Title of Invention	"A PROCESS FOR THE EXTRACTION OF AMYLASE RICH FRACTION FROM MALTED CEREALS"
Abstract	The present invention provides a process for the extraction of amylase rich fraction from malted cereals. The process of the present invention enables to obtain maximum amylase activity in ragi as well as cereal malts which can be used for improving the dough characteristics of hypo-diastatic flours for various wheat based product preparations.

Full Text

The present invention relates to a process for the extraction of amylase rich fraction from malted cereals. The present innovation deals with obtaining maximum amylase activity in ragi as well as cereal malts which can be used for improving the dough characteristics of hypo-diastatic flours for various wheat based product preparation. Ragi as well as cereal malts are used in India to prepare infant, geriatric and health foods in addition to their usage in brewing and baking industries. Cereals are known to be rich in amylase activity. Amylases are globular proteins and can be extracted with buffers and salt solutions at physiological pH. Malted cereals are rich sources of alpha amylases and they occur in multiple forms. Reference may be made to the studies of M. Nirmala, M.V.S.S.T. Subba Rao and G.Muralikrishna. Food chemistry 69 (2000) 175-180, wherein 72 hr ragi malt had maximum amylase activity. Reference may be made to the work of Kruger and Tkachuk, R Cereal Chem 46, (1969) 217-219 Warchaleswski, J.R and Tka chuk . R Cereal chem. 55(1978) 146-150 Greenwood and MacGregor, A.W. J. Inst. Brew 71(1965) 405- 417, wherein wheat and barley alpha amylases were extracted with 0.2% calcium acetate at ambient temperature as well as with 0.001 M calcium acetate at 40°C. Another reference may be made to the work of Marchylo, B; Kruger J.E and Irvine, G.N. Cereal chem. 51(1974). 508-529, wherein 0.2% Calcium Chloride was also used for the isolation of amylases from Hard spring wheat. Reference may be made to the studies of Machiah, J.P and Vakil, V.K. J. Bio. Sci. 6(1984) 47-59, wherein acetate buffer containing 1mM CaCI2 was employed for the extraction of alpha amylases from immature barley and wheat. Yet another reference may be made to the work of Zawistowski, L; Langstaff, A. D and Friesen. Cereal Chem..65(1988)413-416, wherein anionic buffer such as Tris -HCI was employed for the extraction of a-amylases from malted barley and wheat. Reference may be made to the work of Botes, DP; Joubert, F. J; Novellie, J. Sci. Food Agri. 18(1967) 409-415,wherein water was also used as an extractant in isolating sorghum malt alpha amylases. The main object of the present invention is to provide a process for the extraction of amylase rich fraction from malted cereals which obviates drawbacks outlined above. Another object of the present invention is to optimize the parameters to obtain maximum amylase activity from the malt. Accordingly, the present invention provides a process for the extraction of amylase rich fraction from malted cereals characterized in that the extraction is done in phosphate buffer wherein the process steps comprising: (a) dispersing the malted flour in a 0.049-0.005 M phosphate buffer at a pH ranging between 5.5 and 6.6 to obtain a slurry; (b) stirring the slurry obtained in step [a] at a temperature in the range of 4-6°C,for a period ranging from1-2 hr; (c) centrifuging the stirred slurry obtained in step [b] at speed ranging between 5000-6000 rpm for a period ranging from 20-30 min at a temperature in the range of 2-4°C and collecting the supernatant; (d) repeating the steps [a] to [c] for the pellet as obtained in step [c] to collect the second supernatant and mixing these two supernatants by using a stirrer and (e) dialyzing the supernatant as obtained in step [d] against the buffer of molarity ranging between 0.049-0.005 for a period ranging between 2 to 4 hr to obtain the desired amylase rich fraction In an embodiment of the present invention , the malt source used may be selected from ragi, rice and wheat. In another embodiment of the present invention , the amylase activity of cereal malt obtained may be selected in the range of 230-240 units/g for ragi, 725-735 units/g for wheat and 170-180 units/g for rice. Even though there are many extractants used in the literature for isolating amylases there is no single method mentioned to be the best compared to others. There has always a need to define the condition for extracting maximum amylase activity with respect to cereal malts, since they are used in geriatric, infant brewing and health food/ beverage formulations. Accordingly, following parameters have been chosen to select the best conditions a) extractant and temperature b) ionic strength c) number of times required to extract the maximum activity from malt. Ragi malt was chosen for defining the above parameters for extracting maximum amylase activity in order to use in various wheat based products. 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 Four ml each of Sodium acetate buffer (50 mM) of pH 4.6, Sodium phosphate buffer (50 mM) of pH 6.0, Tris-HCI buffer (50 mM) of pH 6.0, NaCI (50mM) and Water (control) were taken in individual beakers and to each of them was added 1 gm of malt flour, individually and was extracted both at 27°C and 4°C and was finally centrifuged. The activities were determined in the supernatants after dialysis. Extractant Total Activity* 27°C 4°C Sodium Acetate buffer pH 4.6, 50 mM T50226 Sodium Phosphate buffer pH 6.0,50 mM 101 235 Tris -Hcl pH 6.0,50 mM 154 218 NaCI, 50 mM 139 226 Water 138 218 (Maltose units/g malt) Example 2 Keeping in view of high amylase activity as well as specific activities, phosphate and acetate buffers were chosen for standardizing the condition with respect to buffer ionic strength. Sodium phosphate (50 mM) extractant was found to be the best condition compared to 100mM phosphate buffer as well as 50 mM and 100 mM acetate buffer as well as 50 and 100 mM acetate buffer extractants. Buffer Sodium acetate, pH 4.6 Sodium phosphate, pH 6.0 Ionic strength 50 100 50 100 Total activity 226 174 235 205 Example 3 To achieve quantitative isolation of amylases, 72 hr malt was extracted with phosphate buffer repeatedly for three times. More than 90% of the activity was obtained in the first two extractions and subsequent (3rd) did not yield any significant activity. Effect of repeated extractions on amylase activity from ragi malt Extractant (50mM) No. of extraction Total activity Sodium acetate pH 4.6 Sodium phosphate pH 6.0 1 2 1 2 226 175 235 176 Keeping the above results in proper perspective the following method is chosen for the extraction to obtain amylase activity from ragi malt which is of immense value in the malting and brewing industries. 50mM Sodium Phosphate buffer with a PH 6.0 at 4°C with number of extractions restricted to two will give maximum amount of amylase activity. Above experimental conditions can be extrapolated for other Cereal malts and need not be confined to ragi malt alone. Accordingly wheat and rice malts were tried with the above extractants and concurrent results were obtained and are presented as follows. Example 4 These experiments were carried out at 4°C Extractant Wheat malt Rice malt 50mM acetate buffer, pH 4.6 697.17 137.02 50mM Phosphate buffer, pH 6.0 729.60 176.60 50mM Sodium Chloride 672.60 140.93 50mM Tris buffer 660.04 140.93 Water 516.80 126.0 From the above experiments it can be clearly stated that phosphate buffer 50 mM at 4°C is ideally suited to extract maximum amylase activity from Cereal malts. Phosphate buffer is permitted in the food industry and can be used without any deleterious effects and side reactions. The main advantages of the present invention: The activities obtained by phosphate buffer extracts from the above malts will be very useful in maintaining appropriate dough characteristics in hypo diastatic flours (low amylase activity) in place of bacterial alpha amylases due to their low temperature stability facilitating an improved product. On the other hand bacterial amylases gives reddish brown color to the bread due to their high activity even at high temperatures. We claim: 1. A process for the extraction of amylase rich fraction from malted cereals characterized in that the extraction is done in phosphate buffer wherein the process steps comprising: [a] dispersing the malted flour in a 0.049-0.005 M phosphate buffer at a pH ranging between 5.5 and 6.6 to obtain a slurry; [b] stirring the slurry obtained in step [a] at a temperature in the range of 4-6°C,for a period ranging from 1-2 hr; [c] centrifuging the stirred slurry obtained in step [b] at speed ranging between 5000-6000 rpm for a period ranging from 20-30 min at a temperature in the range of 2-4°C and collecting the supernatant; [d] repeating the steps [a] to [c] for the pellet as obtained in step [c] to collect the second supernatant and mixing these two supernatants by using a stirrer and [e] dialyzing the supernatant as obtained in step [d] against the buffer of molarity ranging between 0.049-0.005 for a period ranging between 2 to 4 hr to obtain the desired amylase rich fraction. 2. A process as claimed in claim 1, wherein the said malt source used is selected from ragi, rice and wheat. 3. A process as claimed in claims l and 2, wherein the said amylase activity of cereal malt obtained is 230-240 units/g for ragi, 725-735 units/g for wheat and 170-180 units/g for rice. 4. A process for the extraction of amylase rich fraction from malted cereals substantially as herein described with reference to examples.

Documents:

339-DEL-2002-Abstract-(11-08-2008).pdf

339-del-2002-abstract.pdf

339-DEL-2002-Claims-(11-08-2008).pdf

339-del-2002-claims.pdf

339-DEL-2002-Correspondence-Others-(11-08-2008).pdf

339-del-2002-correspondence-others.pdf

339-del-2002-correspondence-po.pdf

339-del-2002-description (complete)-11-08-2008.pdf

339-del-2002-description (complete).pdf

339-DEL-2002-Form-1-(11-08-2008).pdf

339-del-2002-form-1.pdf

339-del-2002-form-18.pdf

339-del-2002-form-2.pdf

339-del-2002-form-3.pdf