-Glucan (mixed linkage), colorimetric method

Transcription

1 -Glucan (mixed linkage), colorimetric method Catalogue number: AK0027, 00 tests Introduction -Glucans are common components in cereals, bacteria, yeasts and mushrooms. Mixed linkage -glucans are naturally found in cereals, namely in barley and oats. Although contributing for the benefit of fibre consumption for human health, soluble mixed linkage -glucans are associated to anti-nutritive effects in animal nutrition and to process problems in brewing industry. Application This accurate and convenient enzymatic method is used for the determination of mixed linkage -glucans in barley, malt, wort and beer, as well as in oats, fibre and other food products. Principles Linearity and precision Linearity of the determination exists from 0 to 00 μg D- glucose per assay (see Figure ), which corresponds to 0.85 to 8.5% of β-glucan in powder samples. Standard errors below 3% are reached routinely. Kit composition Suspension. Lichenase ( ml). Stable for 2 years at 4 C. Swirl bottle before use. Dilute the contents of bottle to 20.0 ml with 20 mm sodium phosphate buffer (ph 6.5). Divide into appropriately sized aliquots and store in PP tubes at -20 C between use (stable for 2 years) and keep cool during use. Suspension 2. -Glucosidase ( ml). Stable for 2 years at 4 C. Swirl bottle before use. Hydrolysis of -glucans Lichenase -glucan + H 2O ph 6.5 -gluco-oligosaccharides + H 2O -gluco-oligosaccharides -glucosidase D-Glucose ph 4.0 Dilute the contents of bottle 2 to 20.0 ml with 50 mm sodium acetate buffer (ph 4.0). Divide into appropriately sized aliquots, store in PP tubes at -20 C between use (stable for 2 years) and keep cool during use. Solution 3. 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. Dilute the contents of the bottle to.0 L with distilled water and use immediately. D-glucose + O 2 + H GOD 2O D-Gluconate + H 2O 2 2 H 2O 2 + p-hydroxybenzoic acid + 4-aminoantipirine POD Quinoneimine dye + 4H 2O Mixed linkage -glucans are hydrolyzed to -glucooligosaccharides using lichenase. -gluco-oligosaccharides are hydrolyzed to D-glucose with -glucosidase, and measured as D-glucose. Free D-glucose in the sample is determined directly with GOD-POD Reagent by conversion to a red coloured quinoneimine dye compound through the combined action of glucose oxidase and peroxidase. On storage, salt crystals may form in concentrated buffer. Be sure that all of these are completely dissolved before dilution to L with distilled water. Mixture 4. GOD-POD reagent enzymes. Freeze-dried powder of glucose oxidase (GOD), peroxidase (POD) and 4- aminoantipyrine. Stable for 5 years at -20 C. Dissolve the contents of one bottle 4 in approx. 20 ml of solution 3 and quantitatively transfer this to the bottle containing the remainder of solution 3. Cover this bottle with aluminum foil to protect the enclosed reagent from light. Stable for 3 months at 2-5 C or 2 months at -20 C. For storage at -20 C, this reagent should be divided in smaller aliquots that should be freeze/thawed only once during use. Solution 5. D-Glucose standard solution (5 ml,.0 mg/ml) in 0.2% (w/v) benzoic acid. Stable for 5 years at room temperature. Use as supplied.

2 50 nm Powder 6. Control barley flour. -Glucan content shown on vial label. Use as supplied. Prepare as a sample (see Examples of sample preparation) Powder 7. Control oat flour. -Glucan content shown on vial label. Use as supplied. Prepare as a sample (see Examples of sample preparation) Figure. Linearity of the assay. Standard curve relating D-Glucose concentration ( g/test) to absorbance at 50 nm (40 ºC) Safety g/test The general safety measures that apply to all chemical substances should be followed. For more information regarding the safe usage of this kit please refer to the MSDS available at Precautions and controls D-Glucose Include reagent blanks and D-glucose controls (00 g, in quadruplicate) with each set of assays: reagent blank = 0.2 ml of distilled water ml GOPOD Reagent. D-glucose control = 0. ml of D-glucose standard solution (00 μg) + 0. ml of distilled water ml GOPOD Reagent. Analyse at least one extract from the control barley flour with each set of assays. The time of incubation with GOD-POD reagent is not critical, but should be at least 20 min. However, the time for maximum colour formation with 00 μg of D-glucose standard should be checked, for each new batch of GOD- POD reagent (usually 5 min). The colour formed through the reaction is stable at room temperature for at least 2 hours after development. It is indispensable that lichenase preparation is not crosscontaminated with -glucosidase preparation. Buffers preparation 20 mm Sodium phosphate buffer, ph 6.5 Dissolve 3.2 g of sodium dihydrogen orthophosphate dihydrate (NaH 2PO 4.2H 2O) in 900 ml of distilled water and adjust ph to 6.5 by the addition of M NaOH. Adjust the volume to L. Add 0.2 g of sodium azide. Stable for 2 months at 4 C. 50 mm Sodium acetate buffer, ph 4.0 Add 2.9 ml of glacial acetic acid to 900 ml of distilled water. Adjust to ph 4.0 by the addition of M NaOH solution. Complete the volume to L. Add 0.2 g of sodium azide. Stable for 2 months at 4 C. 200 mm Sodium acetate buffer, ph 4.0 Add.6 ml of glacial acetic acid to 900 ml of distilled water. Adjust to ph 4.0 by the addition of M NaOH solution. Adjust the volume to L. Add 0.2 g of sodium azide. Stable for 2 months at 4 C. Procedures Streamlined Methods I. Assay method for Barley and Oat flours and Fibre Samples This method is recommended for all dry samples, particularly those containing high levels of β-glucan (e.g. processed oat bran products).. Mill sample (barley, oats or fibre; approx. 50 g) to pass a 0.5 mm screen using a centrifugal mill. 2. Accurately weight flour sample (80-20 mg) to a glass centrifuge tube (7 ml capacity). Tap the tube to ensure that all sample falls to the bottom of the tube. 3. Add 0.2 ml of aqueous ethanol (50% v/v) to the sample to aid dispersion. Add 4.0 ml of sodium phosphate buffer (20 mm, ph 6.5) and stir the contents on a vortex mixer. 4. Once mixed, immediately place the tube in a boiling water bath and incubate for min. Vigorously stir the mixture on a vortex mixer, incubate in boiling water bath for a further 2 min and stir again. 5. Incubate the tube at 50 C for 5 min, to equilibrate. 6. Add 0.2 ml lichenase and stir the tube contents. Seal the tube with Parafilm and incubate for h at 50 C. Regularly (i.e. 3-4 times) stir vigorously on a vortex mixer. 7. Add 5.0 ml sodium acetate buffer (200 mm, ph 4.0) and vigorously mix the tube contents on a vortex mixer. 8. Allow the tube to equilibrate to room temperature (5 min) and centrifuge (,000 g, 0 min). 9. Carefully and accurately dispense aliquots (0. ml) into the bottom of three test tubes (2 ml capacity) 0. Add 0. ml of β-glucosidase preparation to two of these tubes (the reaction). To the third (the reaction blank), add 50 mm acetate buffer (0. ml, ph 4.0). Incubate all tubes at 50 C for 0 min.

3 . Add 3.0 ml of GOD-POD Reagent to each tube and incubate at 50 C for a further 20 min. 2. Remove the tubes from the water bath and measure the absorbance at 50 nm against reagent blank within h. Notes: The amount of D-glucose present in the test tube should range from 0 to 00 μg. Therefore, before Step 6 (βglucosidase treatment) the sample solution must be diluted sufficiently with 200 mm sodium acetate buffer (ph 4.0) to yield a sugar concentration between 0.0 and.0 g/l, which is equivalent to approx and 8.5% of β-glucan in the original sample. E.g. a sample containing 20% of beta-glucan should be diluted 3-fold with 200 mm sodium acetate buffer (ph 4.0) before dispensing aliquots for incubation with β- glucosidase. In other way, for samples containing high β-glucan, (>50% β- Glucan), the sample size should be reduced to 50 mg and the volume should be adjusted to 00 ml with 200 mm sodium acetate buffer (ph 4.0) after lichenase treatment. II. Assay method for Cooked, Toasted or Extruded Cereal Products Samples This method is recommended for cooked, toasted or extruded samples. The method includes a pre-extraction with ethanol to remove free sugars and to reduce the levels of fats and oils. Mill sample (barley, oats or fibre; approx. 50 g) to pass a 0.5 mm screen using a centrifugal mill. 2. Accurately weight flour sample (200 mg) to a glass centrifuge tube (7 ml capacity). Tap the tube to ensure that all sample falls to the bottom of the tube. 3. Add 5.0 ml of aqueous ethanol (50% v/v) and incubate the tubes in a boiling water bath for 5 min. Mix the contents on a vortex stirrer and add a further 5.0 ml of 50 % (v/v) aqueous ethanol. 4. Centrifuge the tubes for 0 min at,800 g ( 3,000 rpm). Reject the supernatant. 5. Resuspend the pellet in 5.0 ml of 50% (v/v) aqueous ethanol and stir on the vortex mixer. Add a further 5.0 ml of 50% aqueous ethanol. Stir on the vortex mixer, centrifuge and discard the supernatant (as in step 4). 6. Suspend the pellet in 4.0 ml of sodium phosphate buffer (20 mm, ph 6.5) and incubate the tube at 50 C for 5 min. 7. Add 0.2 ml lichenase and stir the tube contents. Seal the tube with Parafilm and incubate for h at 50 C. Regularly (i.e. 3-4 times) stir vigorously on a vortex mixer. 8. Add 2.0 ml sodium acetate buffer (200 mm, ph 4.0) and vigorously mix the tube contents on a vortex mixer. 9. Proceed from Step 8 of Assay I. III. Assay method for Yogurt, Milkshake and other Liquid Samples This method is recommended for liquid samples. The method includes a precipitation with ethanol.. Weigh accurately a glass centrifuge tube (7 ml capacity). 2. Add 3 ml of sample to the tube and heat in a boiling water bath for 5 min. Allow cooling to room temperature. 3. Add 3 ml of 95% aqueous ethanol to the tube and mix the contents on a vortex stirrer. Add a further 5.0 ml of 95% aqueous ethanol. Mix well on a vortex stirrer. 4. Centrifuge the tubes for 0 min at,800 g ( 3,000 rpm). Reject the supernatant. 5. Resuspend the pellet in 8.0 ml of 50% (v/v) aqueous ethanol and stir on the vortex mixer. Centrifuge and reject the supernatant (as in step 4). 6. Adjust the volume to 4.0 ml (by weight) with sodium phosphate buffer (20 mm, ph 6.5) from the known weight of the empty tube. Resuspend the pellet. Incubate the tube at 50 C for 5 min. 7. Proceed from Step 6 of Assay I. Note: If the sample absorbance values exceed the absorbance values obtained for glucose standard, the samples must be diluted with 200 mm sodium acetate buffer (ph 4.0) to bring them on assay linearity range before dispensing aliquots for incubation with β-glucosidase. Calculation Streamlined Methods Solid samples (assays I and II) The concentration of -glucans of solid samples are calculated as follows: -Glucans, % w/w (fresh weight basis) Where, A = sample GOD-POD absorbance (after lichenase and -glucosidase treatment) minus reaction blank absorbance F = = = A x F x FV x x 00 x 62 x D W 80 = A x F x FV x D x 0.9 W factor to convert from absorbance to mol of glucose 00 ( g of D Glucose) absorbance of 00 g of D Glucose FV = final volume (i.e. Assay I 9.4 ml; Assay II 6.4 ml; samples containing >50% -glucans 00 ml)

4 0. = volume of sample analyzed W = conversion from g to mg = factor to express -glucans as a percentage of sample weight W = weight of the sample analyzed in mg ( as is ) = conversion from g to mg = conversion from g to g = factor to convert free D-glucose, as determined, to anhydro-d-glucose, as occurs in -glucans = factor to convert free D-glucose, as determined, to anhydro-d-glucose, as occurs in -glucans D = further dilution previous to incubation with - glucosidase, if necessary -Glucans, % w/w (dry weight basis) = -Glucans, % w/w ( as is ) x Note: Dry weight = fresh weight x 00 moisture content (% w w) 00 Moisture content is usually determined by NIR reflectance. Alternatively this can be determined by recording weight loss on storage of flour samples (0.5 g) at 80⁰C for 20 h. or until weigth stabilization. The moisture content of cereal flour samples is regularly in the range of 0-4%. Liquid samples (assay III) The concentration of -glucans of liquid samples are calculated as follows: -Glucans, g/00 ml Where, A = sample GODPOD absorbances (after lichenase and -glucosidase treatment) minus reaction blank absorbance F = = moisture content (% w w) = A x F x x 000 x 000 x 000 x x D = A x F x D x factor to convert from absorbances to mol of glucose 00 ( g of D Glucose) absorbance of 00 g of D Glucose = factor for volume correction (3.0 ml aliquots of sample are treated with ethanol and the volume was adjusted to 9.2 ml: 4.0 ml ml lichenase ml acetate buffer) D = further dilution previous to incubation with - glucosidase, if necessary Procedures Conventional Methods (Brewing industry) IV. Assay method for Barley. Mill barley fine and uniformly ( 50 g) to pass a 0.5 mm screen using a centrifugal mill. 2. Accurately weigh barley flour samples ( 0.5 g) of known moisture content into polypropylene tubes. 3. Add an aliquot (.0 ml) of aqueous ethanol (50% v/v) to each tube, to aid dispersion of samples. 4. Add 5.0 ml of sodium phosphate buffer (20 mm, ph 6.5) and stir the tubes on a vortex mixer. 5. Incubate the tubes in a boiling water bath for 2 min. Remove the tubes and vigorously stir them on a vortex mixer. Heat the tubes for a further 3 min in the boiling water bath. If the solution is very viscous, add 5.0 ml distilled water (it will avoid problems with the diffusion of lichenase). 6. Cool the tubes to 40 C and add 0.2 ml of lichenase to each tube. Cap the tubes, stir and incubate at 40 C for h. 7. Adjust the volume in each tube to 30.0 ml by the addition of distilled water (24.0 ml, if standard procedure was followed, or 9.0 ml, when 5.0 ml of distilled water was added at Step 5). 8. Mix the contents of the tubes carefully and centrifuge an aliquot at,000 g for 0 min (or filter an aliquot from each tube through a Whatman No. 4 filter circle) 9. Carefully and accurately transfer 0. ml aliquots from each filtrate to the bottom of three test tubes. 0. Add 0. ml of sodium acetate buffer (50 mm, ph 4.0) to one of these (reaction blank). Add 0. ml of β- glucosidase preparation to each one of the other two (reaction duplicates). Incubate the tubes at 40 C for 5 min.. Add 3.0 ml of GOD-POD Reagent to each tube and incubate at 40 C for 20 min (see Controls and Precautions section). 2. Remove the tubes from the water bath and measure the absorbance at 50 nm for each reaction and reaction blank within h. 000 = factor for volume adjustment from 0. ml of analysed sample to 00 ml (as presented)

5 V. Assay method for Malt. Mill malt (or lyophilized barley samples removed during the malting process) to pass a 0.5 mm screen using a centrifugal mill. 2. Accurately weigh malt flour samples (.0 g) of known moisture content into polypropylene tubes. 3. Add 5.0 ml of aqueous ethanol (50% v/v). Incubate in a boiling water bath for 5 min. Mix the contents on a vortex stirrer and add a further 5.0 ml of 50% (v/v) aqueous ethanol. Mix tube contents. 4. Centrifuge for 0 min at,000 g. Remove the supernatant. 5. Resuspend the pellet in 0.0 ml of aqueous ethanol (50% v/v), centrifuge and discard the supernatant (as in Step 4). 6. Resuspend the pellet in 5.0 ml of sodium phosphate buffer (20 mm, ph 6.5). 7. Proceed from Step 5 of Assay IV. VI. Assay method for Spent Grain Wash spent grain with hot water (approx. 75 C), and then lyophilize (or lyophilize without washing). Mill this material to pass a 0.5 mm screen and analyze for β- glucan content and perform calculations as described for the malt samples VII. Assay method for Beer or Wort. De-gas beer/wort by heating an aliquot to approx. 80 C in a boiling water bath. Allow to cool. 2. Weigh accurately a glass centrifuge tube (7 ml capacity). 3. Add 5 ml of sample to the tube and add 2.5 g of finely milled ammonium sulphate crystals. 4. Seal the tube with Parafilm and dissolve the ammonium sulphate by careful inversion (to avoid foaming). 5. Let the tube to stand for approx. 20 h at 4 C. 6. Centrifuge at,000 g for 0 min on a bench centrifuge. 7. Discard the supernatant. 8. Resuspend the pellet with.0 ml of aqueous ethanol (50% v/v) by thoroughly vortexing. Add a further 0.0 ml of aqueous ethanol (50% v/v) and mix well by inversion of the tube. 9. Centrifuge at,000 g for 5 min. Reject the supernatant. 0. Repeat the ethanol-washing procedure by resuspending the pellet, etc. as described in steps 8 and 9.. Remove the supernatant. 2. Resuspend the pellet in sodium phosphate buffer (20 mm, ph 6.5): for wort, adjust the volume to 4.8 ml (by weight), for beer, adjust the volume to.8 ml (by weight). 3. Add 0.2 ml lichenase and incubate at 40 C for 5 min. 4. Centrifuge at,000 g for 0 min then proceed from Step 9 of Assay IV. Note: For wort samples containing low levels of β-glucan, incubate a larger aliquot of sample solution (up to 0.5 ml) with β- glucosidase. Use the same aliquot size also for the blank and adjust the D-glucose standard accordingly with distilled water. Take this modification in account when performing calculations. Calculation Conventional Methods (Brewing industry) For Barley, Malt and Spent Grain (Assays IV, V and VI) -Glucans, % w/w For Beer (Assay VII) For Wort (Assay VII) Where, = A x F x 300 x = A x F W x 27 -Glucans, mg/l -Glucans, mg/l = A x F x 0000 x = A x F W x 3.6 = A x F x 0000 x = A x F W x 9 x 00 x W 80 x 2 x x 5 5 x A = sample GOD-POD absorbance (after lichenase and -glucosidase treatment) minus reaction blank absorbance F = factor to convert from absorbance to mol of glucose

6 = 00 ( g of D Glucose) absorbance of 00 g of D Glucose 300 = factor for volume correction (i.e. 0. ml taken from 30.0 ml) 0000 = factor for volume adjustment (from 0. ml of analyzed sample to L, as presented) W = conversion from g to mg = factor to express β-glucan content as a percentage of dry flour weight. W = the calculated dry weight of the sample analyzed, in mg 5 volume correction factor for wort samples. = 5 Sample aliquots of 5.0 ml were treated with precipitant (ammonium sulphate). After washing steps, the volume was readjusted to 5.0 ml (i.e. 4.8 ml ml lichenase) = volume correction factor for beer samples. Sample aliquots of 5.0 ml were treated with precipitant (ammonium sulphate) After washing steps; the volume was readjusted to 2.0 ml (i.e..8 ml ml lichenase). = factor to convert free D-glucose, as determined, to anhydro-d-glucose, as occurs in -glucans References McCleary, B. V. & Glennie-Holmes, M. (985). Enzymic quantification of (-3) (-4)-β-D-glucan in barley and malt. J. Inst. Brew., 9, McCleary, B. V. & Nurthen, E. J. (986). Measurement of (-3) (-4)-β-D-glucan in malt, wort and beer. J. Inst. Brew., 92, McCleary, B. V. & Codd, R. (99). Measurement of (-3) (-4)- β-d-glucan in barley and oats: A streamlined enzymic procedure. J. Sci. Fd. Agric., 55, Released 09/6 Interferences With each new batch of GOD-POD Reagent, the time of maximum colour formation with 00 µg of D-Glucose standard should be checked. This is approximately 5 min. (See Figure 2)..00 Maximum colour formation Incubation time Figure 2. Colour formation on incubation of 00 g of D-glucose, performed with NZYTech kit at 40 ºC using cm pathlength cuvettes. Time for maximum colour formation: 5 min.

7 Certificate of Analysis Test Criteria Result Test Performance Reaction completed within time stated Meets specification Target value for recommended standard material +/- 0% Meets specification Blank reaction absorbance +/- 0% of the blank value Meets specification Approved by: José Prates Senior Manager, Quality Systems Please enquire to obtain any additional information about this kit, including additional specific applications.

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