NSP parameters
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Parameters:
- Arabinoxylan
- Beta-glucan
- Total insoluble NSP
- Total soluble NSP
- Lignin
- Soluble arabinoxylan
Wet chemistry reference methods:
Reference methods used are as outlined in Premium Grains For Livestock Program. Component 1: Co-ordination. An overview of outcomes from PGLP 1 & 2. Final report. September 2008. Prepared by John Black.
- Arabinoxylan
AOAC 995.16
- Beta-glucan
AOAC 995.16
- Total insoluble NSP
- Total soluble NSP
Running reagent blank: Duplicate reagent blanks must be run with each batch of samples.
Pipetting: Always standardise pipettes and pipetting technique using ultra-pure water and an accurate balance. For minute and precise amounts of solutions such as aliquots and standards use either glass or positive displacement pipettes.
Internal standard: See notes 2(4) and 3(5). Allose (and inositol); these should be present at a concentration comparable to that of the main components, or the majority of the components of the sample being assayed.
Extraction of fat: This is not strictly necessary if the fat level is less than 5% but if a free sugar assay is to be done using the supernatant from step 1(5), then a fat extraction is essential. Fat extraction, see note 1(4A).
Removal of proteins: For high-protein ingredients such as lupins, soybeans, peas, faba beans, canola, chickpeas and other legumes, removal of proteins is necessary. Do this by adding 0.1mL of pancreatin solution (add 0.2g pancreatin to mL H2O, centrifuge at 3000g and use the supernatant) at step 1(8).
Preparation of acetate buffer (pH 5.0):
Solution A: 0.2M acetic acid; 2.86mL glacial acetic acid (17.4M, specific gravity 1.051) per 250mL of water.
Solution B: 0.2M sodium acetate; 13.6g sodium acetate trihydrate per 500mL of water.
Mix 148mL solution A with 352mL solution B. Add 4mL of 1M CaCl2 and adjust to 1L. Check pH.
Step 1. Sample preparation
1. Grind the sample so that it passes through a 0.5mm screen.
2. Store in an airtight container.
3. Take 1-2g to determine its dry matter content for future calculations.
4. Accurately weigh approximately 200mg of sample (e.g. wheat) and place it in a 30mL screw-capped culture tube (Note: sample weight should be adjusted according to the NSP estimate of the sample to be determined). Fat extraction - add about 10mL of hexane (or petroleum ether) to the sample. Cap, vortex and sonicate for 15 mins. Centrifuge at 2000g at 20°C for 15 mins and discard supernatant.
5. Add 5mL 80% ethanol to the residue and heat to 80°C for 10 mins. Centrifuge at 2000g for 10 mins and remove the supernatant to an 8mL vial. This step is to remove free sugars and to deactivate the endogenous enzymes. If free sugars are not required, discard supernatant. Dry the residue to a slurry using nitrogen.
6. Add 10mL of acetate buffer (pH 5.0) and heat in water bath at 100°C for 30 mins. Stir after 15 mins. This is to gelatinise the starch so that the enzymes can attack the starch efficiently.
7. Remove one tube at a time from boiling and immediately add 50µL of Sigma a-amylase (Note: mix amylas by inversion). Quickly return tube to 95°C water bath and hold for 30 mins; vortex after 15 mins or mix rack of tubes by inversion.
8. Cool to 55°C and add 50µL of amyloglucosidase (gently mix AMG six times before pipetting). Incubate overnight (16 h) at 55°C either in a heating block with stirring or in a water bath with shaking.
9. Centrifuge at 2000g for 30 mins. Take an aliquot of supernatant (take 4mL if using 2M TFA hydrolysis and take 8mL if using 1M sulphuric acid) for soluble NSP measurement, 1mL for soluble uronics and take another aliquot (about 1mL) for starch determination. Keep the residue for insoluble NSP determination.
Step 2 - Determination of soluble NSP
Hydrolysis with 2M TFA:
1. Transfer 4mL of supernatant into a 30mL screw-capped culture tube. Add 16mL of absolute ethanol (this makes it to 80% ethanol). Leave on ice for at least 30 mins or at 4°C for at least one hour. Centrifuge at 2000g for 20 mins at 4°C. Discard the supernatant and add 10mL 80% ethanol, mix and leave on ice for at least 30 mins. Centrifuge and discard the supernatant. Add 10mL absolute ethanol, mix and leave it on ice for at least 30 mins. Centrifuge and discard the supernatant. This step is to remove sugars released by the a-amylase and amyloglucosidase.
2. Dry the precipitate under a N2 stream at 40°C and add 1mL 2M trifloroacetic acid (made by adding 2mL TFA to 11mL H2O).
3. Heat at 125°C for one hour with stirring, make sure all of the sample is exposed to the acid.
4. Cool to room temperature, add precisely 50µL of both internal standards, (inositol, 4mg/mL and allose 4mg/mL), vortex and remove magnetic fleas.
5. Evaporate to dryness using nitrogen on the heating block at 40-45°C. Wash twice with 0.2mL H2O. (If slow, transfer to a 8mL vial).
6. Dissolve the residue in 0.2mL of water, transfer to a 30mL culture tube. Add 0.2mL absolute ethanol and 1 drop of 3MNH4OH. Mix well and add 0.3mL freshly prepared NaBH4 (prepared by dissolving 50mg sodium per mL 3M NH4OH). Cap and incubate in 40°C water bath for one hour.
7. Add 5-7 drops of glacial acetic acid to decompose the excess amount of NaBH4 (or add dropwise until gas evolution ceases. CAUTION: excess amount of acid interferes with acetylation).
8. Add 0.5mL 1-methylimidazole mix (TOXIC - handle carefully in fume hood) and 5mL acetic anhydride and mix. Leave 10 mins at room temperature.
9. Add 0.8mL of dry absolute ethanol, mix and leave for 10 mins.
10. Place the sample mixture in an ice bath and add 5mL H2O to decompose excess amount of acetic anhydride.
11. Add 5mL of 7.5M KOH (to 3 tubes at a time), cap and gently mix it 6 times by inversion and add another 5mL of 7.5M KOH, cap and mix again, (a clear ethyl acetate top layer should be visible).
12. Leave until phases are clearly formed or spin at 2000g for 5 mins at 5°C. Transfer the top layer into a 4mL vial (use a Pasteur pipette and take about 75% of top layer, take no water from bottom phase).
13. Add about 1mL of water to the vial to wash the ethyl acetate, vortex. Leave it until the phases are separated (add ethyl acetate if necessary). Remove the top layer into another 4mL vial.
14. Evaporate to dryness under N2. This step takes a very short period of time. Do not leave it for extended period of time if heating block is used.
15. Add about 3 drops of ethyl acetate and inject 0.3µL-1µL into the GC. Calibrate the GC using allose as the internal standard. Polymerisation factors; pentoses 0.88, hexoses 0.9 and deoxysugars 0.89.
Step 3 - Determination of insoluble NSP
1. Wash the residue from Step 1 (9) with water and centrifuge at 2000g for 15 mins. Discard supernatant. Repeat this twice to make sure glucose released from starch digestion is completely removed. Add 2mL of acetone and vortex, centrifuge. Discard supernatant and dry the residue under N2.
2. Add 1mL of 12M H2SO4. Stir at 35°C for one hour (use a spin bar to break up big lumps).
3. Cool and add 10mL of water + 1mL inositol (3mg/mL solution) and hold at 100°C for two hours.
4. Cool to room temperature and centrifuge to sediment insoluble materials.
5. Transfer an aliquot of 0.8mL to a 30mL culture tube and add 0.20mL of 28% NH3.
6. Add precisely 50µL of allose (4mg/mL); mix well.
7. Evaporate to dryness at 40-45°C. Add 0.2mL of water, 0.2mL of absolute ethanol and 1 drop of 3M ammonia.
8. Reduce and acetylate the sugars as described for the soluble NSP [at step 2 (6)].
Step 4 - Determination of free sugars
1. Evaporate extract from step 1 (5) with N2 at 40°C.
2. Hydrolyse for two hours at 100°C with stirring using 3mL of 1M H2SO4.
3. Follow steps 3 (5) to 3 (8).
Step 5 - Determination of enzyme-digestible starch
1. Glucose standard: Make up a 0.5mg/mL stock solution in a 200mL flask using 50% saturated Benzoic acid. Then make up working standards as follows:
| Stock | 0 | 0.2 | 0.4 | 0.6 | 1.0 |
| Water | 1 | 0.8 | 0.6 | 0.4 | 0.0 |
| µg glucose / 0.1ml | 0 | 10 | 20 | 30 | 50 |
2. Sample: A 0.1mL aliquot containing about 5-15µg glucose should be prepared. For cereal grains, take 0.1mL of supernatant starch hydrolysate and add 5mL of H2O.
3. Boehringer Kit method for glucose determination: Take 0.2mL of distilled water and 0.1mL of standards and samples. Add 0.1mL of water to samples and standards. Add 5mL of the Boehringer buffer with glucose oxidase/peroxidase. Mix well and incubate at 37°C for 15 mins. Read the absorbance (OD) at 610nm.
4. Calculation: I. Work out the intercept (a), slope (b) and correlation coefficient (r) of the standard curve. II. Amount of glucose in 0.1mL of aliquot (G) = (OD - a)/b. III. Starch in sample (g/kg)=G x 255 (dilution factor) x 0.9 / sample weight. (See separate note for total starch determination).
References:
Englyst, H.N. and Hudson, G.J. (1993). In: Dietary Fiber and Human Nutrition, 2nd edition. Pages 53-71. Ed. Spiller, G.A. (CRC Press, Inc. Boca Raton, Florida).
Theander, O. and Westerlund, E. (1993). In: Dietary Fiber and Human Nutrition, 2nd edition. Pages 77-98. Ed. Spiller, G.A. (CRC Press, Inc., Boca Raton, Florida).
- Lignin
The fibre residue from the acid detergent fibre was reacted with 72% sulphuric acid according to the Official Methods of Analysis of the AOAC (1995).
References:
AOAC (1995) Official Methods of Analysis of the Association of Official Analytical Chemists. Volume 1 and 2, 16th edition, Editor P. Cunniff, Published AOAC International, Arlington, Virginia, 4.1.16, 4.1.10, 4.2.04, 4.5.01, 4.6.01, 4.6.03, 4.6.03D, 32.5.18, 41.1.28, 41.1.29.
Tecnicon, Method Manual, Method 334-74
- Soluble Arabinoxylan
AOAC 994.13