made up of protein- chitosan complex. In the present invention, focus is laid on peeling off the calcium rich aragonite structure, otherwise known as lamellar matrix without organic material from the cuttlefish skin and other scaffolding macromolecular compounds. 3.3 Objective of the invention The concept of'being organic' is getting popular as a consequence of deleterious side effects caused by synthetic chemicals and its derivatives. Inorganic substances derived from rocks, subsoil, calcareous sediments and marine water mostly happened to contain hazardous contaminants that remain intractable to eliminate during the purification process. In addition, as the sources mentioned above often contains the mineral in miniscule amount, it is often proven to be cost inefficient for the extraction of minerals of interest. Products obtained from biological sources gaining importance due to their pristine quality and their exquisite ability to get readily integrated into living system. Calcium, an important alkaline metal is one of the most abundant elements found on earth. Calcium plays crucial role in cellular metabolism of living organisms. Deficiency of these minerals has been found to compromise the normal growth and development of all living organisms. Calcium is the most abundant mineral in the human body. Calcium is essential in bones and tooth architecture, blood coagulation, impulse transmission in brain, muscle contraction, and homeostasis within cells. Constant supplementation of calcium is necessary for the survival of all living organisms. USFDA (United States Food & Drug Administration) recommends dietary allowance of lOOOmg calcium per day for an adult. The calcium drain in pregnant and breast-feeding ladies is prevalent and often lead to tooth decay, osteoporosis, arthritis and other blood related disorders. Considering the importance of calcium for human and other livestock, modern food, pharma and nutraceutical industry learnt to fortify their products with calcium. Human and animal physiological system has a better absorption of calcium extracted from organic sources rather than inorganic calcium when used as a dietary supplement. The OC extracted from CFBs is an excellent source of calcium and could be used in pharma, neutraceutic and cosmetic applications. 4. DESCRIPTION The protocol we followed for the extraction of OC from CFB is as follows: 1. The CFBs may be thoroughly washed in deionised water. The CFBs may be immersed in water to effectively flush the pores of CFBs to eliminate silica content originated from sea sand. It would be ideal to place the CFBs under pressurized water from a tap to completely flush the pores to get rid of sand and other debris present in the CFB sieves. Thereafter, the CFBs may be soaked in deionised water for at least 2 hrs. 2. The water soaked CFBs may be immersed again in O.IN sodium hydroxide (NaOH) (Sigma, USA) for 30 min and then washed with deionised water to remove the sodium hydroxide completely. 3. The wet CFBs may be placed in an oven and incubated at 65°C for an hour followed by 75°C in an oven for 6 hours. The organic matrix and the residual skin of the cuttlefish rich in organic material may be peeled off from the calcium carbonate rich lamellar matrix. The removal of organic matrix from the lamellar matrix may be done manually. 4. The lamellar matrix may be broken into small pieces and pulverized in a mortar and pestle. After getting in granulated form, liquid nitrogen may be poured onto it and a fine powder may be made by homogenization. Liquid nitrogen may be poured on the pulverized pieces or granules of CFB at least thrice to achieve fine powder of calcium carbonate. The fine calcium carbonate powder may be passed through a sieve with a mesh size of 600 micron and may be called as organic calcium (OC) hereafter. 5. The OC may be clarified with 0.5N NaOH and 1% SDS (Sodium dodecyle sulpahate) (Sigma, USA) for 30 minutes. The clarified OC may be washed thoroughly with deionised water to remove the residual NaOH and SDS and dried at 65°C for 2 hours. 6. The dried OC may be subjected to another cycle of washing with acetone (Sigma, USA). The acetone-OC mixture may be clarified thoroughly and passed through Buchner funnel. The filtrate may be dried at 65°C for overnight. 7. The OC may be subjected to atomic absorption spectrometry to determine the presence of calcium and other elements. The calcium in the form of calcium carbonate in OC is found to be 90-95%. The quantity of various elements present in the OC are tabulated below. *ND stands for 'not detected' 8. The protein and carbohydrate content present in OC is 2.72% and 1.4 %? respectively. 5. CLAIMS Marine organisms are excellent source of calcium that could be exploited for commercial application. The cuttlefish bones (CFB) are made up of calcium carbonate with biological macromolecules such as proteins, carbohydrates and other elements. In this document, we explain a protocol to extract the calcium carbonate from CFB by eliminating other organic materials. We claim: 1. The organic calcium (OC) extracted from CFB contains 90-95% calcium carbonate in which 40% remain as elemental calcium, and fractions of other elements (magnesium, silicon, phosphate, sodium and potassium) and macromolecular compounds (protein and carbohydrates). 2. The OC of claim 1 could be extracted by treating water soaked CFBs with 0.1N sodium hydroxide that disintegrates the proteinaceous layer connecting lamellar matrix with organic matrix. 3. The two-step gradient temperature treatment i.e. 65°C for an hour and 75°C for 6 hours could accelerate the disintegration of lamellar matrix from the CFB as mentioned in claim 2. 4. Washing of OC of claim 1 with the anionic detergent 1% SDS and 0.5N sodium hydroxide could remove the organic material i.e protein-chitin complex from the OC. 5. Clarification of OC of claim 1 could remove the residual organic materials and water content from the OC. 6. The OC of claim 1 after the processes mentioned in claim 2, 3, 4, and 5 is an excellent source of either organic calcium or calcium carbonate for pharma, cosmetic and neutarceutic application. 7. The OC of claim 1 is an amorphous white crystalline substance with a specific gravity of 0.17 and having a particle size ranging from submicron to 1000 microns.

Full Text

The raw material used for the extraction of organic calcium is a byproduct of marine food processing industry where these CFBs are discarded as a waste. The CFBs are available all over the world and inexpensive raw material of biological origin.
2. The percent bioavailability of calcium from an organic source is higher than the calcium extracted from limestone and other mineralogical sources. Moreover, the calcium derived from CFBs is devoid of hazardous contaminants like mercury, arsenic and lead.
3. Our method of using CFB as a source of OC could easily replace eco-unfriendly procedures such as exploiting corals, atolls, calcite deposits and sedimentary rocks as a source of OC.
3.2 Background of invention
We have focused our research on extraction of OC from a marine organism viz. Cuttlefish (Sepia sp.). Cuttlefish is one of the predominant species that was caught for human consumption all over the world. Cuttlefish has unique physical organization where the bone architecture composed of calcium carbonate that provides buoyancy for the organism to maneuver aound. The uniform perforations found across CFB facilitate seawater to enter into it by means of a capillary action displacing gas found inside the pores of CFB. By this virtue, CFB acts as a 'buoyancy tank' by maintaining an appropriate gas to liquid ratio within the perforated bone. The size, texture and serrated ridges of CFB vary from species to species albeit the general architecture remains same across species.
The structural organization of CFB has been described elsewhere in the literature. The dorsal side of CFB is coated with the specialized nacreal cells. In fact, these nacreal cells with the assistance of other scaffolding organic contents present in the cuttlefish skin responsible for the nucleation of aragonite to build CFB. The nacres of cuttlefish produce a hydrothermal solution that flows in a defined orientation with the assistance of an organic matrix and construct the calcium carbonate crystallites to produce CFB. In this mode of physical organization, almost the whole CFB is exposed to absorb seawater into it except the organic matrix which is robustly cemented with the ventral side skin of the cuttlefish. The ventral portion of cuttlefish skin that encapsulates CFB is predominantly made up of protein-

chitosan complex. In the present invention, focus is laid on peeling off the calcium rich aragonite structure, otherwise known as lamellar matrix without organic material from the cuttlefish skin and other scaffolding macromolecular compounds.
3.3 Objective of the invention
The concept of'being organic' is getting popular as a consequence of deleterious side effects caused by synthetic chemicals and its derivatives. Inorganic substances derived from rocks, subsoil, calcareous sediments and marine water mostly happened to contain hazardous contaminants that remain intractable to eliminate during the purification process. In addition, as the sources mentioned above often contains the mineral in miniscule amount, it is often proven to be cost inefficient for the extraction of minerals of interest. Products obtained from biological sources gaining importance due to their pristine quality and their exquisite ability to get readily integrated into living system.
Calcium, an important alkaline metal is one of the most abundant elements found on earth. Calcium plays crucial role in cellular metabolism of living organisms. Deficiency of these minerals has been found to compromise the normal growth and development of all living organisms. Calcium is the most abundant mineral in the human body. Calcium is essential in bones and tooth architecture, blood coagulation, impulse transmission in brain, muscle contraction, and homeostasis within cells. Constant supplementation of calcium is necessary for the survival of all living organisms. USFDA (United States Food & Drug Administration) recommends dietary allowance of lOOOmg calcium per day for an adult. The calcium drain in pregnant and breast-feeding ladies is prevalent and often lead to tooth decay, osteoporosis, arthritis and other blood related disorders. Considering the importance of calcium for human and other livestock, modern food, pharma and nutraceutical industry learnt to fortify their products with calcium.
Human and animal physiological system has a better absorption of calcium extracted from organic sources rather than inorganic calcium when used as a dietary supplement. The OC extracted from CFBs is an excellent source of calcium and could be used in pharma, neutraceutic and cosmetic applications.

4. DESCRIPTION
The protocol we followed for the extraction of OC from CFB is as follows:
1. The CFBs may be thoroughly washed in deionised water. The CFBs may be immersed in water to effectively flush the pores of CFBs to eliminate silica content originated from sea sand. It would be ideal to place the CFBs under pressurized water from a tap to completely flush the pores to get rid of sand and other debris present in the CFB sieves. Thereafter, the CFBs may be soaked in deionised water for at least 2 hrs.
2. The water soaked CFBs may be immersed again in O.IN sodium hydroxide (NaOH) (Sigma, USA) for 30 min and then washed with deionised water to remove the sodium hydroxide completely.
3. The wet CFBs may be placed in an oven and incubated at 65°C for an hour followed by 75°C in an oven for 6 hours. The organic matrix and the residual skin of the cuttlefish rich in organic material may be peeled off from the calcium carbonate rich lamellar matrix. The removal of organic matrix from the lamellar matrix may be done manually.
4. The lamellar matrix may be broken into small pieces and pulverized in a mortar and pestle. After getting in granulated form, liquid nitrogen may be poured onto it and a fine powder may be made by homogenization. Liquid nitrogen may be poured on the pulverized pieces or granules of CFB at least thrice to achieve fine powder of calcium carbonate. The fine calcium carbonate powder may be passed through a sieve with a mesh size of 600 micron and may be called as organic calcium (OC) hereafter.
5. The OC may be clarified with 0.5N NaOH and 1% SDS (Sodium dodecyle sulpahate) (Sigma, USA) for 30 minutes. The clarified OC may be washed thoroughly with deionised water to remove the residual NaOH and SDS and dried at 65°C for 2 hours.
6. The dried OC may be subjected to another cycle of washing with acetone (Sigma, USA). The acetone-OC mixture may be clarified thoroughly and passed through Buchner funnel. The filtrate may be dried at 65°C for overnight.

7. The OC may be subjected to atomic absorption spectrometry to determine the
presence of calcium and other elements. The calcium in the form of calcium
carbonate in OC is found to be 90-95%. The quantity of various elements present in
the OC are tabulated below.

*ND stands for 'not detected'
8. The protein and carbohydrate content present in OC is 2.72% and 1.4 %? respectively.

5. CLAIMS
Marine organisms are excellent source of calcium that could be exploited for commercial application. The cuttlefish bones (CFB) are made up of calcium carbonate with biological macromolecules such as proteins, carbohydrates and other elements. In this document, we explain a protocol to extract the calcium carbonate from CFB by eliminating other organic materials. We claim:
1. The organic calcium (OC) extracted from CFB contains 90-95% calcium carbonate in
which 40% remain as elemental calcium, and fractions of other elements
(magnesium, silicon, phosphate, sodium and potassium) and macromolecular
compounds (protein and carbohydrates).
2. The OC of claim 1 could be extracted by treating water soaked CFBs with 0.1N
sodium hydroxide that disintegrates the proteinaceous layer connecting lamellar
matrix with organic matrix.
3. The two-step gradient temperature treatment i.e. 65°C for an hour and 75°C for 6
hours could accelerate the disintegration of lamellar matrix from the CFB as
mentioned in claim 2.
4. Washing of OC of claim 1 with the anionic detergent 1% SDS and 0.5N sodium
hydroxide could remove the organic material i.e protein-chitin complex from the OC.
5. Clarification of OC of claim 1 could remove the residual organic materials and water content from the OC.
6. The OC of claim 1 after the processes mentioned in claim 2, 3, 4, and 5 is an excellent source of either organic calcium or calcium carbonate for pharma, cosmetic and neutarceutic application.
7. The OC of claim 1 is an amorphous white crystalline substance with a specific gravity
of 0.17 and having a particle size ranging from submicron to 1000 microns.

Documents:

1167-CHE-2007 AMANDED CLAIMS 24-11-2009.pdf

1167-CHE-2007 AMANDED PAGES OF SPECIFICATION 24-11-2009.pdf

1167-CHE-2007 EXAMINATION REPORT REPLY RECIEVED 24-11-2009.pdf

1167-che-2007-abstract.pdf

1167-che-2007-claims.pdf

1167-che-2007-correspondnece-others.pdf

1167-che-2007-correspondnece-po.pdf

1167-che-2007-description(complete).pdf

1167-che-2007-form 1.pdf

1167-che-2007-form9.pdf

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Patent Number

243905

Indian Patent Application Number

1167/CHE/2007

PG Journal Number

46/2010

Publication Date

12-Nov-2010

Grant Date

10-Nov-2010

Date of Filing

07-Jun-2007

Name of Patentee

VICTORIA BIOTECH PRIVATE LIMITED

Applicant Address

VICTORIA FRESH SEAFOOD PVT LIMITED 3, 3RD FLOOR, GOKUL ARCADE, NO.2, SARDAR PATEL ROAD, ADYAR, CHENNAI-20.

Inventors:

#	Inventor's Name	Inventor's Address
1	M.L STEPHEN RAJ	VICTORIA FRESH SEAFOOD PVT LIMITED 3, 3RD FLOOR, GOKUL ARCADE, NO.2, SARDAR PATEL ROAD, ADYAR, CHENNAI-20.
2	A. DASNAVIS FERNANDO	VICTORIA FRESH SEAFOOD PVT LIMITED 3, 3RD FLOOR, GOKUL ARCADE, NO.2, SARDAR PATEL ROAD, ADYAR, CHENNAI-20. INDIA

PCT International Classification Number

A23L3/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA