The standard enzymatic–gravimetric method for the mea-surement of total dietary fiber (method 985.29, AOAC 2005) includes enzymatic treatments for starch and protein removal, isolation and weighing of the dietary fiber residue. In cereals, the enzymatic removal of starch with α-amylase and amylo-glucosidase is an essential step. The method is designed to simulate the digestive process of the small intestine. Protein and ash are determined on the residue and values are cor-rected. This is the official analytical technique for the esti-mation of total dietary fiber for labeling purposes.
(a) (c)
(b) (d)
FIGure 2.3 Detergent fiber analysis of foods. (a) Digestion with detergent solution; (b) filtration of detergent fiber; (c) gravimetric deter-mination of detergent fiber residue; (d) incineration of detergent fiber for ash correction.
55 Determination of Chemical and Nutritional Properties of Cereal Grains and Their Products
2.7.2.1 determination of total dietary Fiber A. Samples, Ingredients, and Reagents
• Test samples
• Heat-resistant α-amylase (Termamyl)
• Amyloglucosidase
• Graduated cylinder (1 L)
• Manifold filtration unit for crucibles
• Air-forced convection oven
• Muffle furnace
• Water bath
• Beakers (400 mL)
• Laboratory mill
• Volumetric flasks (1 L)
• Vacuum pump
• Tongs
• Desiccator
• Thermometer
• Fritted crucibles (coarse porosity) C. Procedure
1. Prepare the following reagents:
a. 78% and 95% ethanol solutions.
b. Phosphate buffer. In a volumetric flask, dis-solve 1.4 g of anhydrous dibasic sodium phosphate and 9.68 g of monobasic and mono-hydrated sodium phosphate in approxi mately 700 mL of distilled water. Then, adjust vol-ume to exactly 1 L with distilled water. The pH should be 6 ± 0.2.
c. Sodium hydroxide solution (0.275 N). In a 1-L volumetric flask, dissolve 11 g of NaOH in approximately 700 mL of distilled water.
Then, adjust volume to exactly 1 L with dis-tilled water.
d. Hydrochloric acid solution (0.325). Prepare a 1 M HCl acid solution by adding 83.3 mL of concentrated HCl in distilled water to a 1-L volume. Then, in a volumetric flask, mix 325 mL of 1 M HCl with distilled water to exactly 1 L.
2. Place cleaned fritted crucibles for 1 hour in a muffle furnace set at 525°C and after cooling in a desiccator, add 0.5 g celite and dry at 130°C for at least 1 hour. Allow fritted crucible with celite to cool down for approximately 30 minutes in a
desiccator. Then, record the filter weight with an accuracy of 0.0001 g. It is recommended that two or three repetitions should be run for each test sample. One will be used to obtain a residue for protein content and the other for ash content.
3. Weigh approximately 1 g of dried and finely ground sample (through a 0.5-mm mesh) with an accuracy of 0.0001 g and transfer to a 400 mL beaker (Figure 2.4). If the sample is rich in fat (contains >10% fat), remove the fat with petroleum ether before milling (refer to pro-cedure in Section 2.2.4.1). Record loss of fat weight and make appropriate corrections to final dietary fiber found after the analysis. Run blank through the entire process.
4. Add 50 mL of phosphate buffer (pH 6) to each beaker and then add 0.1 mL of heat-resistant α-amylase (Termamyl). Cover beaker with alu-minum foil and place it in a boiling water bath at 95°C to 100°C. Shake contents at 5-minute intervals for 30 minutes.
5. Cool solution to room temperature, adjust the pH to 7.5 by adding 10 mL of 0.275 N NaOH. Add 5 mg of protease that was previously diluted in phosphate buffer (0.1 mL of a 50 mg protease in 1 mL phosphate buffer). Cover beaker with alu-minum foil and incubate for 30 minutes at 60°C with continuous agitation. Cool, and then add 10 mL of 0.325 M HCl. The final pH should be adjusted to 4 to 4.6. Add 0.3 mL of amylogluco-sidase and incubate for 30 minutes at 60°C with continuous agitation.
6. Add 280 mL of 95% ethanol preheated to 60°C.
After the addition of alcohol, allow contents to cool down for 1 hour at room temperature to enhance the precipitation of the residue.
7. Filter contents through the previously weighed crucible containing the celite. Apply suction until all of the solution passes the filter. Then, wash the residue successively three times with 20 mL of 78% ethanol, twice with 10 mL 95%
ethanol, and twice with 10 mL acetone. Apply vacuum during the whole filtering procedure.
8. Dry crucibles with the residue until constant weight is achieved or dry overnight at 105°C.
Cool crucibles in a desiccator for 20 to 30 min-utes and then weigh to nearest 0.1 mg. Subtract crucible and celite weight of step 1 to calculate the weight of the residue (Figure 2.4).
9. Analyze protein of the residue contained in one of the crucibles and incinerate the other to cal-culate ash. The protein and ash contents of the residue will be used to correct dietary fiber val-ues. Subtract the protein and ash contents from the residue weight to obtain the dietary fiber weight. Express dietary fiber as a percentage
56 Cereal Grains: Laboratory Reference and Procedures Manual
using the following equation: % total dietary fiber
= (dietary fiber weight/sample weight) × 100. If sample was previously defatted, make sure to cor-rect values according to the original fat content.
2.7.2.2 determination of Insoluble and soluble dietary Fiber
This method was previously described by Asp et al. (1983).
A. Samples, Ingredients, and Reagents
• Test samples
• Protease
• NaOH
• Celite (acid washed)
• Acetone
• Aluminum foil
• Desiccant
• Heat-resistant α-amylase (Termamyl)
• Amyloglucosidase
• HCl
• Ethanol
• Phosphate buffer
• Distilled water
B. Materials and Equipment
• Analytical scale
• Graduated cylinder (1 L)
• Manifold filtration unit for crucibles
• Air-forced convection oven
• Muffle furnace
• Water bath
• Beakers (400 mL)
• Laboratory mill
• Volumetric flasks (1 L)
• Vacuum pump
• Tongs
• Desiccator
• Thermometer
• Fritted crucibles (coarse porosity) C. Procedure
1. Prepare the following reagents:
a. 78% and 95% ethanol solutions.
b. Phosphate buffer. In a volumetric flask, dis-solve 1.4 g of anhydrous dibasic sodium phosphate and 9.68 g of monobasic and monohydrated sodium phosphate in approxi-mately 700 mL of distilled water. Then,
(a) (b)
(c) (d)
FIGure 2.4 Dietary fiber analysis of foods. (a) Enzyme hydrolysis; (b) filtration of dietary fiber residue; (c) dehydration of dietary fiber residue; (d) incineration of dietary fiber for ash correction.
57 Determination of Chemical and Nutritional Properties of Cereal Grains and Their Products
adjust volume to exactly 1 L with distilled water. The pH should be 6 ± 0.2.
c. Sodium hydroxide solution (0.275 N). In a 1 L volumetric flask, dissolve 11 g of NaOH in approximately 700 mL of distilled water.
Then, adjust volume to exactly 1 L with dis-tilled water.
d. Hydrochloric acid solution (0.325 M).
Prepare a 1 M HCl acid solution by adding 83.3 mL of concentrated HCl in distilled water to a 1 L volume. Then, in a 1 L volu-metric flask, mix 325 mL of 1 M HCl with distilled water to exactly 1 L.
2. Place cleaned fritted crucibles for 1 hour in a muffle furnace set at 525°C and, after cool-ing in a desiccator, add 0.5 g celite and dry at 130°C for at least 1 hour. Allow fritted cruci-ble with celite to cool down for approximately 30 minutes in a desiccator. Then, record the filter weight with an accuracy of 0.0001 g. It is recommended that two or three repetitions should be run per test sample. One will be used to obtain a residue for protein content and the other for ash content.
3. Weigh approximately 1 g of dried and finely ground sample (through a 0.5-mm mesh) with an accuracy of 0.0001 g and transfer to a 400 mL beaker. If the sample is rich in fat (contains >10%
fat), remove the fat with petroleum ether before milling (refer to procedure in Section 2.2.4.1).
Record loss of fat weight and make appropriate corrections to final dietary fiber found after the analysis. Run blank through the entire process.
4. Add 25 mL of phosphate buffer (pH 6) to each beaker and then add 0.1 mL of heat-resistant α-amylase (Termamyl). Cover beaker with alu-minum foil and place it in a boiling water bath at 95°C to 100°C for 14 minutes. Make sure to shake contents every 3 to 4 minutes.
5. Cool solution to room temperature, add 20 mL of distilled water and adjust pH to 1.5 with 0.1 N HCl. Then, add 100 mg of pepsin. Cover beaker with aluminum foil and incubate for 60 minutes at 40°C with continuous agitation.
6. Cool and then add 20 mL of distilled water and adjust pH to 6.8 with 0.1 N NaOH. Add 100 mg of pancreatin. Cover beaker with aluminum foil and incubate for 60 minutes at 40°C with con-tinuous agitation.
7. Adjust pH to 4.5 with 0.1 M HCl.
8. Filter contents through the previously weighed crucible containing the celite. Apply suction until all the solution passes the filter. Wash twice with 10 mL distilled water. Make sure to recover all the filtrate for soluble fiber analysis.
9. Then, wash the residue retained by the crucible twice with 10 mL of 95% ethanol and twice
with 10 mL acetone. Apply vacuum during the whole filtering procedure.
10. Dry crucibles with the residue until constant weight is achieved or dry overnight at 105°C.
Cool crucibles in a desiccator for 20 to 30 minutes and then weigh to the nearest 0.1 mg.
Subtract crucible and celite weight to calculate the weight of the insoluble residue.
11. Incinerate the crucible to calculate the ash asso-ciated with the residue. Subtract the ash content from the insoluble residue weight to obtain the insoluble dietary fiber weight. Express insoluble dietary fiber as a percentage using the following equation: % insoluble dietary fiber = (dietary fiber weight/sample weight) × 100. If sample was previously defatted, make sure to correct values according to the original fat content.
12. To recover the soluble fiber, the total volume of the filtrate and wash waters of step 8 should be adjusted to exactly 100 mL. Then add 400 mL of warm (60°C) 95% ethanol.
13. Allow soluble fiber to precipitate for 1 hour and then filter the solution through a dried and weighed crucible containing celite. Then, wash the soluble fiber retained by the crucible twice with 10 mL of 78% ethanol, twice with 10 mL of 95% ethanol, and twice with 10 mL of ace-tone. Apply vacuum during the whole filtering procedure.
14. Dry crucibles with the soluble fiber until con-stant weight is achieved or dry overnight at 105°C. Cool dry crucibles in a desiccator for 20 to 30 minutes and then weigh to nearest 0.1 mg.
Subtract crucible and celite weight to calculate the weight of the soluble residue. Incinerate the crucible to calculate the ash associated with the soluble residue. Subtract the ash content from the soluble residue weight to obtain the soluble dietary fiber weight. Express soluble dietary fiber as a percentage using the follow-ing equation: % soluble dietary fiber = (soluble dietary fiber weight/sample weight) × 100. If the sample was previously defatted, make sure to correct values according to the original fat content.
2.7.3 researCh suggestions
1. Determine and compare crude fiber, detergent fiber, and dietary fiber values of a whole wheat bread sample.
2. Determine the acid and neutral detergent fiber and dietary fiber of regular barley and hull-less barley.
3. Determine and compare dietary fiber values of wheat, white-polished rice, and oats.
4. Determine the insoluble and soluble dietary fiber of whole wheat processed into refined flour.
58 Cereal Grains: Laboratory Reference and Procedures Manual 5. Investigate the Englyst-Cummings procedure to
determine dietary fiber and compare the method with the official AOAC (2005) dietary fiber procedure.
6. Determine the insoluble and soluble dietary of bar-ley before and after sprouting or malting.
2.7.4 researCh Questions
1. What are the principles behind fiber determination using the detergent fiber and official dietary fiber procedures?
2. What are the main fiber components associated with crude fiber, acid-detergent fiber, neutral detergent fiber, insoluble dietary fiber, and soluble dietary fiber?
3. Explain the purpose(s) of each of the following steps of the official AOAC (2005) dietary fiber method:
a. Heating sample with thermoresistant α-amylase b. Treating sample with protease
c. Adding warm ethanol d. Washing with acetone
4. Why is resistant starch associated with cereal-based foods considered as part of dietary fiber?
5. Explain the rationale of using bromidic acid in the acid-detergent fiber analysis.
6. What are the positive health effects of the insoluble and soluble fiber components?
7. What are the main chemical components of the sol-uble dietary fiber of oats or groats? How do these components affect blood cholesterol?
2.8 methods For nonFIBrous