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纤维分析
参考文献:
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
试剂:
1. 饱和高锰酸钾 –50 g高锰酸钾和0.05 g硫酸银用去离子水定容到一升容量瓶中。
2. 缓冲液- 将6.0g9水和硝酸铁和0.15g硝酸银溶解于100ml去离子水中，再加入500ml冰醋酸，5g乙酸钾和400ml叔丁醇溶剂。
3. 高锰酸钾组合溶液-将两份饱和高锰酸钾溶液和一份缓冲液混合。此混合液可在冰箱中保存一周。在溶液呈现紫色且无沉淀出现时可用。
4. 去矿物质溶液-将50g二水合已二酸溶解于700ml95%乙醇中，加入50ml浓盐酸，用去离子水定容至一升 。
5. 丙酮
6. 80% 乙醇溶液 – 向845 ml 95% 乙醇中加入155 ml 去离子水。
7. 浓HBr - 48% HBr。
8. 浓H2SO4 – 在20oC时将其容重校正到1.634。
9. 72% H2SO4 – 将容量瓶中加入 383.9 ml 水，放置到凉水中，缓慢向其加入616.1 ml浓硫酸。
10. 淀粉酶- 由Novo 实验室购进热稳定淀粉酶。.
11. 中性洗涤溶液 – 见下页。
12. 酸性洗涤溶液-见下页。
Fib. Detr., pg.2
Reagents:
NDF 溶液:
去离子水体积 L 1 20 40
十二醇硫酸钠, USP g 30 600 1200
乙二醇一乙基醚(2-乙氧基乙醇) ml 10 200 400
,分析纯
乙二胺四乙酸钠, 二水合, 试剂 g 18.61 372.2 744.4
四硼酸钠, 十二水, 试剂 g 6.81 136.2 272.4
*磷酸氢二钠, 无水 g 4.56 91.2 182.4
注意: 配制溶液时需带口罩和手套，且在通风橱中进行。
在一容器中加入1/3水量，依次加入以上各种试剂，待前一试剂完全溶解后再加入下一种试剂。使用匀速搅拌器使溶液混合均匀，同时为了提高溶解性可采用热水溶解。最后一种试剂磷酸氢二钠需分开加，先加入1/10，等其溶解后再将剩余量加入到热水中。混合均匀并确定其PH为6.9-7.1，不过一般需要调整。
ADF 溶液:
去离子水体积 L 1 20 40
浓H2SO4 ml 26.7 533 1066
CTAB (十六烷基三甲基季铵 g 20 400 800
溴化物), USP
注意: 配制溶液时需带口罩和手套，且在通风橱中进行。
在适宜的容器中加入1/2水量，小心加入H2SO4并使用搅拌器搅拌均匀，缓慢加入CTAB，混合均匀。
Fib. Detr., pg.3
步骤:
中性洗涤纤维 (纤维素, 木质素, 及半纤维素)
1. 将每个样品放入有空坩埚或者放在No. 541色层分析滤纸上，105oC干燥。
2. 将0.5-1.0 g 样品放入没有喷嘴的500 ml贝采利乌斯烧杯中，加入100 ml NDF溶液和0.1 ml 淀粉酶， 放在纤维机架上。
3. 在煮沸瓶中回流60min。
4. 用真空过滤器或者滤纸过滤到适宜的坩埚中。
5. 用热的去离子水冲洗两次后用丙酮冲洗两次。
6. 105oC下干燥过夜。
7. 转移到干燥器中冷却，称重。
酸性洗涤纤维 (纤维素和木质素)
1. 将每个样品放入有空坩埚或者放在No. 541色层分析滤纸上，105oC干燥。
2. 将0.5-1.0 g 样品放入没有喷嘴的500 ml贝采利乌斯烧杯中，加入100 ml ADF 溶液。
3. 在煮沸瓶中回流60min。
4. 用真空过滤器或者滤纸过滤到适宜的坩埚中。
5. 用热的去离子水冲洗两次后用丙酮至少冲洗两次。
6. 105oC下干燥过夜。
7. 转移到干燥器中冷却，称重。
酸性洗涤木质素
1. 坩埚中盛放干燥的ADF ，放置到派热克斯浅玻璃盘中。
2. 加入72% H2SO4且没过坩埚中内容物。
3. 用玻璃棒搅拌仪打碎结块。
4. 加入一半体积的 72% H2SO4 ，每隔3小时搅拌20 minute ，温度必须控制在20-23oC。
5. 当液体耗尽时再次加满酸，过滤并用热的去离子水冲洗直至没有酸为止，冲洗玻璃棒。Refill with acid as it drains away. Filter and wash with hot dd water until free
of acid. Rinse and remove rod.
6. 105oC下坩埚干燥过夜。转移到干燥器中冷却，称重。
7. 灰分在马弗炉中500-550oC 过夜。
8. 冷却，干燥，称重。
Fib. Detr., pg.4
高锰酸钾法测木质素
1. 坩埚中盛放干燥的ADF ，放置到派热克斯浅玻璃盘中，盘中放有大约1 cm 凉水 Filtrate in crucibles should remain dry.
2. 加入约25 ml高锰酸钾组合溶液。
3. 用玻璃棒搅拌仪打碎结块。
4. 置 坩埚于20-25oC 众90 + 10 分钟，可适当多加混合的高锰酸钾溶液，必须自始至终呈现紫色。
5. 在过滤器中甩干，不要冲洗. 放置在干净的派热克斯浅玻璃盘中，加入一半体积的去矿物质水。甩干5min，再次加入去矿物质水，若纤维仍呈现 棕色则重复三次，用去矿物质水冲洗。
6. 用80%乙醇溶液冲洗两次后用丙酮冲洗两次。
7. 105oC下干燥过夜，转移到干燥器中冷却，称重。
二氧化硅含量
1. 高锰酸钾木质素或者ADF 500oC过夜残留的灰分，冷却，干燥，称重。
2. 加入足量的48% 溴化氢 使样品湿润，溴化氢不要超过4ml 。静置2小时，若形成了红色，可逐滴加入溴化氢。
3. 真空虑干并用丙酮冲洗。
4. 105oC下干燥过夜，转移到干燥器中冷却，称重。
Fib. Detr., pg.5
Calculations:
% NDF = (Cruc. + residue wt.) - (Cruc. tare wt.) x 100
Dry Sample wt.
% ADF = (Cruc. + residue wt.) - (Cruc. tare wt.) x 100
Dry Sample wt.
% Hemicellulose = % NDF - % ADF
% ADL = (Cruc. + H2SO4 residue wt.) - (Cruc. + Ash wt.) x 100
Dry Sample wt.
% Cellulose = % ADF - % ADL
% PL = (Cruc. + ADF res. wt.) - (Cruc. + Perm. res. wt.) x 100
Dry Sample wt.
% Si = (Cruc + Si ash wt.) - (Cruc. Tare wt.) x 100
Dry Sample wt.
Fib. Detr., pg.6
烧结玻璃坩埚的清洗
步骤:
1. 灰化坩埚，如果它没有没有像样品一样被灰化了的话。
2. 将灰化过的，冷的坩埚放置到派热克斯浅玻璃盘中，加入一半体积的去矿物质水，静置30min。
3. 将坩埚放在抽吸器上面，用手扶住，用去矿物质水冲洗坩埚有孔的底部，反复几次。
4. 将坩埚和抽吸器正常位置放置，抽吸器在上用热的去离子水彻底冲洗，如果坩埚没有过滤干净的话，将硅藻土和水混合薄薄的在坩埚底部铺一层。若坩埚仍没有过滤干净的话，重复步骤1。（重新灰化坩埚）
5. 将干净的，铺有硅藻土的坩埚放置在干燥的容器柜中或者其他地方57oC 过夜。
Total Dietary Fiber
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.
Calculations:
Corrected Spl. Abs. = (Spl. Abs.) - (Reag. Blk. Abs.)
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.
Starch Frac., pg. 3
Calculations:
Corrected Spl. Abs. = (Spl. Abs.) - (Reag. Blk. Abs.)
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 DFs
% FG = Total mg glucose X 100
Spl. wt. mg (DMB)
% DS = Total mg glucose X 100 - Ave % FG for sample X 0.9
Spl. wt. mg (DMB)
% RS = % Total Starch - % DS - (% FG X 0.9) |
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