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Proximate parameters - Cereals

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Parameters:

  • 1000 grain weight

  • Acid detergent fibre (ADF)

  • Ash

  • Crude fat

  • Crude fibre

  • Crude protein

  • Englyst NDF

  • Linoleic acid

  • Oleic acid

  • Oligosaccharides

  • Palmitic acid

  • Specific weight

  • Total free sugars

  • Total starch

  • Moisture

 

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.

  • 1000 grain weight

(grams)

  • ADF

The finely ground (<1.0mm) seed sample (1g) was simmered in 100mL of 0.5M sulphuric acid / cetyltrimethylammonium bromide solution for one hour. The mixture was filtered through a course sintered glass crucible and the fibre was determined gravimetrically (AOAC 1995, 4.6.03). 

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

  • Ash

The ground (<1.0mm) sample (2g) was weighed into a pre-weighed silica basin and ashed at 600°C for two hours. The ash content was determined gravimetrically (AOAC, 1995, 4.1.10).

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

  • Crude Fat

A sample (2g) of ground seed was weighed into a soxhlet extraction thimble and extracted for eight hours with a constant boiling range (40-60°C) hydrocarbon solvent. The hydrocarbon solvent was removed and the amount of fat / oil was determined gravimetrically (AOAC, 1995, 4.5.01). 

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

  • Crude Fibre

A ground (<1.0mm) seed sample (2g) was extracted with 10% w/v sulphuric acid and 10% w/v sodium hydroxide and the residue was collected in a course sintered glass crucible. The crude fibre was determined gravimetrically (AOAC, 1995, 4.6.01).

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

  • Crude Protein

The finely milled (<1.0mm) seed sample (0.8g) was pressed into a pellet and underwent combustion in pure oxygen at 850°C. The amount of nitrogen was measured by thermal conductivity (Dumas Nitrogen, AOAC, 1995, 4.2.04). Crude protein was calculated by multiplying the nitrogen value by 6.25,

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

  • Englyst NDF

P.J. van Soest, J.B. Robertson, B.A. Lewis

  • Linoleic Acid

The lipids and triglycerides in the extracted fat / oil were saponified in alkali-methanol, the methyl ester of the fatty acids were prepared using boron triflouride in methanol. The fatty acids were determined by gas chromatography using a flame ionisation detector (Conacher, 1975; Firestone and Horwitz, 1979; AOAC, 1995, 41.1.28, 41.1.29).

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.

Conacher, H.B.S. (1975) Gas-liquid chromatographic determination of docosenoic acid in fats and oils. Collaborative study. Journal of the Association of Analytical Chemists 58: 488-491.

Firestone, D. and Horwitz, W. (1979). IUPAC gas chromatographic method for determination of fatty acid composition. Journal of the Association of Analytical Chemists 62: 709-721.

Note: Fatty acid profiles of samples analysed in year 1 were determined using similar methods, but in a different laboratory at the University of Newcastle. Commercial services were not available from this laboratory in year 2 of the project. Stepwise regression analysis will only utilise fatty acids for which there is a measurement for all grains (i.e. palmitic, stearic, oleic and limoleic acids). 

  •  Oleic Acid

The lipids and triglycerides in the extracted fat / oil were saponified in alkali-methanol, the methyl ester of the fatty acids were prepared using boron trifluoride in methanol. The fatty acids were determined by gas chromatography using a flame ionisation detector (Conacher, 1975; Firestone and Horwitz, 1979; AOAC, 1995, 41.1.28, 41.1.29).

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.

Conacher, H.B.S. (1975) Gas-liquid chromatographic determination of docosenoic acid in fats and oils. Collaborative study. Journal of the Association of Analytical Chemists 58: 488-491.

Firestone, D. and Horwitz, W. (1979). IUPAC gas chromatographic method for determination of fatty acid composition. Journal of the Association of Analytical Chemists 62: 709-721.

Note: Fatty acid profiles of samples analysed in year 1 were determined using similar methods, but in a different laboratory at the University of Newcastle. Commercial services not available from this laboratory in year 2 of the project. Stepwise regression analysis will only utilise fatty acids for which there is a measurement for all grains (i.e. palmitic, stearic, oleic and limoleic acids). 

  •  Oligosaccharides

The ground (<1.0mm) sample (1g) that was previously defatted was extracted with 70% v/v ethanol at 65°C for 30 minutes. The solvent was removed under reduced pressure. The residue was redissolved in water and quantified by HPLC using a Dextropak and refractometer detection (Quemener, 1988; Knudson, 1986; Cheetham and Teng, 1984 and Wright and Dalel, 1986).

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.

Bos, K.D. and Jetten, J. (1989). Determination of tannin in faba beans. Recent Advances of Research in Anti-nutritional factors in legume seeds. Huisman, van der Poel and Liener, Eds. Pudoc, Wageningen p.168-171.

Cansfield, P.E., Marquardt, R.R. and Campbell, L.D. (1980) Condensed proanthocyandins of faba beans. Journal of the Science of Food and Agriculture, 31: 802-812.

Carmona, A., Seidl, D.S. and Jaffe, W.G. (1991). Comparison of extraction methods and assay procedures for the determination of the apparent tannin content of some beans. Journal of the Science of Food Agriculture 56: 291-301.

Cheetham, N.W.H. and Teng, G.J. (1984). Some applications of reverse phase high performance liquid chromatography to oligosaccharide determinations. Journal of Chromatography 336: 161-172.

Clegg, K.M. (1956). The applications of the anthrone reagent to the estimation of starch in cereals. Journal of the Science of Food and Agriculture, 7: 40-44.

Desphande, S.S., Cheryan, M. and Salunkhe, D.K. (1986) Tannin analysis of food products. CRC Critical Reviews in Food Science and Nutrition, 24: 402-449.

Ellis, R. and Morris, E.R. (1983) Improved ion-exchange phytate method. Cereal Chemistry 60: 121-124.

Harland, B.F. and Oberleas, D. (1977) A modified method for phytate analysis using an ion exchange procedure: application to textured vegetable proteins. Cereal Chemistry 54: 827-832.

Harland, B.F and Oberleas, D. (1986) Anion exchange method for determination of phytate in foods. Journal of the Association of Analytical Chemists 69: 667-670.

Harris, D.J. and Ayliffe, W. (1997) unpublished results.

Knudsen, I.M. (1986). HPLC determination of oligosaccharides in leguminous seeds. Journal of the Science of Food and Agriculture 37: 560-166.

Kuhla, S. and Ebmeir, C. (1981). Untersuchungen zum tanningrhalt in ackerbohhnen. Archives Tierernaehrung 31: 573-588.

Marquardt, R.R., Ward, A.T., Campbell, L.D. and Cansfield, P.E. (1977) Purification, identification and characterisation of a growth inhibitor in faba beans (Vicia faba L. var minor), Journal of Nutrition, 107: 1313-1324.

Quemener, B. (1988). Improvement in the high performance liquid chromatographic determination of amino sugars and alpha-galactosides in faba bean, lupine and pea. Journal of Agriculture and Food Chemistry 36: 754-759.

Wright, W. and Dalil, J.M. (1986). Evaluation of reversed phase HPLC column for estimation of legume seed oligosaccharides. Food Chemistry 21: 167-181.

  • Palmitic Acid

The lipids and triglycerides in the extracted fat / oil were saponified in alkali-methanol, the methyl ester of the fatty acids were prepared using boron trifluoride in methanol. The fatty acids were determined by gas chromatography using a flame ionisation detector (Conacher, 1975; Firestone and Horwitz, 1979; AOAC, 1995, 41.1.28, 41.1.29).

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.

Conacher, H.B.S. (1975) Gas-liquid chromatographic determination of docosenoic acid in fats and oils. Collaborative study. Journal of the Association of Analytical Chemists 58: 488-491.

Firestone, D. and Horwitz, W. (1979). IUPAC gas chromatographic method for determination of fatty acid composition. Journal of the Association of Analytical Chemists 62: 709-721.

Note: Fatty acid profiles of samples analysed in year 1 were determined using similar methods, but in a different laboratory at the University of Newcastle. Commercial services were not available from the laboratory in year 2 of the project. Stepwise regression analysis will only utilise fatty acids for which there is a measurement for all grains (i.e. palmitic, stearic, oleic and limoleic acids). 

  •  Specific weight

(kg/hl)

  • Total free sugars

By Luff-Schoorl titration. AOAC 974.06.

  • Total starch

Reagents:

1. 3-N(N-Morpholino) propanesulfonic acid (MOPS) buffer- pH 7.0 in 1L volumetric flask dissolve 11.55g MOPS in 900ml H2O and adjust pH to 7.0 with 1M HCl. Add 0.74g calcium chloride dihydrate and 0.2g sodium azide. Dilute to volume with H2O. Buffer is stable at room temperature. 

2. Thermostable a-amylase solution - 10ml; 3000U/ml. Dilute 1ml a-amylase (in 50% glycerol) to 30ml with MOPS buffer.

3. Amyloglucosidase solution - 10ml; 200U/ml.

4. Glucose oxidase-peroxidase-aminoantipyrine buffer mixture - mixture of glucose oxidase, 12000U/L; peroxidase, >650U/L; and 4-aminoantipyrine, 0.4mM.

5. Prepare buffer concentrate by dissolving 13.6g potassium dihydrogen orthophosphate, 4.2g sodium hydroxide, and add 3.0g 4-hydroxybenzoic acid in 96ml distilled H2O. Adjust to pH 7.4 with either 2M HCl or 2M NaOH. Dilute solution to 100ml, add 0.4g sodium azide and mix until dissolved. Buffer concentrate is stable up to 3 years when stored at 4°C.

6. Prepare glucose oxidase-peroxidase-antiantipyrine buffer mixture, dilute 50ml buffer concentrate to 1.0L. Use part of diluted buffer to dissolve entire content of vial containing freeze-dried glucose-peroxidase mixture. Transfer contents of vial to 1L volumetric flask containing diluted buffer.

7. Sodium acetate buffer - 200mM, pH 4.5 - pipet 11.8ml glacial acetic acid (1.05g/ml) to 900ml H2O. Adjust pH to 4.5 with 1M NaOH solution (ca 60ml is required). Add 0.2g sodium azide and dilute to 1L with H2O.

8. Dimethyl sulfoxide (DMSO) - analytical grade.

9. Glucose standard stock solution - 1mg/ml. Dry powdered crystalline glucose (purity 97%) for 16 h at 60°C under vacuum, before preparing the solution. 

Preparation of test samples, standards and reagent blanks

1. Test sample - grind sample to pass 0.5mm sieve.

2. D-Glucose standard working solutions - 50 and 100µg. Add 50 and 100ul D-glucose standard stock solution, to separate test tubes, and adjust volume in each test tube to 100ul with H2O. Prepare solutions immediately before use.

3. Reagent blank - transfer 0.1ml H2O into test tubes and proceed with total starch determination.

Detemination of Total Starch

1. Weigh 90-100mg ground test sample into test tubes. (Ensure that all sample drops to the bottom of the tube). 

2. Add 0.2ml 80% aqueous ethanol to tube and stir on a vortex mixer.

3. Add 3.0ml thermostable a-amylase and mix contents of tube on a vortex mixer to ensure complete dispersion of sample.

4. Immediately place the tube in a boiling water bath. Incubate for 2 mins, remove from the water bath, and mix vigorously on a vortex mixer. Return tube to the boiling water bath for an additional 3 mins and then mix contents vigorously.

5. Add 4.0ml 200mM sodium acetate buffer and 0.1ml amyloglucosidase solution and vigorously mix contents on a vortex mixer. Incubate at 50°C for 30 mins.

6. Quantitatively transfer the entire contents of the test tube to 100ml volumetric flask. Dilute to 100ml H2O. (If sample contains <10% starch adjust volume to 10.0ml instead of 100ml.)

7. Centrifuge portion of the suspension for 10 mins at 1000g.

8. Transfer 1ml aliquot of each filtrate to another test tube.

9. Add 3.0ml glucose oxidase-peroxidase-aminoantipyrine buffer mixture to each test tube and incubate for 20 mins at 50°C.

10. Measure the absorbance of each sample at 510nm against reagent blank.

Determination of Enzyme Resistant Starch

1. Accurately weigh 90-100mg ground test samples. (Ensure all particles of the sample drop to the bottom of the tube). 

2. Add 0.2ml 80% aqueous ethanol to the tube and stir on a vortex mixer to ensure all samples are wet.

3. Add 2ml DMSO solution and stir tube on a vortex mixer. Place the tube into a vigorously boiling water bath and remove after 5 mins. Add 3.0ml thermostable a-amylase solution, and mix contents of the tube to ensure complete dispersion of the sample.

4. Immediately place the tube into a boiling water bath. Incubate for 2 mins, remove from water bath and mix vigorously on a vortex mixer. Return tube to the boiling water bath for an additional 3 mins and then mix contents vigorously.

5. Add 4.0ml 200mM sodium acetate buffer and 0.1ml amyloglucosidase solution and vigorously mix contents on a vortex mixer. Incubate at 50°C for 30 mins.

6. Quantitatively transfer entire contents of test tube to 100ml volumetric flask. Dilute to 100ml of H2O (if sample contains 10% starch adjust volume to 10.0ml instead of 100ml).

7. Centrifuge portion of the suspension for 10 mins at 1000g.

8. Transfer 1ml aliquot of each filtrate to another test tube.

9. Add 3.0ml oxidase-peroxidase-aminoantipyrine buffer mixer to each test tube and incubate for 20 mins at 50°C.

10. Measure absorbance of each sample at 510nm against reagent blank.

Reference

McCleary, B.V., Gibson, T.S. and Mugford, D.C. (1997). Measurement of total starch in cereal products by amyloglucosidase-alpha-amylase method: Collaborative study. J. AOAC International. 80 (3):571-579.

  • Moisture

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