TECHNICAL BULLETIN METHOD 1: DETERMINATION OF TOTAL DIETARY FIBRE
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1 TOTAL DIETARY FIBER KIT Cat N 32 v TECHNICAL BULLETIN METHOD 1: DETERMINATION OF TOTAL DIETARY FIBRE Introduction This procedure for the determination of total dietary fiber is based on the method published in the 16th Edition of the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC, method ). This assay determines the total dietary fiber content of food using a combination of enzymatic and gravimetric methods. Samples of dried, fat-free foods are gelatinized with heat stable a-amylase and then enzymatically digested with protease and amyloglucosidase to remove the protein and starch present in the sample. Ethanol is added to precipitate the soluble dietary fiber. The residue is then filtered and washed with ethanol and acetone. After drying, the residue is weighed. Half of the samples are analyzed for protein and the others are ashed. Total dietary fiber is the weight of the residue less the weight of the protein and ash. Reagents and Apparatus TOTAL DIETARY FIBER Heat Stable, a-amylase, incubation at ph 6.0, 15 min., 95 C Protease incubation at ph 7.5, 30 min., 60 C Amyloglucosidase incubation at ph 4.5, 30 min., 60 C Ethanol Precipitation Alcohol and Acetone Washes Drying Kjeldahl Protein Ash Determination Determination 5 hour, 525 C Calculation of Total Dietary Fiber Reagents provided: Total Dietary Fiber Assay Kit: this kit contains enough reagents to perform 200 assays. 1. -Amylase, Heat Stable; 20 ML; ready-to-use. 2. Protease; 20ML; ready to use. 3. Amyloglucosidase; 20 ML; ready-to-use. Fax : (33) /9
2 Reagents Required but Not Provided; 1. Ethyl Alcohol, ACS reagent; 2. Acetone, ACS reagent; 3. Sodium Phosphate, Dibasic, anhydrous; 4. Sodium Phosphate, Monobasic, anhydrous; 5. Sodium Hydroxide, 1.0 N; 6. Hydrochloric Acid, 1.0 N; 7. Petroleum ether; 8. Celite (available on demand). Apparatus 1. Fritted crucible-porosity #2 (coarse microns) 2. Vacuum source. 3. An air oven capable of operating at 105 C or a vacuum oven set at 70 C. 4. Desiccator 5. Muffle furnace 6. Boiling water bath 7. Constant temperature water bath adjustable to 60 C with a multi-station agitation system to provide agitation of the digestion flasks during enzymatic hydrolysis. 8. Beakers ml or 600 ml tall form 9. Analytical balance capable of weighing to 0.1 mg. 10. ph meter - standardized at ph 4.0 and ph 7.0. Preparation Instructions Crucibles Wash crucibles thoroughly. Heat one hour at 525 C and cool. Soak and rinse crucibles in water and then air dry. Add 0.5 grams of Celite to each crucible and dry at 130 C (one hour or more). Cool in desiccator and weigh to nearest 0.1 mg. Record this as "Celite + Crucible Weight" W1. Store in desiccator until needed. Reagents Use distilled or deionized water to prepare solutions % Ethanol Place 207 ml of water into a one liter volumetric flask. Dilute to volume with 95% ethanol. Mix and bring to volume again with 95% ethanol if necessary. Mix. 2. Phosphate Buffer, 0.08 M, ph 6.0 Dissolve 1.4 g of Na 2 HPO 4 and 8.4 g of NaH 2 PO 4 in approximately 700 ml of water. Dilute to one liter with water. Check ph and adjust if necessary with either NaOH or H 3 PO 4. Store in tightly capped container at room temperature. 3. Sodium Hydroxide Solution, N Dilute 275 ml of 1.0 N NaOH solution to one liter with water in a volumetric flask. Store in a tightly capped container at room temperature. 4. Hydrochloric Acid Solution, N Dilute 325 ml of 1.0 N HCl solution to one liter with water in a volumetric flask. Store in a tightly capped container at room temperature. Fax : (33) /9
3 Sample If the fat content of the sample is greater than 10%, defat with petroleum ether. Record the loss of weight due to fat removal and make the appropriate correction to the final % dietary fiber. When dealing with unknowns, fat-extract all samples. Homogenize each sample, if necessary, and dry overnight in an air oven at 105 C (70 C in vacuum oven). Cool in desiccator and dry mill sample to mm mesh. If apparatus is unavailable for milling, grinding in a mortar will be sufficient. If sample cannot be heated, freeze dry before milling. Store dry sample in a desiccator until analysis is carried out. Determination Run blanks along with samples through the entire procedure to measure any contributions to residue from reagents. Samples and blanks to be tested for dietary fiber content should be run at least in quadruplicate so that duplicate protein and ash values are available for improved accuracy. 1. Weigh out four 1 gram samples of each material to be tested into tall form beakers. Sample weights should not differ by more than 20 mg. Record weights to 0.1 mg. 2. Add 50 ml of ph 6.0 phosphate buffer to each beaker. Homogenize. 3. Add 0.1 ml -Amylase to each beaker and mix well. 4. Cover each beaker with aluminum foil and place in a boiling water bath. Agitate beakers gently at 5 minute intervals. Incubate for 15 minutes after the internal temperature reaches 95 C. 5. Allow solutions to cool to room temperature. 6. Adjust the ph of the solutions to 7.5 ± 0.2 by adding 10 ml of N NaOH to each beaker. Check ph, adjust if necessary with either NaOH or HCl. 7. Pipette 0.1 ml Protease (5 mg Protease) into each beaker. 8. Cover each beaker with aluminum foil and place in 60 C water bath. With continuous agitation, incubate for 30 minutes after the internal temperature of the beakers reaches 60 C. 9. Allow solutions to cool to room temperature. 10. Adjust the ph of the solutions to between ph 4.0 and 4.6 by adding 10 ml of M HCl to each beaker. Check ph, adjust if necessary with either NaOH or HCl. 11. Add 0.1 ml Amyloglucosidase to each beaker. 12. Cover each beaker with aluminum foil and place in 60 C water bath. With continuous agitation, incubate for 30 minutes after the internal temperature of the beakers reaches 60 C. 13. Add 280 ml of 95% ethanol to each beaker. 14. Let solutions set at room temperature to allow complete precipitation (time will be dependant on the matrix). Fax : (33) /9
4 15. Filtration: Wet and redistribute the bed of Celite in each crucible using 78% ethanol. Apply gentle suction to draw Celite onto frit as an even mat. Maintain gentle suction and quantitatively transfer the precipitate and suspension from each beaker to its respective crucible. Wash the residue with three 20 ml portions of 78% ethanol, two 10 ml portions of 95% ethanol, and two 10 ml portions of acetone. A gum may form with some samples, trapping liquid. Breaking the surface film with a spatula will improve the rate of filtration. Be sure to rinse any material adhering to the spatula into the crucible. The time for filtration and washing will vary from few minutes to 6 hours per crucible, averaging about 30 minutes per crucible. 16. Dry crucibles containing residues overnight in a 105 C air oven or 70 C vacuum oven. 17. Cool all crucibles in a desiccator, weigh to nearest 0.1 mg. Record this weight as Residue + Celite + Crucible Weight" W Analyze the residues from two samples and two blanks for protein by Kjeldahl nitrogen analysis as specified in the AOAC procedure 2. Use 6.25 as the factor to convert ammonia determined in the analysis to protein except where nitrogen content in the protein sample is known. 19. Ash the residue in the crucibles from two samples and two blanks for 5 hours at 525 C. Cool in desiccator, weigh to nearest 0.1 mg. Record this weight as "Ash + Celite + Crucible Weight" W3. Calculations Residue Weight = W2-W1 Ash Weight = W3-W1 BLANK : B = R BLANK - P BLANK - A BLANK % TDF = [ (R SAMPLE P SAMPLE A SAMPLE ) B ) / SW ] X 100 Where : TDF = Total Dietary Fiber R = Average Residue Weight (mg) P = Average Protein Weight (mg) A = Average Ash Weight (mg) SW = Average Sample Weight (mg) Reference 1. Official Methods of Analysis of AOAC International,16 th Edition, Volume II, Section , Method (1997). 2. Official Methods of Analysis of AOAC International,16 th Edition, Volume I, Section , Method (1997). CELITE is a trademark of World Minerals, Inc for its diatomaceous silicia product. Fax : (33) /9
5 METHOD 2: DETERMINATION OF TOTAL, SOLUBLE AND INSOLUBLE DIETARY FIBRE Introduction This procedure for the determination of dietary fiber is based on the AOAC Method and the AACC Method Briefly, 1 gram dried food samples (duplicate) are subjected to sequential enzymatic digestion by heat-stable amylase, protease, and amyloglucosidase. Soluble/insoluble dietary fibre determination. Insoluble dietary fibre (IDF) is filtered, and then residue is washed with warm distilled water. Combined solution of filtrate and water washings are precipitated with 4 volumes of 95 % ethanol (EtOH) for soluble dietary fibre (SDF) determination. Precipitate is then filtered and dried. Both SDF and IDF residues are corrected for protein, ash, and blank for the final calculation of SDF and IDF values Total dietary fibre determination. SDF is precipitated with EtOH, and residue is then filtered, dried, and weighed. Total dietary fibre (TDF) value is corrected for protein and ash content. FLOW PLAN FOR DIETARY FIBER DETERMINATIONS 1 g Sample + 40 ml MES/TRIS buffer solution + 50 µl Heat Stable -Amylase, incubation 30 min. at 95 C Cool to 60 C µl Protease, incubation 30 min. at 60 C Ajust ph ( ) µl Amyloglucosidase incubation 30 min. at 60 C Filter, wash Total Dietary Fiber Soluble Dietary Fiber Insoluble Dietary Fiber Precipitate with Combine filtrate and rinsings, Rince residue, Ethanol and determine the weight dry, weigh Allow to settle, Precipitate with Protein Ash filter, dry, weigh Ethanol Protein Ash Allow to settle, Filter, dry, weigh Protein Ash Fax : (33) /9
6 Reagents and Apparatus Reagents provided: Total Dietary Fiber Assay Kit: this kit contains: 1. -Amylase, Heat Stable; 20 ml; ready-to-use. 2. Protease; 20ml; ready to use. 3. Amyloglucosidase; 20 ml; ready-to-use. Reagents Required but Not Provided: 4. Ethanol 95% v/v (95 ml ethanol absolute + 5 ml distilled water) 5. Ethanol 78% v/v (78 ml ethanol absolute + 22 ml distilled water) 6. Acetone, ACS reagent 7. MES/Tris buffer solution 0.05M ph 8.2 at 24 C: Dissolve g 2(N-morpholino) ethanesulfonic acid (MES) (Sigma, M 8250) and 1.42 g tris(hydroxymethyl)aminomethane (TRIS) (Sigma,TI503) in 0.17 L deionised water. Adjust ph to 8.2 with 6.0 N NaOH. Dilute to 0.2 L with water. It is important to adjust ph of buffer to approximately 8.3 at 20 C (8.1 at C). The ph change with temperature 8. Hydrochloric Acid, N (Add 93.5 ml of HCl 6N to 700 ml of distilled water. Complete to 1L) 9. Celite (available on demand). Apparatus: 1. Analytical balance capable of weighing to 0.1 mg. 2. Beakers ml or 600 ml tall form 3. Fritted crucible-porosity #2 (coarse microns). Prepare as follows: - Wash crucibles thoroughly. Heat overnight at 525 C and cool. Soak and rinse crucibles in water and then air dry. - Add 1 gram of Celite to each crucible and dry at 130 C (one hour or more). Cool in desiccator and weigh to nearest 0.1 mg. - Record this weight. - Store in desiccator until needed. 4. Vacuum source. 5. Filtering flask 6. An air oven capable of operating at 105 C or a vacuum oven set at 70 C. 7. Desiccator with drying agent 8. Muffle furnace, adjustable to 525 C 9. Waterbath, adjustable to 60 C and 100 C with an agitation system to provide agitation of the digestion flasks during enzymatic hydrolysis. 10. ph meter - standardized at ph 4.0 and ph 7.0. Sample Preparation Instructions If the fat content of the sample is greater than 10%, defat with petroleum ether. Record the loss of weight due to fat removal and make the appropriate correction to the final % dietary fiber. When dealing with unknowns, fat-extract all samples. Homogenize each sample, if necessary, and dry overnight in an air oven at 105 C (70 C in vacuum oven). Cool in desiccator and dry mill sample to mm mesh. If apparatus is unavailable for milling, grinding in a mortar will be sufficient. If sample cannot be heated, freeze dry before milling. Store dry sample in a desiccator until analysis is carried out. Fax : (33) /9
7 Determination procedure A. Enzymatic Breakdown Run blanks along with samples through the entire procedure to measure any contributions to residue from reagents. Samples and blanks to be tested for dietary fiber content should be run at least in quadruplicate so that duplicate protein and ash values are available for improved accuracy. 1. Weigh 1 gram samples of each material to be tested into 400 ml beakers. Sample weights should not differ by more than 20 mg. Record weights to 0.1 mg. 2. Add 40 ml of ph 8.2 MES/TRIS buffer to each beaker. Homogenize. 3. Add 0.05 ml -Amylase to each beaker and mix well. Cover each beaker with aluminium foil and place in a boiling water bath. After the internal temperature reaches 95 C, incubate for 30 minutes with continuous agitation. 4. Allow solutions to cool to 60 C. 5. Pipette 0.1 ml Protease (5 mg Protease) into each beaker. Cover each beaker with aluminium foil and place in 60 C water bath. After the internal temperature reaches 60 C, incubate for 30 minutes with continuous agitation. 6. Adjust the ph of the solutions to between ph 4.0 and 4.7 by adding 5 ml of N HCl solution to each beaker. Check ph, adjust if necessary with either 5% NaOH solution or 5% HCl solution. 7. Add 0.2 ml Amyloglucosidase to each beaker. Cover each beaker with aluminium foil and place in 60 C water bath. After the internal temperature reaches 60 C, incubate for 30 minutes with continuous agitation. B. Determination of Insoluble and Soluble Dietary Fiber 8. Prepare the crucible: Tare crucible containing Celite to nearest 0.1 mg. Wet and redistribute bed of Celite in crucible using approximately 3 ml distilled water. Apply suction to crucible to draw Celite onto fritted glass as even mat 9. Following enzymatic breakdown (step 7), filter the liquid through crucible into the filtering flask. 10. Wash residue with 2 10 ml distilled water preheated to 70 C. Use water to rinse beaker before washing residue in crucible. Save filtrate and water washings for determination of SDF. Transfer solution to a pretared 600 ml beaker. (For SDF determination, go to Step 11 of SDF procedure) a) Determination of Insoluble Dietary Fiber 11. Wash residue 2 10 ml of 95% ethanol and 2 10 ml of acetone. 12. Dry crucible containing residue overnight at 105 C. 13. Cool crucible 1 hour in a dessicator. Weigh crucible containing dietary fibre residue and Celite to nearest 0.1 mg. To obtain residue weight, subtract tare weight, i.e., weight of dried crucible and Celite. Fax : (33) /9
8 14. Protein and Ash determination: One residue from each type of fibre is analysed for protein, and the second residue of the duplicate is analysed for ash. - Perform protein analysis on residue using Kjeldahl method. Use 6.25 factor for all cases to calculate g of protein. - For ash analysis, incinerate the second residue for 5 hours at 525 C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract crucible and Celite weight to determine ash. b) Determination of Soluble Dietary Fiber Follow the step 1-10 of the method. 11. Weigh combined solution of filtrate and water washings in pretared beaker from Step Precipitation of SDF: - Add 4 vols 95 % EtOH preheated to 60 C. Use a portion of EtOH to rinse filtering flask from the step 10 of the procedure. Alternatively, adjust weight of combined solution of filtrate and water washings to 80 g and add 320 ml of preheated (60 C) 95 % EtOH. - Allow the precipitate to form at room temperature for 60 minutes. 13 Filtration setup: - Tare crucible containing Celite to nearest 0.1 mg. - Wet and redistribute the bed of Celite in the crucible, using 15 ml of 78 % EtOH from wash bottle. - Apply suction to crucible to draw Celite onto fritted glass as an even mat 14. Filtration - Filter precipitated enzyme digest from SDF Step 12 through crucible. - Using a wash bottle with 78 % EtOH and a rubber spatula, quantitatively transfer all remaining particles to crucible. 15. Wash: Using a vacuum, wash residue successively with two 15 ml portions of the following: 78 % EtOH, 95 % EtOH, Acetone 16. Dry crucible containing residue overnight at 105 C 17. Cool crucible 1 hour in a dessicator. Weigh crucible containing dietary fibre residue and Celite to nearest 0.1 mg. To obtain residue weight, subtract tare weight, i.e., weight of dried crucible and Celite. 18. Protein and Ash determination: One residue from each type of fibre is analysed for protein, and the second residue of the duplicate is analysed for ash. - Perform protein analysis on residue using Kjeldahl method. Use 6.25 factor for all cases to calculate g of protein. - For ash analysis, incinerate the second residue for 5 hours at 525 C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract crucible and Celite weight to determine ash. C. Determination of Total Dietary Fiber Following the enzymatic breackdown (steps 1-7): 8. Precipitation of dietary fiber with EtOH - To each sample, add 225 ml 95 % EtOH preheated to 60 C. Measure volume after heating. Ratio of EtOH volume to sample volume should be 4:1. If 95 % EtOH is accidentally overheated to 65 C, add 228 ml for expanded alcohol volume adjustment. - Cover all samples with large sheets of aluminium foil. - Allow precipitate to form at room temperature for 60 min. Fax : (33) /9
9 9. Filtration setup - Tare crucible containing Celite to nearest 0.1 mg. - Wet and redistribute the bed of Celite in the crucible, using 15 ml of 78 % EtOH. - Apply suction to crucible to draw Celite onto fritted glass as an even mat 10. Filtration - Filter precipitated enzyme digest from Step 8 through crucible. - Using a wash bottle with 78 % EtOH and a rubber spatula, quantitatively transfer all remaining particles to crucible. 11. Wash Using a vacuum, wash residue successively with two 15 ml portions of the following: 78 % EtOH, 95 % EtOH, Acetone 12. Dry crucible containing residue overnight at 105 C 13. Cool crucible 1 hour in a dessicator. Weigh crucible containing dietary fibre residue and Celite to nearest 0.1 mg. To obtain residue weight, subtract tare weight, i.e., weight of dried crucible and Celite. 14. Protein and Ash determination: One residue from each type of fibre is analysed for protein, and the second residue of the duplicate is analysed for ash. - Perform protein analysis on residue using Kjeldahl method. Use 6.25 factor for all cases to calculate g of protein. - For ash analysis, incinerate the second residue for 5 hours at 525 C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract crucible and Celite weight to determine ash. D. Calculations % dietary fiber = [ ((R SAMPLE P SAMPLE A SAMPLE ) B ) / S ] X 100 Where : R = Average Residue Weight P = Average Protein Weight A = Average Ash Weight S = Average Sample Weight And B = Blank: B = R BLANK P BLANK A BLANK Reference 3. J.Assoc.Off.Anal.Chem. 75: (1992). 4. J.Assoc.Off.Anal.Chem. 71: (1988). 5. J.Assoc.Off.Anal.Chem. 75: (1992). CELITE is a trademark of World Minerals, Inc for its diatomaceous silicia product.
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