Phosphate estimation - (May/05/2005 )
I am working on Phytase production. I am getting problems in the estimation of Phosphate. The background phosphate showing interference. How can I come over from that?
Give suggestion for me 
Dear friend.
I am also working on phytase. I am very happy to see your list on bioprotold.
Coming to the work. I have some question in my mind. Exactly where are you getting the phosphate back ground.
1. are you getting the phosphate background when you are preparing the media for bacterial or fungal growth?.
2. are you getting the phosphate back ground during your enzyme assay using the fermented media.
3. are you getting the phosphate back ground after you are autoclaving the media.
First thing is that phytic acid sodium salt is highly heat unstable. When you autoclave it at 121oc the phytic acid will release the phosphate in to the medium. Hence the phytic acid is to dissolved in buffer and then sterilize it will 0.22 micron filter ( they are cheaply available in torson company for about a volume of 10 –20 ml. This company item I have used for my work) after the media is sterilized the filter sterilized phytic acid is to be added.
Coming to next topic. If you have want to remove the traces of the phosphate that is present in the medium that you are preparing. Normally the yeast extract( YE) and peptone are contaminated with phosphates. In that case normally I remove the phosphate by precipitation of the phosphate by using magnesium sulfate. The protocol is prepare 10% solution of YE or peptone and then add magnesium sulfate such that the concentration of it reaches 0.01M. Then the solution is kept in 4 oc ( in fridge) for overnight. Then next day the solution is checked for any precipitation. If there is any precipitation then is the phosphate complex that are formed. This is removed by centrifugation at 10000 RPM for 10 min. the supernatant is dispensed as aliquots and the used in the medium preparation.
Coming to phosphates that are coming in the medium after the fermentation .during the crude enzyme assay. There are two different methods to over come this problem
Fist once is cumber some but give a perfect result. In this method 5 ml of the fermented culture medium is taken in dialysis membrane ( cut off mark 10,000 or 12,000 daltons ) and it is dialysed against the buffer for over night. This dilaysate is used as the test sample for the analysis of the enzyme yield
The second method is when we are doing the enzyme assay, normally the procedure I follow is for the test sample I will be taking 1 ml of the fermented culture broth +0.5 ml of the buffer ( sodium acetate buffer) +0.5 ml of 0.01 M of phytic acid solution .
Where as for the blank I will be taking 1 ml of the fermented culture broth (heated for 10 min at 80 oc to in activate the enzyme ) +0.5 ml of the buffer ( sodium acetate buffer) +0.5 ml of 0.01 M of phytic acid solution . The logic behind this approach is very simple.
In the fermented media there will be some phosphate, which is left over after it is released and is not utilized. This might give a background. Then I will take this utilized phosphate into account by taking it as control. So whatever values I get for the test sample is due to the phosphate liberated by the enzyme when acted upon the newly added the phytic acid. As the enzyme is inactivated due to heat denaturation there is no logic in expecting an increase in the value.
I also want to know what is method you ar following to estimate the phosphate there are 4 methods that are currently available for the estimation of the phoate
1. AOAC method.
2. fiske subbarow
3. copper and gowing method
4. wyss method
our analysis have show that AOAC methods is good for analysis to estimate the phosphate in the presence of the phytic acid. As there are reports that state that phytic acid interferers with phosphate estimation method.
I hope that you have enough information to solve you problem if have still problem please fell free to contact me at this email ID mukesh_p78@yahoo.com
I think so.
thank you very much!
Hi all,
How can i extract, quantify,and detect phytic acid from rice grains.
please give a working protocole or reference .