Title of Invention

"A PROCESS FOR THE PREPARATION OF PROTEIN FROM FISH WASTE"

Abstract

The present invention relates to a process for preparation of fish protein from fish waste. The present invention particularly relates to a process of recovery of proteins from fish waste using aqueous two-phase extraction (ATPE). The waste being generated in fish markets in India and all over the world is being just dumped as such and no utilization of this waste is being done in any form. Further, this fish creates foul smell and acts as a source for the growth of various microorganisms. It has a high nutritive value and this can be used for constructive purpose, which is being considered here for extraction of protein by using Aqueous two phase extraction (ATPE).

Full Text

The present invention relates to a process for preparation of protein from fish waste. The present invention particularly relates to a process of recovery of proteins from fish waste using aqueous two-phase extraction (ATPE). Fish proteins are easily digested and have excellent metabolic efficiency. giving the protein a high biological value. Fish contains l6%-22% high biological value proteins. In addition, Protein Efficiency Ratio (PER) of fish is 3.55, which is higher compared to beef and milk proteins. These properties highlight the nutritional quality of fish protein both as a direct dietary supplement, and as an ingredient in a variety of formulated foods and beverages. The fish market has a lot of waste being generated every day. Every year over 91 million tons of fish are harvested but only 29.5% is transformed into fishmeal. Possibly more than 50% of the remaining fish tissue is considered to be processing waste and not used as food (Irineu Batista. 1999, Eur Food Res Technol 210: 84-89). Major type of waste generated in processing operations are fish body parts which include head, scales, fins, bones, mollusc shell, crustacean head and exoskeletons, blood, offal, viscera, skin, and small amount of meat particles generated during processing of fish. These types of waste contribute significantly to a solid fraction of the waste stream and the relative volume of solid and liquid waste generated may vary depending upon the size of processing operation, method used and the species. The waste being generated in fish markets in India and all over the world is being just dumped as such and no utilization of this waste is being done in any form. Further, this fish creates foul smell and acts as a source for the growth of various microorganisms. It has a high nutritive value and this can be used for constructive purpose, which is being considered here for extraction of protein by using Aqueous two phase extraction (ATPE). Aqueous two phase extraction (ATPE) comprising a pair of polymer and salts is used for recovery of fish protein and also as an alternate method for fish protein concentrate (FPC). Aqueous two-phase extraction (ATPE) is gaining popularity for its utility in the extraction and purification of biological materials such as enzymes/proteins, nucleic acid, viruses, cell organelles etc.( Albertsson et al., 1986 3rd Ed., Wiley, New York. 1986 and Raghavaro et al., 1998 Adv. Appl. Microbiol . 1995,4), 97-171). Reference may be made to recovery of proteins from fish waste products by alkaline extraction (Irineu Batista. 1999, Eur Food Res Technol 210: 84-89). This reference mainly deals with recovery of proteins by chemical extraction and precipitation with HCL and sodium hexametaphosphate. Another reference may be made to the quality of fish protein concentrates prepared by direct extraction with various solvents ( M.N.Moorjani et al., 1968, Food Technol. 22, 1557). This reference mainly deals with fish protein concentrates by solvent extraction. Another reference may be made to the preparation of protein hydrollysate from fish waste (Tarky et al., 1973, J. Food Science. 38, 917-918). This reference mainly deals with protein hydrolysate obtained by concentrated acid/alkali along with heat treatment. Another reference may be made to recovery of protein from veal bones by enzymatic hydrolysis, (Michel linder et al., 1995, J. Food Science. 5, 949-952) which discusses use of enzymatic hydrolysis for the purpose. Another reference may be made to the study of some factors influencing the production of protein isolates from whole fish (Wilmon w. Meinke et al., 1972, J. Food Science. 37, 195-198). This reference mainly deals with utilization of whole fish and not fish waste. It may be noted that all these references involved processes that are often complicated, besides adversely affected in the fish proteins, in terms of the nutritional and functional properties. The main object of the present invention is to provide a process for preparation of protein from fish waste. Another object of the present invention is to concentrating the proteins into a smaller volume. Still another object of the present invention is to regenerate the PEG phase, which can be reused for the next batch. Yet another object of the present invention is to employ aqueous two phase extraction (ATPE) for waste treatment and recovery of valuable byproducts. Accordingly the present invention provides a process for preparation of fish protein from fish waste comprising; a) grinding raw fish waste of the kind as herein described obtained from fish market to obtain paste; b) diluting the paste with water in the ratio of 1:10; c) centrifuging the viscous liquid as obtained in the above step b) to separate the protein rich top layer; d) filtering protein rich top layer using cotton filter to separate residual suspended solids; e) adding polyethylene glycol and ammonium salt in filtrate in the ratio of 12/10(% w/w) phase composition; f) mixing the mixture as obtained from the above step for a period of 20-40; g) phase separation of the mixture as obtained from the above step f) for a period ranging 5-12 hrs.; h) separating the top and bottom phase using separating device; i) adding polyethylene glycol to the protein containing top polyethylene glycol rich phase and mixing thoroughly ; j) phase separation of the mixture as obtained from the above step i) to obtain fish protein. In an embodiment of the process the phase system containing commercially available polyethylene glycol of molecular weight 6000 as one of the phase system. In an another embodiment of the process the phase system containing ammonium sulphate salts or di-potassium hydrogen phosphate and potassium di-hydrogen phosphate at a ratio of 1:82. In yet an another embodiment of the process the yield of protein in top phase is 50-94% and overall recovery of protein is 40-55%. In yet an another embodiment of the process the the enrichment of protein is 2-3 fold. In the present invention, the raw fish waste from fish market waste is taken and made into paste by grinding and the paste is diluted with water in 1:10 ratio. The viscous liquid fish waste is centrifuged using a refrigerated centrifuge and separated the protein rich top layer. The protein rich top layer is filtered through the cotton filter. Adding PEG and ammonium salts in filtrate in desired composition (w/w %) to the fish waste of desired quantity (decided by the total composition of the phase system, in other words water is replaced by fish waste). The entire mixture was stirred in a mechanical agitated contactor for 20-40 minutes and then the phases were allowed to separate for over night in separating funnel which led to a distinct phase separation, the samples were removed from both the phases and analyzed for protein concentration. Adding PEG to the protein containing phase, mixing thoroughly and phase separating to obtain concentrated protein phase (of lower volume) and PEG phase. Protein concentration was determined by Bradford method (1976) using coomassie brilliant blue G-250 taking bovine serum albumin as standard. The fractions were assayed for proteolytic enzyme activity by the tyrosine method using denatured hemoglobin as substrate and casein digestion method. The novelty of the present invention is that the aqueous two-phase extraction (ATPE) was successfully employed for the recovery of fish proteins. After clarification remove the suspended solids, ATPE was successfully employed for the concentration of fish proteins by selectively partitioning the proteins into low volumes of aqueous solution and also enabling to regenerate the PEG rich phase, which can be reused for the next batch. The novelty of the present invention 1. Enables to over come the draw backs of recovery of fish proteins by using conventional methods like chemical extraction and precipitation with HCL and sodium hexametaphosphate. 2. Provides a simple alternative method based on the differential partitioning technique for concentrating the fish protein. 3. Offers higher yield and low cost over conventional processes used for fish protein recovery. 4. Enables to regenerate the PEG rich phase, which can be recycled and reused for the next batch. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention. Example-1 1 kg of raw fish waste product from fish market is taken and made in to paste by grinding and the paste is diluted with water in 1:10 ratio. The viscous liquid fish waste is centrifuged at 6000rpm using a refrigerated centrifuge and separated the protein rich top layer. The protein rich top layer is filtered through the cotton filter. 17.88 gm of polyethylene glycol of molecular weight 6000, 14.26gm of ammonium sulphate were taken in the beaker and to this the fish waste extract of 67.86 gm was added. The entire mixture was stirred in a mechanical agitated contactor for 30 minutes then the phases were allowed to separate for over night in a separating funnel, which led to a distinct phase separation. The samples were removed from both the phases and analyzed for protein concentration. Adding 7.15 gm PEG to the protein containing phase, mixing thoroughly and separating the phase to obtain concentrated protein phase (2.6 fold enriched) and PEG phase. In this system, the phase composition of 17.88/14.26 shows the partition coefficient of 1.66 and volume ratio 55/36. Most of the protein is partitioned in top protein rich phase with 96.17% yield of protein. The overall recovery of protein was 55%. The recovered protein showed a 3.1 fold purification of proteolytic enzymes assayed by casein digestion method. Example-2 500 gm raw fish waste from fish market is taken and made in to paste by grinding and the paste is diluted with water in 1:10 ratio. The viscous liquid fish waste is centrifuged at 6000 rpm using a refrigerated centrifuge and separated the protein rich top layer. The protein rich top layer is filtered through the cotton filter. 15.3 gm of polyethylene glycol of molecular weight 6000, 12.46 gm of ammonium sulphate were taken in the beaker and to this the fish waste extract of 72.24gm was added. The entire mixture was stirred in a mechanical agitated contactor for 30 minutes and then the phases were allowed to separate overnight in separating funnel, which led to a distinct phase separation. The samples were removed from both the phases and analyzed for protein concentration. Adding 6.12 gm of PEG to the protein containing phase, mixing thoroughly and phase separating to obtain concentrated protein phase (3.6 fold enriched) and PEG phase. In this system, the phase composition of 15.3/12.46 shows the partition coefficient of 0.92 and volume ratio 43/39. Most of the protein is partitioned in top protein rich phase with 84.48% yield of protein. The overall recovery of protein was 53.6%. The recovered protein showed a 2.3 fold purification of proteolytic enzymes assayed by casein digestion method. Example-3 750gm of raw fish waste product from fish market is taken and made in to paste by grinding and the paste is diluted with water in 1:10 ratio. The viscous liquid fish waste is centrifuged at 6000rpm using a refrigerated centrifuge and separated the protein rich top layer. The protein rich top layer is filtered through the cotton filter. 22 gm of polyethylene glycol of molecular weight 6000, 4.06gm of di-potassium hydrogen phosphate and 2.24gm potassium di-hydrogen phosphate were taken in the beaker and to this the fish waste of 76.48 gm was added. The entire mixture was stirred in a mechanical agitated contactor for 30 minutes and then the phases were allowed to separate overnight in separating funnel, which led to a distinct phase separation. The samples were removed from both the phases and analyzed for protein concentration. Adding 11gm PEG to the protein containing phase, mixing thoroughly and separating the phases to obtain concentrated protein phase (3.1 fold enriched) and PEG phase. In this system, the phase composition of 22/6.3 shows the partition coefficient of 0.37 and volume ratio 75/15. Most of the protein is partitioned in top protein rich phase with 51.47% yield of protein. The overall recovery of protein was 40.7%. The recovered protein showed a 2.5 fold purification of proteolytic enzymes assayed by casein digestion method. The main advantages of the present invention are > Aqueous two-phase extraction (ATPE) was successfully employed for the recovery of fish proteins. > The yield in top phase is 50-94% and overall recovery of protein is 40-55%. > After clarification ATPE were successfully employed for the concentration of fish proteins by selectively partitioning the proteins into low volumes of aqueous solution (enrichment of protein is 2-3 fold). > The recovered protein showed a 3.1 fold purification of proteolytic enzymes assayed by casein digestion method We claim: 1. A process for preparation of fish protein from fish waste, comprising: a) grinding raw fish waste of the kind as herein described obtained from fish market to obtain paste; b) diluting the paste with water in the ratio of 1:10; c) centrifuging the viscous liquid as obtained in the above step b) to separate the protein rich top layer; d) filtering protein rich top layer using cotton filter to separate residual suspended solids; e) characterized in that adding phase system containing polyethylene glycol and ammonium sulphate salts or di-potassium hydrogen phosphate and potassium di-hydrogen phosphate at a ratio of 1:82 in filtrate in the ratio of 12/10(% w/w) phase composition; f) mixing the mixture as obtained from the above step for a period of 20-40; g) phase separation of the mixture as obtained from the above step f) for a period ranging 5-12 hrs.; h) separating the top and bottom phase using separating device; i) adding polyethylene glycol to the protein containing top polyethylene glycol rich phase and mixing thoroughly ; j) phase separation of the mixture as obtained from the above step i) to obtain fish protein. 2. A process as claimed In claim 1, wherein the phase system containing commercially available polyethylene glycol of molecular weight 6000 as one of the phase system. 3. A process as claimed In claims 1-2, wherein the yield of fish protein in top phase is 50- 94% and overall recovery of fish protein is 40-55%. 4. A process for preparation of fish protein from fish waste, substantially as herein described with reference to the examples.

Documents:

563-DEL-2004-Abstract-(22-07-2009).pdf

563-del-2004-abstract.pdf

563-DEL-2004-Claims-(22-07-2009).pdf

563-del-2004-claims.pdf

563-del-2004-Correspondence-others (16-12-2009).pdf

563-DEL-2004-Correspondence-Others-(22-07-2009).pdf

563-del-2004-correspondence-others.pdf

563-del-2004-correspondence-po.pdf

563-DEL-2004-Description (Complete)-(22-07-2009).pdf

563-del-2004-description (complete).pdf

563-DEL-2004-Form-1-(22-07-2009).pdf

563-del-2004-form-1.pdf

563-del-2004-form-18.pdf

563-DEL-2004-Form-2-(22-07-2009).pdf

563-del-2004-form-2.pdf

563-DEL-2004-Form-3-(22-07-2009).pdf

563-del-2004-form-3.pdf

563-del-2004-form-5.pdf