參考文獻(xiàn):
Goering and Van Soest, 1970, USDA Handbook No. 379.
Jeraci et al., 1988, J. Anim. Sci. 66(Suppl. 1):351(Abstr.)
Official Methods of Analysis of AOAC International, 2002, Vol. 17, Method 2002.04
Official Methods of Analysis of AOAC International, 2002, Vol. 17, Method 973.18
酸性洗滌木質(zhì)素
1. 坩堝中盛放干燥的ADF ，放置到派熱克斯淺玻璃盤中。
2. 加入72% H2SO4且沒過坩堝中內(nèi)容物。
3. 用玻璃棒攪拌儀打碎結(jié)塊。
4. 加入一半體積的 72% H2SO4 ，每隔3小時(shí)攪拌20 minute ，溫度必須控制在20-23oC。
5. 當(dāng)液體耗盡時(shí)再次加滿酸，過濾并用熱的去離子水沖洗直至沒有酸為止，沖洗玻璃棒。Refill with acid as it drains away. Filter and wash with hot dd water until free
of acid. Rinse and remove rod.
6. 105oC下坩堝干燥過夜。轉(zhuǎn)移到干燥器中冷卻，稱重。
7. 灰分在馬弗爐中500-550oC 過夜。
8. 冷卻，干燥，稱重。
References:
Prosky et al., 1984, J. Assoc. Off. Anal. Chem. Vol. 67, No. 6:1044-1052
Official Methods of Analysis of AOAC International, 2002, Vol. 17, Method 985.29
Reagents:
1. 95% Ethanol
2. 78% Ethanol - Place 207 ml of distilled water in a 1 liter volumetric flask. Bring up to volume with 95% ethanol.
3. Acetone
4. Phosphate Buffer, 0.08 M, pH 6.0 - Dissolve 1.4 g of sodium phosphate
dibasic, anhydrous, and 9.68 g of sodium phosphate monobasic, monohydrate in a 1 liter volumetric flask. Bring up to volume with distilled water.
Check by pH measurement. This solution will keep for up to 48 hrs if kept refrigerated.
5. Termamyl (heat stable alpha-amylase) Solution 120 L - Novo Laboratories Inc., Wilton, Conn. 06897. Stored in the refrigerator.
6. Protease P-5380 - Sigma Chemical Co., St. Louis, Mo. 63178. Make up a
5 mg/100 microliter phosphate buffer solution. This solution will keep for up to 48 hrs if kept refrigerated.
7. Amyloglucosidase A-9913 - Sigma Chem. Keep refrigerated.
8. Sodium Hydroxide Solution (0.275 N) - Dissolve 11.0 g of sodium hydroxide in 700 ml distilled water. Bring up to volume in a 1 liter volumetric flask.
9. Phosphoric Acid Solution (0.325 M) - Place 22.0 ml of 85% o-phosphoric acid in a 1 liter volumetric flask. Bring up to volume with distilled water.
10. Chloroform:Methanol Solution (2:1)
TDF, pg.2
Procedure:
1. Fold and number Whatman 541 filter paper. Dry crucibles and filter paper at 105oC overnight. To run samples in duplicate, you will need four filter papers and two crucibles for each sample.
2. Fat extraction: (This does not need to be done on samples with <15% fat. Go to step 4.) Weigh out 1.0 g samples in quadruplicate into 30 ml screw cap test tubes. These samples must not differ by more than 20 mg. (The closer the sample weights are, the better your results will be.) Add
15 ml of the 2:1 chloroform:methanol solution. Place tubes in a metabolic shaker overnight. If the samples are very high in fat, use a 50 ml tube and 30 ml 2:1 chloroform:methanol.
3. Filter samples through dacron under vacuum. Rinse the tubes with the
minimum amount of chloroform:methanol needed to remove the sample
from the tube. Stand these upright in labeled 100 ml beakers. Dry at 55oC
overnight.
4. Transfer the samples, quantitatively, from the dacron filters to tall form berzelius beakers, labeled with tape. If you did not run the fat
extraction, weigh out 0.5-1.0 g samples in duplicate into the same beakers mentioned above. These must not differ by more than 20 mg. (The closer your sample weights are, the better your results will be.) Add 50 ml
of phosphate buffer to each beaker.
5. Add 0.1 ml of Termamyl solution to each beaker.
6. Cover beaker with aluminum foil and place in a boiling water bath. Weight the beakers down with metal washers or watchglasses, if necessary. Heat for 30 minutes.
7. Remove beakers from water bath. Adjust pH to 7.5 by adding 10 ml of 0.275 N NaOH solution.
8. Add 0.1 ml of Protease solution to each beaker.
9. Recover with foil and place in a 60oC water bath. Heat for 30 minutes.
10. Remove beakers from water bath. Adjust pH to 4.5 by adding 10 ml of 0.325 M phosphoric acid solution.
11. Add 0.1 ml of amyloglucosidase solution to each beaker.
12. Recover with foil and place in a 60oC water bath. Heat for 30 minutes.
13. Add 280 ml of 60oC 95% ethanol to each.
Note: Room temperature ethanol will work as well as 60oC ethanol.
14. Allow samples to precipitate for 60 minutes at room temperature.
15. Filter precipitated samples through tared filter paper.
16. Wash residue successively with three 20 ml portions of 78% ethanol, two
10 ml portions of 95% ethanol, and two 10 ml portions of acetone.
17. Dry at 105oC overnight and weigh.
18. Place two of each four in the appropriate crucibles. Ash at 400-600oC overnight, cool, and weigh back. Run the Kjeldahl procedure or the Leco procedure for crude protein on the other two.
TDF, pg.3
Calculations:
g residue = (dry spl. + filter paper wt.) - dry filter paper wt.
% CP = (mls - mls blank) x F value x 100 x 6.25
g residue in sample
% Ash = (Cruc. + ash wt.) - dry cruc. wt. x 100
g residue in sample
g CP = % CP x average g residue of duplicates (one ash, one kjeldahl)
g Ash = % Ash x average g residue of duplicates (one ash, one kjeldahl)
% TDF = average g residue - g CP - g Ash x 100
average spl. wt. of same pair (DMB)
Total Dietary Fiber - Soluble/Insoluble
References:
Prosky et al., 1992, J. Assoc. Off. Anal. Chem. Int. Vol. 75, No. 2:360-367
Reagents:
1. 95% Ethanol
2. 78% Ethanol - Place 207 ml of distilled water in a 1 liter volumetric flask. Bring up to volume with 95% ethanol.
3. Acetone
4. Phosphate Buffer, 0.08 M, pH 6.0 - Dissolve 1.4 g of sodium phosphate
dibasic, anhydrous, and 9.68 g of sodium phosphate monobasic, monohydrate in a 1 liter volumetric flask. Bring up to volume with distilled water.
Check by pH measurement. This solution will keep for up to 48 hrs if kept refrigerated.
5. Termamyl (heat stable alpha-amylase) Solution 120 L - Novo Laboratories Inc., Wilton, Conn. 06897. Stored in the refrigerator.
6. Protease P-5380 - Sigma Chemical Co., St. Louis, Mo. 63178. Make up a
5 mg/100 microliter phosphate buffer solution. This solution will keep for up to 48 hrs if kept refrigerated.
7. Amyloglucosidase A-9913 - Sigma Chem. Keep refrigerated.
8. Sodium Hydroxide Solution (0.275 N) - Dissolve 11.0 g of sodium hydroxide in 700 ml distilled water. Bring up to volume in a 1 liter volumetric flask.
9. Phosphoric Acid Solution (0.325 M) - Place 22.0 ml of 85% o-phosphoric acid in a 1 liter volumetric flask. Bring up to volume with distilled water.
10. Chloroform:Methanol Solution (2:1)
TDF-S/I, pg.2
Procedure:
1. Fold and number Whatman 541 filter paper. Dry crucibles and filter paper at 105oC overnight. To run samples in duplicate, you will need eight filter papers and four crucibles for each sample.
2. Fat extraction: (This does not need to be done on samples with <15% fat. Go to step 4.) Weigh out eight 1.0 g samples into 30 ml screw cap test tubes.
These samples must not differ by more than 20 mg. (The closer the sample
weights are, the better your results will be.) Add 15 ml of the 2:1
chloroform:methanol solution. Place tubes in a metabolic shaker overnight. If the
samples are very high in fat, use a 50 ml tube and 30 ml 2:1 chloroform:methanol.
3. Filter samples through dacron under vacuum. Rinse the tubes with the
minimum amount of chloroform:methanol needed to remove the sample
from the tube. Stand these upright in labeled 100 ml beakers. Dry at 55oC
overnight.
4. Transfer the samples, quantitatively, from the dacron filters to tall form berzelius beakers, labeled with tape. If you did not run the fat
extraction, weigh out 0.5-1.0 g samples in duplicate into the same beakers mentioned above. These must not differ by more than 20 mg. (The closer your sample weights are, the better your results will be.) Add 50 ml
of phosphate buffer to each beaker.
5. Add 0.1 ml of Termamyl solution to each beaker.
6. Cover beaker with aluminum foil and place in a boiling water bath. Weight the beakers down with metal washers or watchglasses, if necessary. Heat for 30 minutes.
7. Remove beakers from water bath. Adjust pH to 7.5 by adding 10 ml of 0.275 N NaOH solution.
8. Add 0.1 ml of Protease solution to each beaker.
9. Recover with foil and place in a 60oC water bath. Heat for 30 minutes.
10. Remove beakers from water bath. Adjust pH to 4.5 by adding 10 ml of 0.325 M phosphoric acid solution.
11. Add 0.1 ml of amyloglucosidase solution to each beaker.
12. Recover with foil and place in a 60oC water bath. Heat for 30 minutes.
13. Add 280 ml of 60oC 95% ethanol to the first four beakers of each sample. Add
280 ml of dd water to the second four beakers of each sample
Note: Room temperature ethanol will work as well as 60oC ethanol.
14. Allow samples with ethanol to precipitate for 60 minutes at room temperature.
15. Filter precipitated and non-precipitated samples through tared filter paper.
16. Wash precipitated residue successively with three 20 ml portions of 78% ethanol,
two 10 ml portions of 95% ethanol, and two 10 ml portions of acetone.
Wash non-precipitated residue with three 20 ml portions of water and two 10 ml
portions of acetone.
17. Dry at 105oC overnight and weigh.
18. Place two of each four in the appropriate crucibles. Ash at 400-600oC
overnight, cool, and weigh back. Run the Kjeldahl procedure or the Leco procedure for crude protein on the other two.
TDF-S/I, pg.3
Calculations:
Each set of eight was divided into two sets of four during the assay. The following
equations must be done for each set.
g residue = (dry spl. + filter paper wt.) - dry filter paper wt.
% CP = (mls - mls blank) x F value x 100 x 6.25
g residue in sample
% Ash = (Cruc. + ash wt.) - dry cruc. wt. x 100
g residue in sample
g CP = % CP x average g residue of duplicates (one ash, one kjeldahl)
g Ash = % Ash x average g residue of duplicates (one ash, one kjeldahl)
% TDF = average g residue - g CP - g Ash x 100
average spl. wt. of same pair (DMB)
% Total DF is the % TDF calculated from the samples that had ethanol added at step 13.
% Insoluble DF is the % TDF calculated from the samples that had water added at
step 13.
% Soluble Fiber = % TDF - % IDF
Note: For the best results, both TDF and IDF need to be run at the same time.
Starch Determination
Reference:
Thivend, Merier and Guilbot, 1972, Methods in Carbohydrate Chemistry. 6:100-105, Academic Press, New York
Official Methods of Analysis of AOAC International, 2002, Vol. 17, Method 979.10
AACC #76-11
Reagents:
1. Sodium Acetate Buffer, 0.15 N, pH 4.5 - Place 20.4 g of sodium acetate trihydrate
(or 12.3 g anhydrous) in a 1 liter volumetric flask. Add enough
distilled/deionized (dd) water to dissolve. Add 10 ml glacial acetic acid. Bring up to volume with dd water. Adjust pH to 4.5 with glacial acetic acid.
2. Amyloglucosidase Solution - Novozyme AMG 3L (Sigma A-7095) (~300 U/ml)
3. Corn Starch
4. Glucose Working Standard - Place 15 ml of glucose standard (Sigma G-3285) in a
100 ml volumetric flask. Bring up to volume with dd water. (0.15 mg/ml)
5. Color Reagent - Reconstitute o-dianisidine dihydrochloride vial (Sigma F-5803) with
20 ml dd water. Stable for 3 months in the refrigerator.
6. Enzyme/Color Reagent - Add contents of 1 PGO capsule (Sigma P-7119) to a brown
glass wide mouth bottle. Add 100 ml dd water. Add 1.6 ml of Color Reagent. Mix
gently. Stable for 1 month in the refrigerator. Discard if color or turbidity develops.
Procedure:
1. Grind samples to 0.5mm. Weigh samples and standard in duplicate into 50 ml screw cap test tubes. Start a reagent blank here.
Sample g/tube
Feed with starch 200 - 300 mg
Duodenal/ileal 600 - 700 mg
Fecal 1000 mg
Forage 1000 mg
Corn Starch Standard 200 mg
2. Add 20 ml of 0.15 N sodium acetate buffer to each tube. Vortex.
3. Place in a boiling water bath for 20 minutes. Vortex tubes once during boiling.
Note: It is critical that the sample goes into suspension. Increase the boil time if needed.
Glass beads may be added to the tubes to help suspend samples.
4. Vortex tubes again. Cap the tubes lightly.
Autoclave for 1 hour between 121oC and 135oC.
Note: Increase the autoclave time as you decrease the temperature from 135oC to 121oC.
5. Let tubes cool to room temperature. Add 0.1 ml of amyloglucosidase solution to
each tube. Vortex gently.
Starch Detr., pg.2
6. Incubate tubes for 24 hours at 55oC. Vortex tubes frequently.
7. Transfer samples to 100 ml volumetric flasks. Bring up to volume with dd water.
8. Centrifuge an aliquot for 10 minutes at 10,000 - 15,000 x g.
9. Save the supernate. Dilute an aliquot of each 30 fold.
Note: If the colorimetric portion of this assay does not work, you can used your saved supernate to rerun the assay from step 10.
9. Prepare glucose standards.
0.12 mg/ml = 4 ml working std + 1 ml dd water
0.09 mg/ml = 3 ml working std + 2 ml dd water
0.06 mg/ml = 2 ml working std + 3 ml dd water
0.03 mg/ml = 1 ml working std + 4 ml dd water
Water Blank = dd water
10. In duplicate, pipet 0.5 ml of glucose standards, water blank, reagent blank, starch standard, and samples into 13 x 100 mm disposable flint glass test tubes.
11. Add 5 ml enzyme/color reagent to each tube. Vortex.
12. Let stand 45 minutes in the dark. Vortex.
13. Read absorbance on a spectrophotometer at 450 nm. Zero the spec. with the water blank.
Determine the glucose concentration of the samples and starch standard using the corrected absorbance against the glucose standard curve.
Total mg glucose in sample = mg glucose from std. curve X DF X 30
% Starch = (Total mg glucose X 0.9) X 100
Spl. wt. mg (DMB)
The starch standard is used to verify that all the starch has been hydrolyzed and measured as glucose. You should get nearly 100% recovery. If you do not, rerun the entire assay.
Starch Fractionation Assay I
Note: You need the total starch value from the Thivend Starch Determination assay in order to calculate the resistant starch fraction.
References:
Muir and O’Dea, 1992, Am. J. Clin. Nutr. 56:123-127
Muir and O’Dea, 1993, Am. J. Clin. Nutr. 57:540-546
Reagents:
1. Pepsin/HCl Solution - Place 400 ml of distilled/deionized water in a 500 ml volumetric flask. Add 0.5 g of pepsin (1:10,000, Sigma P-7000). Add 1.03 ml conc. HCl. Bring up to volume with dd water. Adjust pH to 2.0 with HCl. Stable for 60 days. Store in the refrigerator. Bring up to room temperature before use.
2. Sodium Hydroxide, 0.5M - In a 1 liter volumetric flask, dissolve 20 g of NaOH in
500 ml dd water. Bring up to volume with dd water. Stable.
3. Sodium Acetate Buffer, 0.2M - Place 16.408 g of sodium acetate, anhydrous, in a
1 liter volumetric flask. Dissolve in 500 ml of dd water. Add 3 ml glacial acetic acid. Bring up to volume with dd water. Adjust pH to 5.0 with acetic acid.
4. Enzyme Solution - Place 0.14 g amyloglucosidase (Sigma A -7255 Lot 66H0483) and
1.0 g -amylase (Sigma A-6880) in a 100 ml volumetric flask. Bring up to volume
in 0.2M sodium acetate buffer, pH 5.0. Final solution will be 28U/ml amyloglucosidase & 10 mg/ml -amylase.
Note: The amount of amyloglucosidase needs to be adjusted for each lot number.
The purity varies from lot to lot.
5. Glucose Working Standard - Place 15 ml of glucose standard (Sigma 635-100) in a
100 ml volumetric flask. Bring up to volume with dd water. (0.15 mg/ml)
6. Color Reagent - Reconstitute o-dianisidine dihydrochloride vial (Sigma D-2679) with
20 ml dd water. Stable for 3 months in the refrigerator.
7. Enzyme/Color Reagent - Add contents of 1 PGO capsule (Sigma P-7119) to a brown
glass wide mouth bottle. Add 100 ml dd water. Add 1.6 ml of Color Reagent. Mix
gently. Stable for 1 month in the refrigerator. Discard if color or turbidity develops.
Procedure:
1. Weigh 0.1 - 0.25 g samples into 50 ml pyrex screw cap centrifuge tubes. You will need four tubes per sample. Label two tubes FG and two tubes DS.
You will need to start a set of four blank tubes and a set of tubes for the standards.
Note: Sample should contain no more than 0.1 g carbohydrate.
2. Add 1 ml Pepsin/HCl solution to each FG tube. Vortex gently. Incubate 30 minutes at 37oC.
3. Neutralize each tube with 0.5 ml NaOH.
Note: Test the pH of the neutralization step with a blank that can be discarded. It must
equal pH 5.0. If it doesn’t, adjust the amount of NaOH added at step 3 and 7.
4. Add 5 ml water to each tube. Vortex.
5. Centrifuge at 3,000 x g for 10 min. Pipet off supernate into a 10 ml volumetric flask. Bring up to volume with dd water. Set aside for glucose analysis.
Starch Frac., pg. 2
6. Add 1 ml Pepsin/HCl solution to each DS tube. Vortex gently. Incubate 30
minutes at 37oC.
7. Neutralize each tube with 0.5 ml NaOH.
8. Add 5 ml of 0.2M sodium acetate buffer and 1 ml of enzyme solution to each
tube. Cap tightly. Vortex. Incubate DS tubes for 15 hr at 37oC in a shaking
water bath. Set shaker at 120 or higher.
9. After 15 hr, pull DS tubes from water bath. Centrifuge at 3,000 x g for 10 minutes.
Pipet off supernate into a 25 ml volumetric flask. Label volumetric flasks as DS
samples.
10. Wash pellet with 1.5 ml of sodium acetate buffer.
11. Centrifuge at 3,000 x g for 10 minutes. Pipet off supernate into the same 25 ml
volumetric flask.
12. Wash pellet with 1.5 ml of sodium acetate buffer.
13. Centrifuge at 3,000 x g for 10 minutes. Pipet off supernate into the same 25 ml
volumetric flask.
14. Wash pellet with 1.5 ml of sodium acetate buffer.
15. Centrifuge at 3,000 x g for 10 minutes. Pipet off supernate into the same 25 ml
volumetric flask. Bring up to volume with dd water. Dilute an aliquot of each 50
fold. Set aside for glucose analysis.
Note: The FG samples will probably run without further dilution.
Note: If the colorimetric portion of this assay does not work, you can used your saved supernate to rerun the assay from step 17.
16. Prepare glucose standards.
0.12 mg/ml = 4 ml working std + 1 ml dd water
0.09 mg/ml = 3 ml working std + 2 ml dd water
0.06 mg/ml = 2 ml working std + 3 ml dd water
0.03 mg/ml = 1 ml working std + 4 ml dd water
Water Blank = dd water
17. In duplicate, pipet 0.5 ml of glucose standards, water blank, reagent blank, and all set aside samples into 13 x 100 mm disposable flint glass test tubes.
18. Add 5 ml enzyme/color reagent to each tube. Vortex.
19. Let stand 45 minutes in the dark. Vortex.
20. Read absorbance on a spectrophotometer at 450 nm. Zero the spec. with the water blank.