BRIEF DESCRIPTION OF THE DRAWINGS
[0018] It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:
[0019] Fig. 1 illustrates the Biomass cell concentration for basal medium with 1% corn starch, in accordance with an embodiment of the present invention;
[0020] Fig. 2 illustrates the Enzyme activity for basal medium with 1% corn starch concentration, in accordance with an embodiment of the present invention;
[0021] Fig. 3 illustrates the Biomass concentration for Basal medium with 0.5% defatted Soya flour, in accordance with an embodiment of the present invention;
[0022] Fig. 4 illustrates the Enzyme activity for Basal medium with 0.5% defatted Soya flour, in accordance with an embodiment of the present invention;
[0023] Fig. 5 illustrates the Biomass cell concentration for basal medium with 2.5% corn starch, in accordance with an embodiment of the present invention;
[0024] Fig. 6 illustrates the Enzyme activity for basal medium with 2.5% corn starch concentration, in accordance with an embodiment of the present invention;
[0025] Fig. 7 illustrates the Biomass concentration for Basal medium with 3% defatted Cotton seed, in accordance with an embodiment of the present invention;
[0026] Fig. 8 illustrates the Enzyme activity for Basal medium with 3% defatted Cotton seed, in accordance with an embodiment of the present invention;
[0027] Fig. 9 illustrates the Biomass cell concentration for basal medium with 0.5% corn starch, in accordance with an embodiment of the present invention;
[0028] Fig. 10 illustrates the Enzyme activity for basal medium with 0.5% corn starch, in accordance with an embodiment of the present invention;
[0029] Fig. 11 illustrates the Biomass cell concentration for Basal medium with 2% Mustard seed, in accordance with an embodiment of the present invention;
[0030] Fig. 12 illustrates the Enzyme activity for Basal medium with 2% Mustard seed, in accordance with an embodiment of the present invention;
[0031] The figure depicts embodiments of the present subject matter for the purposes of illustration only. A person skilled in the art will easily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] While the embodiments of the disclosure are subject to various modifications and alternative forms, specific embodiment thereof have been shown by way of example in the figures and will be described below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the scope of the disclosure.
[0033] The terms “comprises”, “comprising”, or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, assembly that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such system, or assembly, or device. In other words, one or more elements in a system or device proceeded by “comprises… a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or device.
[0034] The present subject matter relates to a low cst media composition from then production of alpha-amylase enzyme.
[0035] Reference may be made to Figure 1 illustrating Biomass cell concentration for basal medium with 1% corn starch, in accordance with an embodiment of the present invention;
[0036] Reference may be made to Figure 2 illustrating Enzyme activity for basal medium with 1% corn starch concentration, in accordance with an embodiment of the present invention;
[0037] Reference may be made to Figure 3 illustrating Biomass concentration for Basal medium with 0.5% defatted Soya flour, in accordance with an embodiment of the present invention;
[0038] Reference may be made to Figure 4 illustrating Enzyme activity for Basal medium with 0.5% defatted Soya flour, in accordance with an embodiment of the present invention;
[0039] Reference may be made to Figure 5 illustrating Biomass cell concentration for basal medium with 2.5% corn starc, in accordance with an embodiment of the present invention;
[0040] Reference may be made to Figure 6 illustrating Enzyme activity for basal medium with 2.5% corn starch concentration, in accordance with an embodiment of the present invention;
[0041] Reference may be made to Figure 7 illustrating Biomass concentration for Basal medium with 3% defatted Cotton seed, in accordance with an embodiment of the present invention;
[0042] Reference may be made to Figure 8 illustrating Enzyme activity for Basal medium with 3% defatted Cotton seed, in accordance with an embodiment of the present invention;
[0043] Reference may be made to Figure 9 illustrating Biomass cell concentration for basal medium with 0.5% corn starch, in accordance with an embodiment of the present invention;
[0044] Reference may be made to Figure 10 illustrating Enzyme activity for basal medium with 0.5% corn starch, in accordance with an embodiment of the present invention;
[0045] Reference may be made to Figure 11 illustrating Biomass cell concentration for Basal medium with 2% Mustard seed, in accordance with an embodiment of the present invention;
[0046] Reference may be made to Figure 12 illustrating Enzyme activity for Basal medium with 2% Mustard seed, in accordance with an embodiment of the present invention;
[0047] In accordance with an embodiment of the present invention relates to the production and optimization of high temperature alkaline Alpha-Amylase enzyme by Bacillus lichenoformis using low cost media composition derived from agricultural byproducts.
[0048] Material and Methodology: Microbial Strain Bacillus Lichenoformis NCIM 2051 was Received from National Chemical Laboratory, Pune.
[0049] Chemicals: Beef extract, Peptone, NaC1, MgSO4, Biosar KH2PO4, Biosar CaCl2, Biosar Yeast extract, Agar, Corn Starch, Defatted Cotton Seed, Defatted Soya flour, Mustard Seed.
[0050] Medium Universal Medium for Bacteria Beef extract : 1.0 % Sodium Chloride : 0.5 % Peptone : 1.0 % pH : 7.0 - 7.2 Sterilized the medium, and adjusted the pH at 7.2. Added 2% Agar for making slants.
[0051] Corn starch medium: (Basal Medium) Corn starch : 1 % Yeast extract : 0.2 % Peptone : 0.5 % MgSO4 : 0.05 % KH2PO4 : 0.05 % NaCl : 0.15 % CaCl2 : 0.015 %
[0052] Low cost medium Corn starch : 1 % MgSO4 : 0.05 % KH2PO4 : 0.05 % NaCl : 0.15 % CaCl2 : 0.015 % Soya bean : 0.5 % Mustard Seed : 2 % Cotton seed : 3 % 5.4.
[0053] Example 1: Procedure: Flask cultures were operated at constant temperature of 370C and fixed rpm with 100 ml of medium in a 500 ml Erlenmeyer flask and inoculated with the culture. Fermentation studies were carried out in above described B. Braun Biostat E fermentor with the cultural conditions of 370 C, pH 7, and 300 rpm. Since it was an aerobic fermentation, the aerobic rate was maintained at 1 vvm. 41 For every six hours the sample were collected from the sampling point provided in the top of the culture vessel, and analyzed.
[0054] Example 2: Stock Culture:Bacillus Lichenoformis NCIM 2051 was maintained in an Agar slant at 40 C. Sub Culture Maintenance Subculture was prepared using a universal Bacteria medium and it was maintained in an incubator at 370 C.
[0055] Pre inoculums: Took 100 ml of the Universal medium inoculate this with a stock agar culture in a 500 ml Erlenmeyer flask and kept in a shaker at 300 rpm and 370 C. It was also called as seeding of culture.
[0056] Biomass Biomass was estimated by the method of dry weight for every sample. It was expressed in terms of 1 dry wt. /lit. 5.8. Enzyme Activity One unit of enzyme activity (DUN) is defined as the quantity of enzyme that causes 1 % reduction of blue color intensity of starch-iodine solution in 1 min. The optical density was first measured at 660 nm using an UV spectrophotometer.
[0057] Example 3: ANALYTICAL METHODS: Biomass Estimation: The Biomass for the sample, which got from the fermentation broth was determined by the dry weight method. Took some known amount of liquid from the fermentation of liquid from the fermentation broth in the centrifuge test tube, and kept in a centrifuge for 20 mins at 5000 rpm. The supernatant liquid was collected and kept for a – amylase activity determination. The cells settled in the bottom of the centrifuge tube was transferred to a funnel contains the gravimetric filter paper (ash less), and washed thoroughly with distilled water. Transferred this gravimetric filter paper in to the known weight silica crucible, and incinerated for 30 min. Once the contents cooled down, measured the weight from this calculated cell concentration.
Model Calculation: Weight of empty crucible : 15.8603 gms.
Volume of fermentation liquid taken : 11.4 ml
Weight of crucible with cells : 15.8895 gms.
Cell mass/volume of liquid taken : 15.8895 – 15.8603 0.0292 gms
For 1 liter : 2.57 Cell concentration : 2.57 gm dry wt lit
[0058] Enzyme Activity Determination: Different techniques had been used to measure enzyme activities. Extra cellular amylase activity was determined by measuring the decrease in iodine color reaction showing dextrinization of starch. The reaction contained 1 ml of enzyme (cell free supernatant) and 10ml of 1% starch solution incubated at 400 C for 10 min. The reaction was stopped by adding 10ml of 0.1N HCl. 1 ml of this acidified solution was added to 10ml 0.1N HCl. From this 1ml was added to iodine solution (0.05% iodine in 0.5% Kl). The optical density of the blue colored solution was determined of 660 nm one unit of enzyme activity (DUN) was defined as the quantity of enzyme that causes 1% reduction of blue color intensity of starch iodine solution at 400 C in 1 min. For amylase activity determination required the standard chart for starch iodine solution. Took six test tubes, in that added 0, 0.2, 0.4, 0.6, 0.8 and 1 ml of 1% starch solution respectively and added 1, 0.8, 0.6, 0.4, 0.2 and 0 ml of water reply. Added 10ml of iodine solution (0.05% I2 and 0.5% Kl) in all the six text tubes. The inference was the light blue color formation. The optical density of the blue colored solution was measured at 660 nm in the UV spectrophotometer. The standard graph was drawn by plotting starch concentration Vs absorbance. From the standard graph the enzyme activity was calculated
[0059] Results: Production of Enzyme: The growth pattern of Bacillus Lichenoformis NCIM 2051 and a – amylase production was observed for three days in basal medium with 1% cornstarch as a carbon source. The formation of a – amylase started from 4 hours. The maximum enzyme production was achieved at 24 hours. The pH of the broth increased from 7 at the beginning to 8.9 at the end of fermentation. The maximum yield was achieved at 350 C.
[0060] Effect of Corn Starch Concentration: The effect of corn starch concentration was further studied. The a – amylase production was studied, by changing the Corn starch concentration at 0.5%, 1% and 2.5%. It was found that with an increase of starch concentration in the medium beyond 1%, enzyme production did not increase. At higher starch concentration, enzyme production was comparatively lower and the time required to reach the maximum enzyme level was longer (Figures 1-12).
[0061] Effect of pH: The bacterium was found to grow at pH 3-11, with growth resulting in an increase of the patient e media’s pH. Enzyme production started at 5.0 and ceased at pH 10.0. Maximum enzyme production occurred at pH 6-9. Very little enzyme production in the medium at initial pH of 3 - 4. At higher pH values (10-11), growth was quite high, but the amount of enzyme production was very low.
[0062] Effect of Temperature: The strain was found to grow and produce enzyme at temperatures from 25 to 500C. Maximum enzyme production was observed at 350C. Growth and enzyme production both started decreasing drastically above 400 C.
[0063] a – amylase production in low cost medium: The a – amylase production was further studied by using the low cost medium which containing the carbon and nitrogen sources like flour, mustered seeds. Since the cost of yeast extract and peptone in the Basal medium is very high, we can replace the yeast extract and peptone with the above mentioned things. The low cost medium produced 2 times more enzyme than the high cost synthetic medium (yeast extract and peptone).the medium containing 0.5% defatted, 2% mustered seed in the place of yeast extract and peptone , was found to yield the maximum amount of a – amylase. The experimental data are listed in the below tables(Tables 1-3) for different concentration and compositions of the low cost medium.

Table 1: Enzyme Production using Basal medium + 0.5% defatted Soya Flour

Table 2: Enzyme Production using Basal medium + 3% defatted cotton seed

Table 3: Enzyme Production using Basal Medium + 2% Mustard Seed
[0064] Although embodiments for the present subject matter have been described in language specific to structural features, it is to be understood that the present subject matter is not necessarily limited to the specific features described. Rather, the specific features and methods are disclosed as embodiments for the present subject matter. Numerous modifications and adaptations of the system/component of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the scope of the present subject matter.

We Claim:

1. A media composition from agricultural byproducts for the the production of alkaline Alpha-Amylase enzyme from Bacillus lichenoformis, comprising a cheap source of nitrogen, Soya bean 0.5 %, Mustard Seed 2 % and Cotton seed 3 %.
2. The media composition as claimed in claim 1 wherein, the cheap
nitrogen sources is corn steep liquor.
3. The media composition as claimed in claim 1 wherein, a –amylase shows excellent stability at high temperatures and over a
wide pH range.
4. The media composition as claimed in claim 1 wherein, the composition is inexpensive.