 Dear students in this experiment we will learn about production of enzyme alpha myelase by submerged fermentation. In submerged fermentation the microorganism grow as suspension in fermentation media. The fermentation media contains substrate that is either dissolved or a solid substrate is added to a large amount of liquid media. The organism is grown in liquid medium that is aerated and agitated continuously. Submerged fermentation has been used for the production of different kinds of microbial enzymes. There are several benefits of submerged fermentation including proper agitation and aeration, uniform distribution of nutrients and waste products, uniform distribution of cells and improved process control. However submerged fermentation is costly and there are more chances of contamination. Alpha myelase catalyzes the hydrolysis of alpha 1-4 glycosidic bonds in starch and give products like dextrins and maltose. Alpha myelase is produced by different microbes including bacteria, fungi and yeast. The medium composition and the process parameters required in the fermentation experiment vary according to the type of the microorganism selected for the experiment. The fermentation media is already prepared. This medium contains 1% soluble starch, 0.5% magnesium sulphate, 0.02% potassium nitrate, 0.5% potassium dihydrogen phosphate and 0.1% calcium chloride. The pH of the medium was adjusted to 5.5. 25 ml of the fermentation media was 4 to 250 ml Aranmer flask. The flask is covered with a cotton plug and aluminum foil and is sterilized in an autoclave at 0.21 degrees centigrade for 15 minutes at 15 psi. Conadial suspension of pure culture of a specialist niger will be used as inoculum. For the preparation of conadial inoculum add 10 ml of sterilized distilled water or 0.05% monoxyl OT. Use a sterile inoculating loop to break the clumps of the conadia and shake the tube vigorously to make a homogeneous suspension. Then 1 ml of the suspension will be added to the fermentation media. The conadial count per ml will be done using hemocytometer. Inoculation place the flask in a shaking incubator at 30 degree centigrade 200 rpm for 72 hours. It is already processed and incubated for 72 hours. After 72 hours of incubation transfer the contents of the flask to a 15 ml centrifuge tube and 11 ml of the fermented medium to the centrifuge tubes. Use the contents of the flask at 6000 rpm for 15 minutes. The centrifuge tubes, centrifugation two layers are formed, pellet contains the cells and the cell debris whereas the upper supernatant and clear layer will be used for the estimation of alpha myelies. The next process of the quality check and others. So whenever we have to take the samples we will check the contamination too. After under the examination of the microscope. So gradually after four hours we will go proceed for the estimation of the product is formed or not and then we will continue this fermentation process for about 72 hours because we will proceed the fermentation process on fermenter after optimizing in a shake flask. So during the shake flask study we have optimized that aspergillus niger on this medium is produced maximum myelies at 72 hours. But we will check other parameters that how that agitation, evaporation, pH and other factors will affect as we have studied that we can change the different ingredient of the medium just to optimize this fermentation process. So we can utilize a different approaches as we have studied in our theory modules about placid berman design and other central component design. But at this time after four hours we will check the samples and then we will talk about that either our product in term of myelies is produced or not if produced how much that is produced. So after four hours we will take the samples now as I have already told you while taking the sample first we will discard the first 5 ml we have take out the 5 ml sample. Now we will analyze it for the you can see here the sample. So we will use first of all we will separate the biomass from the so we use 1 ml 1 ml and 2 append off and then we will centrifuge. So we will put into the list we have to put an equal amount in the opposite direction and then we will fix then we will select the so we will put 10 minute at 10,000 rpm and then we will have a start. So after the centrifugation after 10 minute we will use the supernatant for enzyme estimation and then the remaining biomass for the dry cell mass. Now the centrifugation is over so we will take out our samples so you can see that there is a little pallet behind and we will use the supernatant as a enzyme assay. So we to start the estimation of the mileage production we will add 1 ml of the starch solution in in flask in the test tubes. So we have added the 1 ml of the starch into four different test tubes then we will add 1 ml of citrate phosphate buffer having the suitable pH that is 5.5 in each so we have added the citrate phosphate buffer then we add 100 microlitre of the enzyme which we called as supernatant into the two test tubes in one test tube we will add 100 microlitre the buffer in one test tube we will add a standard maltose 0.2 milligram per ml of the standard which we have here already made. So in the third one test tube which will call as a control so this is basically we can call as blank this is for standard in which we add the 0.2 milligram of that and it is the control in which we will add the enzyme after the incubation and two are the experimental so after adding it we will incubate all these test tubes into the shaking incubator in a shaking water bath at temperature about 45 degrees celsius. So Mr. Osman will incubate these test tubes in water bath incubating shaker for 15 minutes. So we will come back again after 15 minutes to check either the enzyme is active or not. So as we incubate our five different test tubes first one are blank and then were standard and then there was a control and two were experiment. So we will check that the amylase is produced or not just we add one percent one ml of one percent of star solution in each and then we add the enzyme extract into these tubes or in the last we will only add which we called as the control later on so because there is no more incubation time. So now we will add there are two methods we check either the starch is hydrolyzed or not one is just adding the iodine if there will be a blue color it means that there will be a starch is present if there will be a no more blue color then we say that the enzyme produced during during the fermentation process that hydrolyze the whole starch. So first we will check it on the iodine just by putting it into this we will take the 100 microliters of the experiment and then we add the iodine you can see that color so by the same way another if we add and we add as a control which we called as the blank so you can see that what is the difference. So in 15 minutes the enzyme produced through this fermentation totally hydrolyzed the starch that how much it hydrolyzed we will quantify it with DNS method. So for the DNS method we will add one ml of DNS dinitro salicylic acid solution after adding the DNS we will put all these five test tube into the boiling water for five minutes the water is already here the brown color formation will show the intensity of the maltose or a reducing sugar produced in result of the starch hydrolyzation so after five minutes we will check that how much is the brown color. So by comparing with the control and the standard on a spectrophotometer at the wavelength of 550 nanometer we can check and quantify the starch hydrolyze in result of the amylase produced. As the iodine test shows that it is the complete hydrolysis of the starch present in that sample it means that the aspergillus niger has produced a potential amount of the amylase in that. So we can also check the protein produced during this fermentation process so by estimating the protein produced we can estimate the specific activity of this fermentation process. As I already told yet we have to put after the DNS for 15 minutes in the boiling water so after the five minutes we will take out all our as we have seen in our iodine test that almost all the starch has hydrolyzed you can see easily here the comparison of the control with the experiment. So in which there was no enzyme and there is the experiment. So here you can see another in which we add the enzyme just without the incubation we have no time for the incubation so there is no more hydrolyzation. So in one test tube we have added the standard of maltose 0.2 milligram per ml. So this standard we can compare the these so this first one is the control in which there was only starch and no enzyme and here is the maltose reducing sugar 0.2 milligram per ml. So here you can see that how much is the red color. So now we can estimate that how much of 100 micro ml of the enzymes in 10 minutes that how much maltose is produced. So we can check and quantify in a spectrophotometer. So let's go on a spectrophotometer. So first we will add the blank first we will put the blank and then make it zero. So we will add the experiment but before adding the experiment we will add the our standard which contain 0.2 milligram per ml of maltose the reading is 0.29 absorbance. Then we add our experiment the reading is 1.68. So as we have seen that while taking the absorbance of the standard 0.2 milligram per ml of maltose we have the reading 0.29. But our experiment that has the reading 1.926. So this experiment as we use 100 micro liter of the enzyme and incubate for 15 minutes 15 minutes. So we can calculate here that how much is the enzyme activity just by putting the formula. So the absorbance of the experiment 1.926 divided by the absorbance of the standard 0.290 multiply by the concentration of the standard 0.2 and then the enzymes volume of the enzyme. So that is micro 100 micro liter that is about 0.1 ml. So if we divide this by the time of incubation it is 15 minutes. So if we calculate this we can calculate here 1.926 multiply by 0.2 is equal to 0.385 and then 0.029 multiply by 0.1 multiply by 15 so is equal to 0.435. So if we divide 0.385 divided by 0.435 so it is 0.88 milligram per ml of the maltose formed by 1 ml of the enzymes in one minute. So if we want to calculate the unit of the enzymes so convert this 0.88 milligram into the molecular into the micromole. So for conversion of the micromole so we have to convert 0.88 multiplied by 1000 and divided by the molecular weight of the maltose there is about 2.36 units per ml per minute. So in 4 hours of fermentation we produce the enzymes 2.3 units per ml. So we can easily say that if the total amount of 505 liter so 5 liter if we convert into 2 ml we can calculate this what is the total amount of the unit which we produced by this fermentation within 4 hours.