Title of Invention

A PROCESS FOR THE PREPARATION OF ZN-ALPHA CASEIN COMPLEX

Abstract

The present invention relates to a process for preparation of heat stable Zn-alpha casein complex. Zn Alpha Casein Complex obtained by mixing solution A containing Caseins in Hepes NaOH buffer fully dissolved at pH 6.8 and solution B containing Zinc at a concentration of 1xl0-4M to 4x10-4M. The complex obtained is dialysed against Hepes buffer so as to remove any unbound Zinc. The complex formed is thermally stable and does not aggregate when heated for extended periods of time. The complex formed has good digestibility for micronutrient zinc.

Full Text

The present invention relates to a process for preparation of heat stable Zn-alpha casein complex. Nutritional significance of zinc is well known. Zinc is an essential micronutrient and has a role in neonatal growth and immunity. The major consequences of zinc deficiency were shown to be growth retardation, retarded sexual maturation, skin lesions iron deficiency anemia, and has neuromodulatory effect. Zinc requirement for cell-mediated immunity is also well established. Zinc is an essential trace element in human nutrition playing a role in structure and function of several enzymes. Zinc deficiency will lead to anorexia, poor growth and wound healing, and skin problems. Zinc according to the World Health Organization reports is considered to be an essential mineral along with Iron and Beta-Carotene. A crude estimate of zinc deficiency in children indicates that it ranks before iron deficiency anemia but after Vitamin A deficiency in the global burden of diseases and disabilities. Zinc deficiencies are also known to cause diarrheal diseases and malaria. Zinc is an essential requirement to all forms of life. Among the trace metals present in higher mammals, Zinc is second to iron and is tightly bound to metalloproteins and enzymes. Decrease in Zinc levels can reduce memory function in brain. To date no programs have been implemented to address iron and zinc deficiencies in children. Such programs can be food based; including fortification of commonly used foods or consists of the provision of supplements. Milk in its natural form is almost unique and as a balanced source of man's dietary need. The various steps in processing and storage have a measurable impact on some specific nutrients. Milk also provides a convenient and useful vehicle for addition of certain nutrients to man's diet and has the following benefits: Milk and milk products are widely consumed regularly in predictable amounts by the people of all age groups. Costs are affordable by target population. The stability and bioavailablility of the added micronutrients are very high. Addition of fortificants usually caused minimum change in color, taste, and appearance. The minerals in bovine milk are present in a solution in which there exists equilibrium between the free ions and complexes with various components such as proteins, carbohydrates, and aminoacids. The mineral content of the milk is not constant. Based on the knowledge that nearly all the twenty minerals that are considered as essential in the human diet are present in higher amount in cow's milk than in human milk. The availability of zinc in human milk is 1.5 mg/L compared to 3.5 mg/L in cows milk. Cow's milk contains about 78% casein, of which 65% is a Casein, a Casein is a major fraction of milk caseins involved in transport of calcium and other metal ions to neonates. It is present in all mammalian milk as random coil protein. The a Casein found to be protective against heat coagulation, having chaperone like activity, binds to membrane receptors and acts as a signal transducer, rheomorphic (plasticity), having poly (1-proline) II (PPII) helical conformation, transporter of other milk proteins to secretary organelles in the mammalian epithelial cells. Much attention has been paid in the last few years to zinc absorption by infants fed on different milks. The average zinc content of bovine milk is 3.5 mg/ L and it is associated with skim milk fraction and over 95% is associated with casein micelles. It has been proposed that the relatively high Zn- binding capacity of casein, together with its relatively poor digestibility, could lead to the formation of complexes of Zn(II) with undigested casein and render a significant portion of Zn(II) unavailable for absorption in the gastrointestinal tract. Despite the apparent nutritional significance of the zinc-casein interaction the nature of Zn(II) binding by caseins has received little study. Milk essentially carried or have the capacity to retain only 4mg/L of zinc but the required amount of Zinc as per RDA values is 15.5 mg/L. So there is a need to increase the capacity of zinc intake so that efficient biofortification is possible. The present invention related to a process for preparation of Zn -alpha casein complex Reference may be made to United States Patent 6,849,595 wherein Calorically dense nutritional composition by Mark, et al. (February 1, 2005) the generated composition is for metabolically stressed people the drawbacks of this patent applicants are that they have used milk fat and whey hydrolysate along with 225% more zinc than specified by USRDA for normal humans was included as this product. This product according to the applicants is only for patients and disorder people. This product cannot be specified for the normal growing children and the infants, because of ingredients present in large excess. This product is not having wider applicability. All the ingredients specified here are in large excess as required for normal human beings based on the specifications of USRDA which can have serious hyper nutrient implication for normal growing children and humans fed on this product. Reference may be made to United States Patent 6,797,293 by Shin, et al. (September 28, 2004) where in there is use of enriched milk with capsules containing nutrients the drawbacks are milk is used as a vehicle for biofortification and with nutrients enclosed in capsules. This patent emphasizes only on the deficient nutrients and method to enrich them. The nutrients are encapsulated and made easy for ingestion. Among the nutrients there is mention of zinc but there are no details about the amount added and the methodology therein. Applicants have made this enriched milk based on Korean population as the recommended daily intake for Korean people is taken for formulations. The capsules are made up of variety of compounds like gelatin, agar, sorbitol where some are digestible in stomach and some in intestine. The effect of these materials on the digestive enzymes or vice versa has not been discussed. Reference may be made to United States Patent 6,150,547 by Sakurai, et al. (November 21, 2000) where in Iron-casein complex hydrolyzate and process for the production thereof. This invention provides carbonic acid- and/or hydrogencarbonic acid-iron-hydrolyzates of casein complexes containing a large amount of iron, without causing precipitation after heating their aqueous solution or astringent taste and the processes for production thereof. Reference may be made to United States Patent 6,124,258 by Sakurai, et al. (September 26, 2000) where in iron-casein complex and process for preparing the same and Iron-casein complexes and methods for preparation thereof without iron characteristic astringent taste even by heat sterilization and having high iron supplying effect. The main objective of the present invention is to provide a process for the preparation of zinc- alpha casein complex that obviates the drawbacks as detailed above. Another objective of the present invention is to provide a thermally stable complex, which does not aggregate on heating and also does not change the characteristics of the milk. Another objective of the present invention is to provide easier method to provide micronutrient zinc for easy bioavailability. Another objective of the present invention is to provide zinc alpha Casein Complex with good digestibility. Accordingly, the present invention provides a Process for the preparation of heat stable Zn Alpha Casein Complex, which comprises the steps of a) preparing solution A containing Caseins in Hepes NaOH buffer fully dissolved at pH in the range of 6.0-8.0, b) preparing solution B containing Zinc at a concentration in the range of 1 x 10-4M - 4 x 10-4M, c) Contacting solution A with solution B followed by thorough mixing using a shaker at an angular speed in the range of 100-150 rpm at a temperature in the range of 35 - 40 deg C for a period in the range of 3-6 hours in order to obtain a complex, and d) dialyzing the complex obtained in step (c) in so as to remove any unbound Zinc in order to obtain heat stable Zn Alpha Casein Complex. In an embodiment of the present invention heat stable alpha casein complex obtained has alpha S1-casein, alpha S2 casein and beta casein. In an embodiment of the present heat stable alpha casein complex obtained has at least one mole of alpha Casein, with 1-17 moles of Zinc. In an embodiment of the present the heat stable alpha casein complex obtained is thermally stable at a temperature in the range of 60-80 deg C for a period in the range of 1-10 minutes. In an embodiment of the present the heat stable alpha casein complex obtained is stable at a PH in the range of 6-8. In an embodiment of the present the heat stable alpha casein complex obtained is stable in the presence of NaCI at the concentration in the range of 10-100mM. In an embodiment of the present the zinc used in the step (b) is a divalent zinc compound selected from the group consisting of zinc chloride, zinc acetate, and zinc sulfate. Flow sheet for the preparation of Zn-alpha casein complex is enclosed. Flow Sheet for the Preparation of Zn-Alpha Casein Complex is provided in fig.l 1. In the embodiment of the present invention the said alpha casein comprises of alpha S1-casein, alpha S2 casein and beta casein. 2. In another embodiment of the present invention the Zinc alpha casein complex comprises of at least one mole of alpha Casein, with 1-17 moles of Zinc. 3. In yet another embodiment of the present invention Zn Alpha Casein Complex is thermally stable complex at 60-80°C for 1-10 minutes. 4. In yet another embodiment of the present invention the Zinc alpha casein complex is stable in the range of 6-8 pH. 5. In yet another the embodiment of the present invention the Zinc alpha casein complex is stable in the presence of 10-100mM NaCI. 6. In still another embodiment of the present invention A method for the preparation of the complex wherein the zinc used in preparing said solution containing zinc is a divalent zinc compound selected from the group consisting of zinc chloride, zinc acetate and zinc sulfate. Novelty of the process Novelty and inventive steps of this invention lies in a process for the preparation of a Zinc alpha Casein Complex. 1. Zn Alpha Casein Complex obtained by mixing solution A containing Caseins in Hepes.NaOH buffer fully dissolved at pH 6.8 and solution B containing Zinc at a concentration of 1x10-4M. The complex obtained is dialysed against Hepes buffer so as to remove any unbound Zinc. 2. The complex formed is thermally stable and does not aggregate when heated for extended periods of time. 3. The complex formed has good digestibility for micronutrient zinc. The process is further illustrated by the examples given below, which should not be however construed to limit the scope of the invention. Example -1 Alpha casein was isolated by known method. The process for the preparation of thermally stable Zn Alpha Casein Complex obtained by mixing 10ml solution A containing alpha Casein in Hepes NaOH buffer fully dissolved at pH 6.0 and solution B containing Zinc at a concentration of 1x10-4M. for 3 hours, at a temperature of 35°C, on a shaker at 100 rpm. The complex obtained is dialysed against Hepes buffer so as to remove any unbound Zinc. Estimation of the bound Zinc to alpha casein was done by Atomic Absorption Spectroscopy. Moles of Zinc bound to alpha casein was estimated to be 17:1 Properties: Thermal Stability upto 60°C, salt stability upto 10mM, pH stability 6.0, enhanced digestibity by proteases. Example -2 Alpha casein was isolated by known method. The Process for the preparation of thermally stable Zn Alpha Casein Complex obtained by mixing 100ml solution A containing alpha Casein in Hepes NaOH buffer fully dissolved at pH 7.0 and solution B containing Zinc at a concentration of 3x10-4M. for 5 hours, at a temperature of 37°C, on a shaker at 120 rpm. The complex obtained is dialysed against Hepes buffer so as to remove any unbound Zinc. Estimation of the bound Zinc to alpha casein was done by Atomic Absorption Spectroscopy. Moles of Zinc bound to alpha casein was estimated to be 17:1 Properties of the complex are thermal stability upto 75°C, salt stability upto 60mM, pH stability 7.0, enhanced digestibity by proteases. Example -3 The Process for the preparation of thermally stable Zn Alpha Casein Complex obtained by mixing 500ml solution A containing alpha Casein in Hepes NaOH buffer fully dissolved at pH 8.0 and solution B containing Zinc at a concentration of 4x10-4M. for 6 hours, at a temperature of 40°C, on a shaker at 150 rpm. The complex obtained is dialysed against Hepes buffer so as to remove any unbound Zinc. Estimation of the bound Zinc to alpha casein was done by Atomic Absorption Spectroscopy. Moles of Zinc bound to alpha casein was estimated to be 17:1 Properties of the complex are thermal stability upto 80°C, salt stability upto 100mM, pH stability 8.0, enhanced digestibity by proteases. Advantages of the invention 1. Zn Alpha Casein Complex is thermally stable even up to boiling temperature. 2. Zn Alpha Casein Complex will not aggregate or precipitate even at boiling temperature. 3. Digestibility of the Zn- Alpha Casein Complex increases for proteases. 4. Alpha Casein binds maximum Zinc compared to other proteins in milk. 5. Zn Alpha Casein Complex useful for prevention and treatment of zinc related disorders. 6. Zn Alpha Casein Complex does not change the characteristic properties of milk. 7. Zn Alpha Casein Complex is stable in the pH range between 6-8. 8. Zn Alpha Casein Complex is stable in presence of 10 mM -100 mM NaCI. We claim: 1. A Process for the preparation of heat stable Zn Alpha Casein Complex, which comprises the steps of: a) preparing solution A containing Caseins in Hepes NaOH buffer fully dissolved at pH in the range of 6.0-8.0, b) preparing solution B containing Zinc at a concentration in the range of 1 x 10-4M - 4 x 10-4M, c) contacting solution A with solution B followed by thorough mixing using a shaker at an angular speed in the range of 100-150 rpm at a temperature in the range of 35-40 deg C for a period in the range of 3-6 hours in order to obtain a complex, and d) dialyzing the complex obtained in step (c) to remove any unbound Zinc in order to obtain heat stable Zn Alpha Casein Complex. 2. The process as claimed in claim 1, wherein the heat stable alpha casein complex obtained has alpha S1-casein, alpha S2 casein and beta casein. 3. The process as claimed in claims 1 and 2, wherein the heat stable alpha casein complex obtained has at least one mole of alpha Casein, with 1-17 moles of Zinc. 4. The process as claimed in claims 1-3, wherein the heat stable alpha casein complex obtained is thermally stable at a temperature in the range of 60-80 deg C for a period in the range of 1-10 minutes. 5. The process as claimed in claim 1-4, wherein the heat stable alpha casein complex obtained is stable at a PH in the range of 6-8. 6. The process as claimed in claim 1-5, wherein the heat stable alpha casein complex obtained is stable in the presence of NaCI at the concentration in the range of 10-100mM. 7. The process as claimed in claim 1-6, wherein the zinc used in the step (b) is a divalent zinc compound selected from the group consisting of zinc chloride, zinc acetate, and zinc sulfate.

Documents:

332-del-2006-Abstract-(22-05-2012).pdf

332-del-2006-abstract.pdf

332-del-2006-Claims-(22-05-2012).pdf

332-del-2006-claims.pdf

332-del-2006-Correspondence Others-(22-05-2012).pdf

332-del-2006-correspondence-others.pdf

332-del-2006-Description (Complete)-(22-05-2012).pdf

332-del-2006-description (complete).pdf

332-del-2006-description (provisional).pdf

332-del-2006-drawings.pdf

332-del-2006-form-1.pdf

332-del-2006-form-18.pdf

332-del-2006-form-2.pdf

332-del-2006-Form-3-(22-05-2012).pdf

332-del-2006-form-3.pdf

332-del-2006-form-5.pdf