Raffinose/Sucrose/D-Glucose, colorimetric method

Catalogue number: AK00261, 100 tests of each analyte

Application Galactosyl-sucrose oligosaccharides or raffinose-series oligosaccharides (RSO) are hydrolysed to sucrose and D- This rapid and simple specific enzymatic method is used for galactose using -galactosidase. Sucrose is hydrolysed to D- the simultaneous determination of sucrose, D-glucose and glucose and D-fructose with -fructosidase, and measured as galactosyl-sucrose oligosaccharides (raffinose, stachyose and D-glucose. This method does not differentiate between verbascose) in flour mixtures, seeds, seed meal as well as raffinose, stachyose and verbascose, nevertheless measure other matrices. these as a group (RSO). Since one mole of raffinose-series oligosaccharides contains one mole of D-glucose, the Introduction concentrations are presented on a molar basis. Leguminous are significant component of both human and livestock diets. Galactosyl-sucrose or raffinose-series Linearity and precision oligosaccharides (RSO) are major components in many food Linearity of the determination exists from 10 to 100 μg D- legumes. The anti-nutricional activity of grain legumes is glucose, raffinose or sucrose per assay (see Figure 1). frequently attributed to these oligosaccharides. Due to the Standard errors below 5% are reached routinely. absence of -galactosidase, RSO are not hydrolysed in the upper part of digestive tract. In the lower intestine, Kit composition metabolization of RSO by bacteria leads to flatulence and diarrhoea. Thus, RSO limit the use of grain legumes in both Solution 1. Buffer (20 mL, pH 4.6). Stable for 2 years at 4 °C. human and animal diets. Dilute the content of bottle 1 to 100 mL with distilled water before use. Stable for > 1 year at 4ºC. Principles Suspension 2. -Fructosidase (2.4 mL). Stable for 2 years at 4 °C. Swirl bottle before use. GOx D-glucose + O2 + H2O D-Gluconate + H2O2 Add 0.02 ml of Suspension 2 plus 0.180 ml of Solution 1, per assay, to a test tube and homogenise (Solution 1+2). This 2 H2O2 + p-hydroxybenzoic acid + 4-aminoantipirine solution should be prepared for each assay day. POD Quinoneimine dye + 4 H2O Suspension 3. -Galactosidase (2.2 mL). Stable for 2 years at 4 °C. Swirl bottle before use.

Add 0.005 of Suspension 3 plus 0.02 ml of Suspension 2 plus 0.175 ml of Solution 1, per assay, to a test tube and Hydrolysis of raffinose (RSO) (at pH 4.6) homogenise (Solution 1+2+3). This solution should be -galactosidase prepared for each assay day. Raffinose Sucrose + D-Galactose Solution 4. GOD-POD reagent buffer (30 mL, pH 7.4), p- hydroxybenzoic acid and sodium azide (0.64% w/v) as a preservative. Stable for 3 years at 4 °C. Hydrolysis of sucrose (at pH 4.6) Dilute the contents of the bottle to 1.0 L with distilled water -fructosidase and use immediately. Sucrose + H2O D-Glucose + D-Fructose Mixture 5. GOD-POD reagent enzymes. Freeze-dried powder of glucose oxidase (GOD), peroxidase (POD) and 4- aminoantipyrine. Stable for 5 years at -20 °C. Free D-glucose in the sample is determined directly with GOPOD Reagent by conversion to a red coloured Dissolve the contents of one bottle 5 in approx. 20 mL of quinoneimine dye compound through the combined action solution 4 and quantitatively transfer this to the bottle of glucose oxidase and peroxidase. containing the remainder of solution 4. Cover this bottle with aluminum foil to protect the enclosed reagent from light. Safety Stable for 3 months at 2-5 °C or 12 months at -20 °C. The general safety measures that apply to all chemical Solution 6. D-Glucose standard solution (5 mL, 1.0 mg or substances should be followed. For more information 0.556 mol/mL) in 0.2% (w/v) benzoic acid. Stable for 5 years regarding the safe usage of this kit please refer to MSDS at room temperature. available at www.nzytech.com. Use as supplied. Precautions and controls Powder 7. Control flour sample. RSO, sucrose and D-glucose contents shown on vial label. Include reagent blanks and D-glucose controls 0.556 moles (i.e. 100 g) in quadruplicate with each set of assays. Use as supplied. Prepare as a sample (see Examples of sample preparation) Analyse an extract from the control powder with each set of assays.

The time of incubation with GOPOD reagent is not critical, 1.00 D-Glucose but should be at least 20 min. However, the time for

maximum colour formation with 100 μg of D-glucose

0.80 standard should be checked, for each new batch of GOPOD Reagent (usually: 15 min).

0.60 The colour formed through the reaction is stable at room temperature for at least 2 hours after development. 0.40

Raffinose Procedure (endpoint analysis) 0.20 Absorbance@ nm 510 Wavelength: 510 nm 0.00 Cuvette: 1 cm light path (glass or plastic) 0 20 40 60 80 100 Temperature: 40 °C g/test Final volume: 3.3 mL Figure 1. Linearity of the assay. Standard curves relating D-Glucose Sample solution: 10-100 μg of total RSO, sucrose and D- and Raffinose concentration (g/test) to absorbance at 510 nm (40 glucose per cuvette ºC) Read against reagent blank

For each sample D-Glucose Pipette into cuvettes (mL) Blank Standard D-Glucose Sucrose + D-Glucose RSO + Sucrose + assay (A) assay (B) D-Glucose assay (C)

Solution 1 - - 0.20 - -

Solution 1+2* - - - 0.20 -

Solution 1+2+3* - - - - 0.20

Sample solution - 0.10 0.10 0.10 0.10

H2O 0.30 0.20 - -

Mix. Incubate for 20 min at 40 ºC. Then add:

GOD-POD reagent (4+5) 3.00 3.00 3.00 3.00 3.00

Mix, incubate at 40 °C for 20 min and read absorbances at 510 nm against the reagent blank to obtain ΔAD-glucose standard, Δ A, ΔB and ΔC

Mixtures can be obtained with a plastic spatula or by gentle inversion after sealing with a cuvette cap or Parafilm®. *Pipette both Solution 1,1+2, 1+2+3 and sample into the bottom of the cuvette and mix by gentle swirling

Where, Calculation 0.180 = MW of D-Glucose (180)/1000 mg of D-Glucose The concentration of D-glucose, sucrose and RSO 0.342 = MW of Sucrose (342)/1000 mg of Sucrose (mmol/100g) are calculated as follows: MW/1000 = MW of main RSO/1000 mg of RSO

D-Glucose, millimoles/100g If the sample has been diluted or a different sample volume 1 = A x F x 500 x 200 x was used during the reaction, the result must be multiplied 1000 = A x F x 100 by the corresponding dilution/concentration factor.

Interferences

An internal standard should be included during sample Sucrose, millimoles/100g analysis if the presence of interfering substances is suspected. A quantitative recovery of this standard should be 1 = (B-A) x F x 500 x 200 x 1000 expected. = (B-A) x F x 100 With each new batch of GOD-POD Reagent, the time of maximum colour formation with 100 µg of D-Glucose standard should be checked. This is approximately 15 min. (See Figure 2). Raffinose-series oligosaccharides, millimoles/100g Maximum colour formation 1 = (C-B) x F x 500 x 200 x 1000 1.00

= (C-B) x F x 100

0.80

Where, 0.60 A = GODPOD absorbances for assay A 0.40 B = GODPOD absorbances for assay B

C = GODPOD absorbances for assay C 0.20 Absorbance@ nm 510 F = factor to convert from absorbances to mol of glucose 0.00 0 5 10 15 20 25 Incubation time

Figure 2. Colour formation on incubation(min) of 100 g of D-glucose, 500 = conversion to 50 mL extract (i.e., 0.5g sample) performed with NZYTech kit at 40 ºC using 1 cm pathlength 200 = conversion from 0.5 to 100g sample cuvettes. Time for maximum colour formation: 15 min. 1/1000 = conversion from moles to millimoles General information on sample preparation

The total amount of RSO, sucrose and D-glucose present in The concentrations of sucrose and D-Glucose can be the cuvette should range between 10 and 100 μg. Thus, if a represented as millimoles/100g or determined as g/100g of sample volume of 0.10 mL is used the sample solution must flour sample, as proposed bellow. However, since RSO are a be diluted to yield a sugar concentration between 100 and mixture of raffinose, stachyose and verbascose, is not 1000 mg/L. possible to express RSO as g/100g. If the main component of To implement this assay use clear, colourless and practically a sample is known, then is possible to calculate an neutral liquid samples directly, or after dilution; filter turbid approximate value in g/100g of flour by using the molecular solutions; degas samples containing carbon dioxide (e.g. by weight of this component. filtration; measure "coloured" samples (against a sample blank; treat "strongly coloured" samples that are used D-Glucose, g/100g undiluted or with a higher sample volume with PVPP (ad 0.2 = D-Glucose (mmoles/100g) x 0.180 g of PVPP/10 mL sample, shake vigorously for 5 min and filter through Whatman nº1 filter paper); crush or Sucrose, g/100g homogenize solid or semi-solid samples, extract with water = Sucrose (mmoles/100g) x 0.342 or dissolve; extract samples containing fat with hot water. RSO, g/100g Sample dilution buffer (Sodium acetate buffer (50 mM, pH = RSO (mmoles/100g) x MW/1000 4.5) Add 2.9 mL of glacial acetic acid (1.05 g/mL) to 900 mL of distilled water. Adjust the pH to 4.5 by careful addition of 1 M (4 g/100 mL) sodium hydroxide solution. Adjust the volume to 1 litre and store the buffer at 4°C. Sodium azide References (0.2 g) can be added as a preservative. Stable for > 6 months at 4°C. McCleary, B. V., Charnock, S. J., Rossiter, P. C., O’Shea, M. F., Power, A. M. and Lloyd, R. M. (2006). Measurement of Examples of sample preparation carbohydrates in grain, feed and food. J. Sci. Food Agric., 86: 1648-1661. Determination of raffinose-series oligosaccharides, Released 06/16 sucrose and D-glucose in flour mixtures (Enzyme Inactivation and Sugar Extraction)

Accurately weigh 0.5 g of milled sample into a glass test-tube and add 5 mL of ethanol (95% v/v). Incubate the tube in a water bath at 85-90°C and allow to reflux for 5 min (This inactivates endogenous enzymes). Quantitatively transfer the tube contents to a 50 mL volumetric flask using Sample dilution buffer from a wash bottle to ensure complete transfer. Adjust to volume (50 mL) with Sample dilution buffer. Allow the sample to extract over 15 min. and then mix thoroughly. Transfer 5 mL of this solution/suspension to a glass test tube (suitable fort centrifugation at 1000 g). Add 2 mL of chloroform, mix vigorously on a vortex mixer for 15 sec and centrifuge (1000 g) for 10 min. Use the upper aqueous solution directly for analysis. (Sample will be at 10 mg solid matter/ml).

Certificate of Analysis

Test Criteria Result

Test Performance Reaction completed within time stated Meets specification

Target value for recommended standard material +/- 10% Meets specification

Blank reaction absorbance +/- 10% of the blank value Meets specification

Approved by: José Prates Senior Manager, Quality Systems

Please enquire [email protected] to obtain any additional information about this kit, including additional specific applications.

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