We are trying to measure xylitol in supernatants after fermentation in YPD-Xilose.
Please check this researchgate link and pdf attachment.
https://www.researchgate.net/publication/12747146_Colorimetric_Assay_of_Alditols_in_Complex_Biological_Samples
Good Luck
Article Colorimetric Assay of Alditols in Complex Biological Samples
Dear Nicolas,
Herein the required protocol:
R-BIOPHARM - Enzymatic BioAnalysis and Food Analysis
D-SORBITOL / XYLITOL (Colorimetric Method)
Colorimetric Method For The Determination Of D-Sorbitol And Xylitol In Foodstuffs Such As Bakery Goods, Chocolate, Diabetic And Dietetic Food, Fruit Products, Soft Drinks, Sweets And Candies, As Well As In Pharmaceuticals, Cosmetics, Paper And Cardboard And In Biological
Samples.
D-Sorbitol, a sugar alcohol, the reduction product of D-fructose, occurs extensively in fruits, e.g. in apples, cherries, pears, plums, but it is not or only in traces contained in grapes, grape juice and wine. D-Sorbitol is used in the food industry as a moisturizing agent and as a sugar substitute for diabetic
products as, in contrast to D-glucose, insulin is not necessary for metabolism.
Xylitol is a sugar alcohol that occurs frequently in fruits, vegetables and mushrooms. It is used in liver infusion therapy.
Test Principle:
D-Sorbitol and xylitol are oxidized by nicotinamide-adenine dinucleotide (NAD) to D-fructose or Dxylulose, respectively, in the presence of the enzyme sorbitol dehydrogenase (SDH, also called polyol dehydrogenase) with the formation of reduced nicotinamide-adenine dinucleotide (NADH) (1a, 1b).
(1a) D-Sorbitol + NAD+ (SDH) give D-Fructose + NADH + H+
(1b) Xylitol + NAD+ (SDH) give D-Xylulose + NADH + H+
Under the assay conditions, the equilibrium of the reactions (1a, 1b) lies on the side of NAD and Dsorbitol or xylitol, respectively. However, they are favourably displaced as the formed NADH is removed in a subsequent reaction in which NADH reduces iodonitrotetrazolium chloride (INT) to a formazan in the presence of diaphorase (2).
diaphorase
(2) NADH + INT + H+ give NAD+ + formazan
The absorbance of the formazan is measured at its maximum at 492 nm.
Materials Provided:
- 1 x Potassium phosphate/triethanolamine buffer solution, pH approx. 8.6; (25 ml) consisting of: Triton X-100 (trademark of Rohm & Haas, Philadelphia, USA).
- 3 x Lyophilizate; (35 mg); consisting of: diaphorase (approx. 4 U); NAD (approx. 28 mg).
- 1 x Iodonitrotetrazolium Chloride Solution, (approx. 2.5 ml).
- 3 x Lyophilizate SDH, (approx. 25 U, each).
- 1 x D-Sorbitol Assay Control Solution, for assay control purposes (measurement of the assay control
solution is not necessary for calculating the results.) Use the assay control solution undiluted.
Kit Specifications:
Detection Limit: - 0.2 mg/l.
Specificity: - Besides D-sorbitol and xylitol, sorbitol dehydrogenase also
oxidizes other polyols, such as iditol, allitol, ribitol, although
with reduced velocity. Other polyols such as mannitol,
arabitol, and dulcitol do not react. Glycerol is not oxidized
under the assay conditions.
Linearity: - Linearity of the determination exists from 0.4 µg D-sorbitol
or xylitol/assay (0.2 mg D-sorbitol or xylitol/l sample solution;
v = 2.000 ml) to 10 µg D-sorbitol or xylitol/assay (0.1 g Dsorbitol
or xylitol/l sample solution; v = 0.100 ml).
Precision: - In a double determination of D-galactose using one sample
solution, a difference of 0.005 to 0.010 absorbance units
may occur. The relative standard deviation is approx. 1% to
2% in the measuring range.
Samples Per Kit: - 3 x 12 tests.
Product ID #: - E0670057.
To view the original protocol, please use the following link:
http://www.xygen.com/pdfs/constituents/enzymatic/D-Sorbitol%20Xylitol.pdf
Hoping this will be helpful,
Rafik
Thank you Arvind and Rafik again!
We want to comparate three different method to measure xylitol: HPLC, enzymatic and colorimetric.
Best regards to both!
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