Invariably distilled water is used. Then one can further proceed to work out the comparative effectiveness of the two extract ants

If you are using enzimes is very important use buffers. You have to do blanks and in the final process, lyophilize then.

I want to know the significance. Answers from Dr. Daljit and Rodriguues are appreciated but looks like hust informative. I expect more of discussion on scientic ground

Water is mostly used but you have to keep the pH constant by frequent addition of NaOH in order to maintain high level of enzyme activity at the optimum pH. As you know, during enzymatic protein hydrolysis, hydrogen ions (H+) are released when peptide bonds are broken. Accumulation of H+ causes reduction in pH, which necessitates NaOH addition to bring it up to the optimum hydrolysis point of the protease being used. For example, Alcalase is highly active at pH 8.0. During protein hydrolysis with Alcalase, the pH will decrease continuously and you must add NaOH to bring it back to pH 8.0. If you don't adjust back to pH 8.o, you will not get efficient hydrolysis of your protein and the desired type of bioactive peptides (quality) or amount of bioactive peptides (quantity) may not be realized. The use of buffers is discouraged because it can add a lot of salt to the protein hydrolysate. Moreover, if you use a buffer, it may be difficult to use the protein hydrolysate at another pH level during determination of bioactive properties.

Mr. Krishnamoorthy , how do you intend do an evaluation of bioactive peptides?

for example, if you utilize human cells, the buffer choice can not contain chemicals that attack the cells. There are incovenients (salts) to use buffer, but there is ways to eliminate then. After the hydrolisys, you can make a dialysis which consists in put the hidrolysates in a membrane and wait that salt pass, for difusion, to the external water.

If you want to sequence the peptides, the amount of salt won't interfere in the analysis because of the sensibility of equipment MS/MS.

On the other hand, to use or not use the buffer depends of the posterior analysis.

Answers from Rodrigues and Aluko added the value to the question

Is the enzyme in powder or liquid form? You could always look at the detail of the product from the manufacturer's website. I used protease in liquid form, and dilute it in Milli-Q water to have the desired enzyme activity. What are your samples ?

I am using four different enzymes of which both liquid and solid kind of enzymes are there. Thing is significance of using distilled water instead of buffer. During the hydrolysis process we may keep the reaction mixture at higher temperature during which the presence of salt may react with the hydrolysed product as well as with the reaction mixture. Another thing as Marcelo mentioned it is sometyhing which should be decided by the end use and as mentioned by Aluko it is related the hydrolysis efficiency which may be affected by the drop in pH during hydrolysis.

My samples are fish meat protein and looking for the preparation of hydrolysates and analysing the antioxidant and Antihypertensive properties in vitro. I hope the form of enzyme whether liquid or solid have a particular optimum pH and should be maintained

Two questions are proposed, the first being related to the successful isolation and potential clevage of a protein macro structure, the second being the the analysis of its bioactive properties.

When considering protein isolates from a raw product a decision must be made whether particular characteristics of the proteins isolated are more highly valued than others and whether groups of proteins have selective benifits over others isolated. For example one group of proteins may have a higher composition of sulfur based amino acids than another which will effect their nutritional, physical and bioactive attributes.

First question should be how pure and stable is your protein isolate in its primary secondary and tertiary structure.

Next question is after isolation is there any benifit in chopping it up enzymatically or using hydrolysis techniques, if you chose this pathway we are begining to get a long way away from any resemblance to the original source product, usually by this stage the proportion of the material showing original bioactive properties has been significantly changed if a comparison is desired.

If no comparison with original product is desired then prehaps your concentration should be on the ability to consistantly produce your peptide hydrolysates and your ability to define their structure and diversity. This should be repeatable and known prior to any bioactive assessment.

If you choose to use your bioactive assessment as a screening tool for a range of treatments/isolations then each protein treatment/isolation must be reproducable and again assessment for primary secondary and tertiary structure made once selection and isolation is acheived otherwise the experiment looses significant value. There is a strong risk if the structural identification is not done that QA/QC in scale up production may never be able to be met.

When considering your treatment process you must understand that due to the zwitterion characteristics of amino acids changes to pH will affect structure and could effect bioactive properties due to the changes in protein conformation cross linkage capacity etc. Chopping up the longer chain proteins will significantly reduce this variability and significance but may also lose bioactivity, this needs to be understood and a structured approach made to decision making based on cause and effect relationships using a range of experimental tools. Working with peptides is much easier than with large proteins or mixes of proteins.

An approach could be to assess bioactivity accross a range of pH for each peptide preparation, this will allow you to specify optimal conditions for activity for each peptide set produced.

As mentioned by others your scope will largly depend on what bioactivite measure you are examining and in what medium and whether the measure is to be made invitro ir invivo.

Basically in the preparation of the hydrolysate distilled water is being used. But is better if you use MilliQ water. As in the hydrolysis we are using enzyme, so we need to control the pH by using NaOH or HCl. So that the enzyme activity will always in optimum pH condition.

Mr. Krishnamoorthy, all opinions are very good and you can take them. The antioxidant and antihypertensive analysis can be altered by the salt amount, because some of them are enzimatic reactions. In my opinion, you can use MilliQ water in your protein isolates. The proteins are natural buffer, so it is possible to control the pH only using NaOH or HCL. After this, as I said, you can make a dialysis, removing all the salt. To end, lyofilize the protein hydrolysates and ressuspend, as you want, to make antioxidant or antihypertensive analysis.

You have to be in mind that, in vitro hydrolysys will not be as same as in vivo and there are conditions that is impossible control at the same time. For this, make a blank with the protein isolates without the enzimes. You will get the information about the hydrolysis caused by the manipulation.

I thank all those involved in the discussion and made it interesting. I have couple of opinions on this discussion. The answers or discussion was on the two line. one is on the optima of enzymatic reaction. The other one on the effects on biological activity.

Is there any work / reference on such aspects like effect of using distilled water and buffer on the biological activity or the or on the hydrolysis process. Though it is well understood on the pH optima of hydrolysis, what will be the drop in the pH during hydrolysis. Can we think of any other effects of presence of salts in the buffer system? interaction of these salts with particular amino acid residue and changing the nature?

It would be better that you use water. Buffer contains salt and since you are working on bioactive peptides, will influence your products quality. pH does not change significantly. I used buffer, then I found that, it is going to be an issue. I just adjust the pH for enzyme.

preferably used buffer