Dear Iman,

The first step of titration involves the free acid groups. In the second step, de-esterification happens in presence of alkali.

I am trying to understand the process. The enzyme hydrolyzed solutions may also have the presence of enzymes and buffer. In this case, changing the native pH of pectin solution may affect your readings. Now, when you digest the pectin samples, one may try to reprecipitate the pectin after digestion. As a result, now I will have a processed pectin with a DE. A solution of this pectin may be prepared and titration may follow.

Another way could be using the controls. How about preparing a blank without the pectin (enzyme + NaOH + whatever you have used for the pectin enzymatic hydrolysis). When you titrate the blank in absence of pectin, you will find values for this. After pectin digestion, if you wish to use this solution for DE determination, you will a set of values for the blank and rest of it for your treated solutions.

Please note that I am not a chemist. This is purely based on my experience with pectin DE determinations.

Good Luck

Hi Iman, this is indeed a problem.

In general, esters are nor very chemically reactive and treatment with NaOH will hydrolyze the glyosidic bonds between the sugars before they hydrolyze the esters.

There is only one way I can think of to get rid of all of the methyl esters in pectin.

Treat the pectin with elastase. Although elastase is a metabolic enzyme that cleaves peptide bonds, it is also an esterase that is just as happy to cleave peptide bonds and ester bonds.

Hi,

I guess you are trying to measure pectine methylesterase activity (or pectineacetylesterase activity, it would be the same). This is the classical method:

- dissolve the pectin in water or 0.1 M salt (NaCl, NaNO3) solution;

- Carefully bring it to pH 7 (to neutralise the free carboxyl groups)

- At the enzyme's reaction temperature (20 to 37°C?) and under constant pH monitoring : ADD the enzyme: pH will decrease as it liberates more carboxyl groups: add NaOH (keeping carefull account of how much and when) to keep the pH at 7.

- This gives you directly the enzyme activity in moles of NaOH used (=moles of COO- liberated) / seconds

- of course it is much more convenient with a titrator...

For examples: Food Bioprocess Technol (2017) 10:662–673

DOI 10.1007/s11947-016-1850-7

or

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE 80 (2000) 1503-1509

And to correct the previous answer: yes, the alkali DO saponify the methyl groups of pectins way before they cleave the normal glycosidic bonds (this is not true for methylated pectins, which are highly susceptible to beta-elimination, see Albersheim's work in the 1960's): polysaccharides are actually usually oxidised fom the reducing end, not cleaved, in alkaline conditions.

To completely demethylate pectins just use pH > 12 and temperatures around 4°C. : look up

Renard C.M.G.C. & Thibault J.-F: Degradation of pectins in alkaline conditions: kinetics of demethylation. Carbohydrate Research 286 (1996) 139-150.

and

Renard C.M.G.C. & Jarvis M.C. Acetylation and methylation of homogalacturonans 1: optimisation of the reaction and characterisation of the products. Carbohydrate Polymers 39 (1999) 201-207. (this one actually has the method to demethylate pectins)

or

Renard C.M.G.C. & Thibault J.-F.: Kinetics of demethylation of pectins in alkaline conditions. in: Pectins and Pectinases, Process in Biotechnology vol 14, J. Visser & A.G.J. Voragen (eds), Elsevier, 1996, 603-608. (it also has the kinetics of betaelimination)

cordially

Catherine Renard

Dear Catherine,

Many thanks for the provided references. Actually, I'm using different enzymes (Lactase, glucosidase, galactosidase, pectinase etc.) to study their efficiency for producing oligogalactosides from pectin where I thought the titration and degree of esterification would give an indirect information about the pectin degradation as a result of enzymes activity during the time intervals. I guess it's not a welcomed methods in this context. I'd be thankful to receive more remarks if your precious time allows !

Thanks again,  

Iman 

Dear Iman,

You can produce oligosaccharides from pectin (oligogalacturonides , oligogalactosides...) but NOT fructooligosaccharides as it is NOT a fructose polymer, you want inulin or another fructan for that. To compare enzyme activities in splitting polysaccharide chains you can try and measure reducing power, there are colorimetric assays.

Cordially

Catherine Renard

Dear Catherine,

I'm so sorry, It was a silly mistake of mine. You are right. I mixed it up with Inulin which I'm also working with.     

By colorimetric assays, you mean ONPG or PNPG? or the liberation of glucose?    

Will you be so kind to lead me to some publication for colorimetric assays if you have in mind or some more keywords! 

I worked with ONPG or PNPG but I'm not quite sure how to apply them in pectin solution! A simple spectrophotometric measurement did not reveal any detectable pattern before and after enzymatic digestion for pectin solution!

Many many thanks again,

Iman

No, by colorimetric assays I mean assays that measure the generation of reducing sugars end groups : the classical Nelson-Somogyi method, dinitrosalicylic acid, or the simpler cyanacetamide method (but it needs UV). They do not require a specific sugar and work with pectin as well as cellulose for example.

Somogyi, M. (1952). "Notes on sugar determination." Journal of Biological Chemistry 195: 19-23.

Honda S. et al.: A manual method fo the spectrophotometric determination of reducing carbohydrates with 2-cyanacetamide Analytical Biochem 119 (1982) 194-199.

Hatanaka C. & Kobara Y. (1980) Determination of Glucose by a

Modification of Somogyi-Nelson Method, Agricultural and Biological Chemistry, 44:12, 2943-2949,

Breuil C. & Saddler J.N. Comparison of the 3, 5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying for reducing sugars and determining cellulase activity. Enzyme and microbial technology, 7 (1985) 327-332

Cordially

Catherine