FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention: STABILIZED BUFFALO COLOSTRUM POWDER
2. Applicant(s):

(a) NAME: G.C. CHEMIE PHARMIE LTD.
(b) NATIONALITY: An Indian Company
(c) ADDRESS: 5/C, Shree Laxmi Industrial Estate, New Link Road,
Andheri (W), Mumbai 400 053, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
The present invention relates to Buffalo Colostrum powder, a dietary supplement comprising stabilized fat and a minimum of 20% IgG immunoglobulin of the total colostral protein, derived from Indian Water Buffalo and process for its preparation.
BACKGROUND OF THE INVENTION
Buffalo Colostrum Powder is a natural food supplement. It is a condensed and dried form of the buffalo's first milk. Buffalo's first milk is the first lacteal secretion produced by the mammary gland of it just after delivery (parturition), prior to the production of transitional milk. Any secretion after the first removal of all or part is then termed Transitional Milk. Only colostrum, collected from the first and second milking within 0-12 hours after parturition maintains nature's perfect balance of immunological and body regulating biologically active proteins.
Colostrum is a very important component of breast milk and lays the foundation for every immune system. It contains protective antibodies to prevent infections in the newborn called passive immunity. It also provides vital nutrients for tissue development.
Human colostrum is not enough for its use at large and at the same time mother donors had problems of abuse of alcohol or disorders etc. Therefore, there is a need for alternate colostrums from animal source with safe and suitable transfer of immunity and growth factors. These properties exist in bovine colostrums as well. Buffalo colostrum is richest in IgG (up to 24%) compared to cow (maximum 20%) and human (2%) colostrums. It is a natural immune system enhancer designed carefully as nature intended it as the first food for new life.

Important Health Factors In Buffalo Colostrum Powder (BCP)
There are ninety known components in the buffalo colostrum. There are two primary components of colostrum: Immune factors and growth factors. Colostrum also contains vitamins, minerals and amino acids according to need of neonates.
Immune Factors: (a) Immunoglobulin: Immunoglobulin helps in defense in both treatment and prevention of viral infections, bacterial infections, allergies, yeast and fungus. There are five types of immunoglobulins i.e. IgA, IgD, IgE, IgG and IgM. Buffalo colostrum powder contains 8%-25% IgG whereas human colostrums contain 2% IgG. Buffalo colostrum also contains IgA, IgD, IgE, IgM. Specific Antibodies: The immune factors obtained from the mother buffalo have been shown to fight against viruses, bacteria, yeast and fungus. There are around 20 specific antibodies in the colostrum to fight microbes like E. Coli, Salmonella, Rotavirus, Candida, Streptococcus, Staphylococcus, Cryptosporidium, H. Pylori, etc. There is adequate transfer of passive immunity against diarrhea; (b) Prolin Rich Polypeptide (PRP); (c) Lactoferrin; (d) Cytokines; (e) Lymphokines; (f) Oligopolysaccharides and Glycoconjugate Sugars; (g) Glycoproteins and Trypsin Inhibitors; (h) Lysozyme; (i) Leucocytes; (j) Lactoperoxidase-thiocynate, Peroxidase and Xanthine Oxidase Enzymes; (k) Lactalbumins.
Growth Factors: The vital growth factors in the human colostrum are similar to that of buffalo colostrum. They stimulate growth, help in regeneration and accelerate the repair of aged original muscle, skin, collagen, bone, cartilage and nerve tissue. Growth factors also stimulate the body to burn fat for fuel instead of the body's muscle tissue in times of fasting and lean built. Growth factors have also been used as an effective topical application for burns, injuries and skin rejuvenation.
Various Growth Factors: (a) Epithelial Growth Factor (EGF); (b) Insulin like growth factors 1 and 2 (IgFl and IgF-2) are the most abundant; (c) Transforming Growth Factors A and B (TGF A and B); (d) Platelet Derived Growth Factor (PDGF); (e) Vitamins and Minerals: There are more than adequate amounts of

vitamins like C, E, and A in the colostrum. These vitamins make colostrum to serve as antioxidant in the body; (f) Amino acids.
In all species of mammals, immunoglobulins are passed from the mother to its young to provide passive immunity to a newborn animal. In humans and apes IgG and its compliment of antibodies pass across the placental barrier to the foetus during the second two-thirds of gestation. This passage appears to be selective in that other immunoglobulins are not transferred. In cattle, passive immunity is provided through the secretion of immunoglobulin, and in particular IgG, in colostrum produced by the buffalo in the first few days after birth of the calf, and the absorption of those immunoglobulins through the gastrointestinal tract of the calf This absorption appears to be selective, in that there is a preferential passage of IgG through the gut wall, in comparison with other immunoglobulins.
Bovine milk antibodies have been shown to be an effective means of providing local protection within the gastrointestinal tract against disease caused by pathogenic micro-organisms. Trials have shown that specific antibodies in bovine milk are effective against enteropathogenic and enterotoxigenic Escherichia coli, Cryptosporidium, rotavirus and Shigella flexneri.
Colostrum has received widespread attention as a dietary supplement since the mid 1990. However, it has a lengthy history of medicinal use. Ayurvedic physicians in India have used colostrum for thousands of years. In the United States, mainstream medical practitioners recommended colostrum as a natural antibiotic before the discovery of penicillin and sulfa drugs. In the 1950s, colostrum was first used to treat rheumatoid arthritis (RA). Dr. Albert Sabin, who developed the vaccine for poliomyelitis, found colostrum contains antibodies against polio and recommended it as a supplement for children who were at risk ofpolio.

Food trend is reverting back to natural foods. Inbuilt immunity is the answer for every disorder. Colostrum seems to be most potent and proven supplement to provide basic immunity.
The colostrum powder thus produced in India, from buffalo's first milk, is unmatchabfe with the leading producers in the rest of the world. There are a lot of differentiations.
The below table reflects that buffalo first milk is more potent in all parameters compared to cow's milk.

A common thing observed in all commercially available colostrums is that the basic raw material is cow's first milk and they are devoid of fat.
Fat is an integral part of the first milk and is always well digested by the newborn. It is not only IgG but the colostrum as a whole which will possesses all the defined benefits. Therefore, commercially available colostrum powders are actually termed as IgG supplement not colostrum powder.

The milk fat in complete first milking colostrum is one of the most under-rated and misunderstood components by many companies that promote bovine colostrum for human consumption.
Casein is a nutritionally valuable complete protein that is broken down in the stomach to small peptides and amino acids so that they can be absorbed and used to build new muscle protein by forming a cottage cheese-like curd in the stomach. This occurs enzymatically in the newborn and the adult and the basis for the curd that forms is the fat in the colostrum. Therefore, without it, in addition to losing some significant biologically active substances that are associated with the fat, one loses most of the nutritional value of the casein. That is part of the reason why the fat content of colostrum increases with time after birth as the amount of casein increases in the secreted fluid.
High quality first milking buffalo colostrum contain 20-30% milk fat. The milk fat in colostrum is also a very important means to deliver some of its beneficial biologically active substances. Dissolved in or associated with the fat in colostrum are vitamins A, D, E and K; steroid hormones; corticosteroids; some growth factors; and insulin. Like this, associated fat, Indian water buffaloes are also under rated, while the colostrum milked from them possesses much potential in almost all parameters compared to any Immunoglobulin supplement.
Table I provides the analytical characteristics of fat of composite mixtures of buffalo colostrum (The Component Fatty Acids of Buffalo Colostrum Fat; C. P. Anantakrishnan, V. R. Bhale Rao, T. M. Paul, and M. C. Rangaswamy; Imperial Dairy Research Institute, Bangalore, India, received for publication, April 10, 1946)

TABLE I Analytical Characteristics of Fat of Composite Mixtures of Buffalo Colostrum

Day of Isctation M.p. Butyroraeter reading Saponification
vsas Iodine value Point ike
value Klndmer
velue
°C.
3 39.1 44.5 218.7 38.6 25.(38 0.00 20.S2
2 38.8 43.6 219.4 33.0 28.71 1.25 25.83
3 36.5 43.4 224.7 32.2 30-91 1.25 26.92
4 36.6 42.5 227.2 26.8 34.21 1.35 30.15
6 36.9 41.8 228.8 27.5 34,87 1.25 30.16
10 36.4 41.5 228.8 26.3 34.85 1.25 30.12
15 36.2 41.5 228.7 26.6 37.91 1.20 31.53
Table II provides the summary of component Fatty Acids of Buffalo Colostrum
(Expressed in Mole Per Cent) [The Component Fatty Acids of Buffalo Colostrum
Fat; C. P. Anantakrishnan, V. R. Bhale Rao, T. M. Paul, and M. C. Rangaswamy;
Imperial Dairy Research Institute, Bangalore, India, received for publication,
April 10, 1946]
TABLE IT
Summary of Component Fatty Acids of Buffalo ColoMlrum (Exprented inMole Per Cent)


The composition of the present invention is a dried form of naturally milked first milk i.e. colostrum (first and second milking within 0-12 hours after parturition) of Indian water buffalo. All other similar products are deprived of milk fat or they are fortified with other proteins, starches as fillers.
Most of the prior art documents relating to dried colostrum powder involve the removal of fat during the process of treating the colostrum to reduce bioburden, which may be added back to the colostrum composition at a later stage of the process. However, the process of the present invention does not involve the removal of fat. Here the present inventors have kept the composition as it is, since nature has designed it so carefully, so balanced that all nutritional elements required by a newborn are there. These nutrients are in easily digestible form. There is no need to disturb the nature created composition. It is the expertised process of handling the raw and intermediate material and finally keeping all active parts in the product as active. So technically, except moisture all the prospective nutrients remains intact in our product. The dried form will help in availability of buffalo colostrums in deprived areas.
An important attribute of the present invention is that the composition is stabilized i.e. has improved shelf life. Generally shelf life of first milk in milk form is few hours, while our prepared product is stable for minimum of a year. The increased shelf life is the heart of the invention. Secondly this developed format allows people to apply/consume in various applications. Thus the colostrum powder of the present invention is providing another way to enjoy its health benefits at any point of time in life.
B-Colostrum™ a product manufactured by Biostrum Nutritech Pvt. Ltd., is the powdered form of first milk of the female of animal i.e. bovine. It is a highly concentrated form of milk containing proteins 60%, immunoglobulin (IgG) antibodies 20%, fat 20%. However, B-Colostrum™ does not contain any

additional constituents such as antioxidants to protect the fat and improve the shelf-life of the colostrum powder.
The present inventors have surprisingly found that an antioxidant or a combination of two antioxidants selected from the group comprising ascorbyl palmitate, standardized pine bark extract, natural tocopherols and rosemary extract and the stabilizer disodium phosphate enhance the shelf life of the buffalo colostrum powder by preventing the colostral fat from going rancid and thereby protecting the active protein.
Further, the buffalo colostrum contains 10 times more vitamin A than milk. Buffalo Colostrum contains 3 times more vitamin D than milk. Buffalo Colostrum contains at least 10 times more iron than milk. Buffalo Colostrum contains more calcium, phosphorous and magnesium than milk.
OBJECT OF THE INVENTION
It is an object of the present invention to provide a stabilized dried buffalo colostrum with a composition of minimum 60% protein of which IgG is minimum 20%, fat is maximum 25% which is stabilized and shielded with an antioxidant or a synergistic combination of two antioxidants selected from the group comprising ascorbyl palmitate, standardized pine bark extract, natural tocopherols and rosemary extract and disodium phosphate as a stabilizer, lactose is maximum 25% and ash maximum 6%.
SUMMARY OF THE INVENTION
According to an aspect of the present invention there is provided a stabilized buffalo colostrum powder composition comprising:
i. at least 60% Protein of which at least 20% is IgG;

ii. upto25%Fat;
iii. an antioxidant or synergistic combinations of two antioxidants in a total concentration of 50-200 mg/kg of total dry matter present in milk;
iv. 0.05% to 0.15 % disodium phosphate as stabilizer;
v. upto 25% lactose; and vi. upto 6% ash.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
Figure 1: The percentage change from the initial value of physical parameters such as moisture, bulk density and pH of Table 4 over the period of 0 - 240 days are graphically represented.
Figure 2: The percentage change from the initial value of HPLC / Chemical Parameters of Table 4 over the test period of 0 - 240 days are graphically represented.
DETAILED DESCRIPTION OF THE INVENTION
Buffalo Colostrum Powder is a natural food supplement. It is a condensed and dried form of buffalo first milk. Buffalo first milk is the first lacteal secretion produced by the mammary gland of it just after delivery (parturition), prior to the production of transitional milk. The colostrum of the present invention is collected from the first and second milking within 0-12 hours after parturition.
Unlike much of the colostrum that is processed from cows in sub-tropical climates (e.g. New Zealand), colostrum from so-called "pasture-fed" cows may not contain the rich nutrients found in colostrum from buffaloes that have been provided a dry period of at least three months. If it is not in the mother cattle, it will not occur in the colostrum. Therefore the colostrum of the present invention is taken from

buffaloes that have been provided a dry period of at last three months. Dry period is that period wherein the mammary gland of the buffalo is provided a non-lactating (dry) period prior to an impending parturition to optimize milk production in the subsequent lactation.
Dry period is regular system followed by Indian dairy farms; this is based on ancient experience that it helps restoring all natural nutrients in best combination in the mother livestock. Since the same is suggested in human being also to restore the previous nutritional level in mother.
Buffalo colostrum has wide industrial applicability.
Industrial Applicability of the Buffalo Colostrum Powder
Colostrum has a very large potential market. Main potential applications are;
• Oral Hygiene;
• Gastro Intestinal Functions as capsules;
• Growth factors (Food Supplements and Over the counter products);
• Infant Formula as chewable tablets, diskettes etc.;
• Specific Drugs;
• Senior people with auto-immune diseases, Rheumatoid arthritis, Multiple sclerosis;
• Cosmetics.
The present invention is stabilized dried buffalo first milk with a composition of minimum 60% protein of which IgG is minimum 20%, fat is maximum 25% which is stabilized and shielded with an antioxidant or a synergistic combination of two antioxidants selected from the group comprising ascorbyl palmitate, standardized pine bark extract, natural tocopherols and rosemary extract and disodium phosphate as stabilizer, lactose is maximum 25% and ash maximum 6%.

The term 'Ash' herein refers to the total mineral content present in the stabilized buffalo colostrum powder of the present invention.
Process Steps For The Production Of Stabilized Buffalo Colostrum powder is as follows:-
• All quantity of first and second milks is collected with 0-12 hours after parturition from salvage farms, which are dedicatedly serving the pregnant mother buffaloes;
• Collection of first and second Milk:- The milk collected is from a buffalo in respect of whom a clear dry period of minimum 90 days has been provided. This is to procure the nutrient rich first milk, known to restore nutrients when proper dry period is maintained;
• The first and second buffalo milk is recovered within 0-12 hours of parturition. The milk is collected in total hygienic conditions to avoid any microbial load;
• Chilling of Milk: - Milk is immediately chilled to below 10° C to avoid curdling because of lowering in pH. Also it helps reaching the milk to the drying plant in proper conditions;
• Salt balancing:- Stabilizer disodium phosphate is added in a concentration of 0.05% to 0.15%, it helps in maintaining pH and does not allow it to be lowered. The disodium phosphate causes a significant shift in all the salt constituents (calcium, magnesium, phosphate and citrate) from the dissolved to the colloidal phase. This is a unique method of keeping all milked nutrients intact until the milk is dried;
• This milk is transported to drying plant in insulated cans;
• Bactofugation: - The process is based on the concept of centrifugation and difference in specific gravities of micro-organisms with food part. Bactofugation is preferred over pasteurization to avoid any excessive heating of milk. Bactofuge was installed and commissioned on H2O. Flow rates were set at approximately: inlet = 1600 lph, outlet = 1440 lph. The

process is carried out at 50° C-55° C and at 4000-4500 rpm. Efficiency achieved is an average of 98 - 99.5 %. This process is most efficient for spores as they are relatively heavy. Maximum density of feed is 1100kg/m3;
alternatively Low-Temperature-Long-Time (LTLT) Pasteurization [Low Heat Pasteurization] can be carried out instead of bactofugation. The LTLT is preferably carried out at 63° C for 30 minutes. However, bactofugation is preferred.
• An antioxidant or a synergistic combination of two antioxidants (total weight is 50-200 mg/kg of total dry matter present in milk) selected from the group comprising Ascorbyl palmitate, Standardized pine bark extract, natural Tocopherol and Rosemary extract is added to milk prior to drying to ensure that it starts working from drying process. This prevents fat, present in the milk and subsequently in powder, from going rancid. The dosage of antioxidant or synergistic combination of two antioxidants is calculated on the basis of total solids present in milk, since on drying the same quantity will be obtained as dry powder. As a regular practice, these micro additives are always added with 10% overages, so as to get the required quantity in the final product of the present invention and to supplement for any loss during the process of preparing the dry powder of the present invention. Antioxidants of the present invention are commercially available. Standardized pine bark extract is extracted from bark of pine (Pinus Pinaster), natural tocopherol is preferably extracted from Soyabean oil, Rosemary extract is extracted from a herb i.e. Rosemary (Rosmarinus officinalis). The natural tocopherols of the present invention are commercially available and may be obtained from a variety of manufacturers such as Sonic Biochem, ADM, BASF. Standardized pine bark extract of the present invention are also commerically available and may be obtained from a variety of manufacturers such as Qingdao Sunrise Biotechnology Co., Hangzhou Joymore Technology Co.
• Low heat spray drying is carried out at conditions mentioned below:

1. Hot air Inlet Temperature:- 175 °C - 185 °C;
2. Outlet Temperature:- 85 °C - 91 °C;
3. Product temperature: - 65 °C - 71 °C. During total drying process, the temperature of dried powder at outlet was in this range;
4. Objective of maintaining these temperatures is to protect the active protein part from denaturation. (Protein gets denatured at 72 °C);

• The product can be stored at room temperature. It should be stored at cool and dry conditions. All the packs are vacuum packed. After filling of material, air is sucked from the LDPE (Low-density polyethylene) packing followed by immediate sealing. This is done by vacuum packing machine. Gauge is not installed to measure levels of vacuum.
• Cumulatively the process is so designed to ensure that all macro and micro-nutrients remain intact except the water present in milk.
THE PROCESS IS OUTLINED BELOW
COLLECTION OF FIRST AND SECOND MILK FROM BUFFALO WITHIN 0-12 OF PARTURITION

SALT BALANCING AND CHILLING OF MILK
TRANSPORTATION OF MILK TO DRYING PLANT

BACTOFUGATION / LOW HEAT PASTEURIZATION OF MILK

ADD ANTIOXIDANT(S)




LOW HEAT DRYING/ VACUUM DRYING OF CODENSED MILK
VACUUM PACKING
STORAGE AT COLD STORE
The powder recovered by low heat drying is of Buffalo first and second milk (within 0-12 hrs. of parturition), so balanced in Ca/P (Calcium/ Phosphorus) ratio, to avoid any denaturation because of decrease in pH during storage and handling.
The antioxidant or synergistic combination of two antioxidants selected from the group comprising ascorbyl palmitate, standardized pine bark extract, natural tocopherols and rosemary extract and the stabilizer disodium phosphate are integral ingredients of powder, which enhance shelf life of powder by six times by preventing the colostral fat part from going rancid and maintaining the active protein. The active protein part is kept intact with the help of stabilized fat and added stabilizer.
The prefered concentration range of the antioxidant in the present invention is 50 -200 mg/kg of total dry matter present in milk (Prevention of Food Adulteration Rules, 1955, Table - 14 provides that 0.5 g/kg or 500 mg is the maximum dosage permitted, and after trials it has been found out that from a minimum of 50 upto a maximum of 200 mg does not affect physical characteristics of the product of the present invention i.e. upto 200 mg is the optimum quantity).
The preferred concentration range of disodium phosphate in the present invention is 0.05% to 0.15% of the total dry matter of milk (Prevention of Food Adulteration Rules, 1995, Table 14, provides that 3g/kg is the maximum dosage

permitted, which after trials the concentration range of 0.05% to 0.15% was found to be optimum quantity for the present invention to get desired results).
Milk solids (Total dry matter) are basically divided into two viz. Fat and SNF (solid not fat). The fat part is derived by a standardized method called Gerber Fat Method which is based on the principle of centrifugal separation if the densities are different. Then SNF is derived via an apparatus called Lactometer. The sum of fat and SNF is equal to 'total solids' present in milk.
The Stabilized Buffalo colostrum powder of the present invention is predominantly dried form of first and second milk (0-12 Hrs) milked from Indian water buffaloes only, after partutition and prepared according to the process dislosed herein and in which at no step of preparation is the colostrum de-fatted. The composition of the present invention has an improved shelf-life of a minimum of 1 year. The objective of the invention is to keep all macro and micro-nutrients same except moisture.
There is proportionate increase in percentage of all macro and micro ingredients, when the physical form of first milk (liquid) is changing to powder. When the moisture content is removed from first milk, all ingredients are increased.
The Buffalo colostrum powder of the present invention contains 5-30% milk fat of which saturated fat is 50-66%, monounsaturated fat is 25-40% and Polyunsaturated Fat is 1-4%. It further contains 1-3% of MCT (Medium Chain Triglyceride) as Laurie acid, which is a human friendly saturated fatty acid. It also contains principally essential fatty acid i.e. Omega 9, 25-35% in total monounsaturated fatty acid. It also contains essential fatty acid omega 6, 1-3% as part of poly unsaturated fatty acids. It also contains carbohydrates as lactose 3-24%. But it does not contain any other mono, di or polysaccharide except lactose.
The Buffalo colostrum powder of the present invention contains 0.5-1.4% of calcium, 0.1-0.3% of magnesium, 0.1-0.4% of sodium, 0.1-0.8% of potassium and 0.1-0.6%) of phosphorus.

The present invention may be illustrated by the following non-limiting example.
Example:
Example 1:
Part A:
Process Steps For The Production Of Buffalo Colostrum powder stabilized
with ascorbvl palmitate is as follows:
i. Collecting of milk i.e. first and second milk (4-5 litres) within 0-12 hours of parturition from a buffalo under total hygienic conditions. As the milk is collected in clean sterilized vessel with the help of a milking machine, so there is no human handling of the buffalo in respect of whom a clear dry period of minimum 90 days is provided;
ii. Chilling the milk to below 10° C, via passing of first milk through a heat exchanger, where there is a cross flow arrangement of milk from one direction and chilled water with 4° C from opposite direction;
iii. Salt balancing of the milk by adding disodium phosphate in a concentration of 40 mg powder of disodium phosphate added for every 50 g of total dry matter present in milk;
iv. Bactofugating the milk. Bactofuge was installed and commissioned on H2O. Flow rates were set at approximately: inlet = 1600 lph, outlet = 1440 lph. The process is carried out at 50° C-55° C and at 4000-4500 rpm, Efficiency achieved is an average of 98- 99.5 %. This process is most efficient for spores as they are relatively heavy. Maximum density of feed isll00kg/m3;
v. Adding antioxidant i.e. llmg of ascorbyl palmitate is added including the overages for every 50 g of total dry matter present in milk;
vi. Low heat spray drying of the milk was carried out using a spray dryer with automizer, carried out at following conditions:
a. Hot air Inlet Temperature:- 175 °C- 185 °C;
b. Outlet Temperature:- 85 °C - 91 °C;

c. Temperature at which product is maintained during the entire process:-65 °C-71 °C; To obtain 500g of dried powder (product); vii. Vacuum packing after filling 50 g of product in each LDPE (Low-density polyethylene) packing followed by immediate sealing. Details of the Vacuum Packing machine used are:
• Outer Dimensions: 550mm x 450 mm x 345 mm
• Packing Chamber: Height S.S. 490 mm x 360 mm x 75 mm depth
• Vacuum Pump - Suction Capacity: 10 cbm (cubic meter) per hour
• Electric Motor: Single Phase, 230 V, 0.5hp
• Package Sealing Length: 480 mm (Maximum)
• Maximum Bag Size: 480 x 300 mm
Part B:
Table 1: Composition of buffalo colostrum stabilized with ascorbyl palmitate
from Part A (Total weight: 50 g)

Component Concentration (%)
Total protein 66.97%
IgG 23.56%
Total Fat 9.85%
Total Carbohydrate 18.72%
Total Ash 3.37%
Moisture 1.14%
Disodium phosphate 0.1%
Ascorbvl Palmitate 0.012%

Part C:
Accelerated stability tests using the composition outlined in Table 1 was conducted for a period of 240 days (1 year) at a relative humidity (RH) of 75% and temperature at 40 °C. The objectives of the studies were as follows:
• Determine the stability of the product with respect to its specifications.
• Determine stability of Immunoglobulin (IgG) present in the product.
• Determine stability of product with respect to bacterial attack and contamination.
Accelerated Stability Studies:
Table 2: Accelerated Stability Study Protocol for the composition outlined in Table 1

No. Particulars Specifl carious
1 Stability Study Accelerated Stability Study
2 Conditions for Stability Study RH= 75%, Temp = 40C
3 Parameters to Measure- 23
Appearance, Color Taste, Moisture, Bulk Density, pH, IgG, Total Protein. Casein, Total fat, SAFA. PUFA, Omega 6, MUFA, Omega 9, Trans Fat, Total carbohydrate. Lactose. Ash. Calorific Value, Total Microbial Count, E. Coli & Salmonella and Yeast and Mould
The stability study protocol was developed based on ICH (International Conference on Harmonisation) Guidelines.

The stability study matrix developed based on Table 2 is outlined in Table 3.
Table 3: Stability Study Matrix

120 ISO 240 days
No. TEST Oday 30 davs 90 davs davs davs
STORAGE
PHYSICAL TESTS Yes Yes Yes Yes Yes Yes
1 Appearance Yes Yes Yes Yes Yes Yes
2 Taste Yes Yes Yes Yes Yes Yes
3 Colour Yes Yes Yes Yes Yes Yes
4 Bulk Deasity Yes Yes Yes Yes Yes Yes
5 pH of 10% w/v solution Yes Yes Yes Yes Yes Yes
6 Moisture
BY HPLC/CHEMICAL Yes Yes Yes Yes Yes Yes
j IgG Yes No Yes No Yes Yes
5 Total Protein Yes No Yes No Yes Yes
o Casein Yes Yes Yes Yes Yes Yes
10 Total fat Yes Yes Yes No Yes Yes
11 Of Which SAFA Yes No Yes No Yes Yes
12 PUFA Yes " No No No Yes Yes
13 Of WHch Omega 6 Yes . No No No Yes Yes
14 MUFA Yes No No No Yes Yes
15 Of Which Omega 9 Yes No Yes No Yes Yes
16 Trans Fat Yes No Yes No Yes Yes
17 Total Carbohydrate Yes Yes Yes Yes Yes Yes
IS Lactose Yes No No No Yes Yes
19 Ash Yes Yes Yes Yes Yes Yes
20 Calorific Value
MICROBIAL Yes No Yes No Yes Yes
21 Total Microbial Couat Yes Yes Yes No Yes Yes
22 E.coli &- salnxmells Yes Yes" Yes No Yes Yes
23 Yeast& molds per s Yes No Yes No Yes Yes
Key: Yes = test conducted; No = test no conducted


Table 4: Results of the comparison of the stability studies of Table 3
Key:
AH 50g samples were put in the stability chambers (maintained according to the
specifications of the Protocol of this example-) at the same time, and are removed
from the chambers on the specific dates mentioned on them.
F20090455-A: Sample used for the stability studies at 0 day;
F20090455-B: Sample used for the stability studies at 30 days;
F20090455-C: Sample used for the stability studies at 90 days;
F20090455-D: Sample used for the stability studies at 120 days;
F20090455-E: Sample used for the stability studies at 180 days;
F20090455-F: Sample used for the stability studies at 240 days.

From the comparison over 240 days (1 year), it is clear that appearance, colour, taste, pH, IgG, total protein etc., do not change significantly. When any food item is tested on accelerated basis i.e. 40°C/70 RH, it is Common practice to extrapolate the data. It means on conservative basis 1.5-2 times of the stability achieved can be claimed as the shelf life of the product.
The maximum variation in moisture content is ~ 32 % of the 0 day value and the bulk density is 16% of the 0 day value, but both the values are very much in the specification limit (ICH guidelines). Results are complying with specified standards (ICH guidelines).
Figures 1 and 2 represent the variations in physical and chemical properties respectively of Table 4.

WE CLAIM:
1. A stabilized buffalo colostrum powder composition comprising;
i. at least 60% Protein of which at least 20% is IgG;
ii. upto 25% Fat;
iii. an antioxidant or a synergistic combination of two antioxidants in a total concentration of 50-200 mg/kg of total dry matter present in milk;
iv. 0.05% to 0.15 % disodium phosphate as stabilizer;
v. upto 25% lactose; and vi. upto 6% ash.
2. The antioxidant as claimed in claim 1 which is selected from the group
comprising ascorbyl palmitate, standardized pine bark extract, natural
tocopherols and rosemary extract.