- 背景資料
現行衛福部對膳食纖維的定義為:⌈人體小腸無法消化與吸收之三個以上單醣聚合之可食碳水化合物及木質素⌋
故膳食纖維涵蓋範圍廣泛，依據其不同的化學性質或分類方式，可大致分成以下幾類:
1️⃣抗性澱粉(Resistant Starch，簡稱RS)，可分成5類，分別為RS1、RS2、RS3、RS4、RS5
2️⃣高分子量膳食纖維 (High Molecular Weight Dietary Fiber，簡稱HMWDF)，此類別又可分成
➡️➡️非水溶性膳食纖維( Insoluble Dietary Fiber，簡稱 IDF)
➡️➡️高分子量水溶性膳食纖維(水溶但78%酒精不可溶)(Soluble Dietary Fiber which precipitates in 78% ethanol，High Molecular Weight Soluble Dietary Fiber，簡稱SDFP或HMWSDF)
3️⃣低分子量水溶性膳食纖維(水溶且78%酒精也可溶)( Soluble Dietary Fiber that remains soluble in 78% ethanol，non-digestible oligosaccharides，Low Molecular Weight Soluble Dietary Fiber，簡稱SDFS或NDO或LMWSDF)
⚠️備註⚠️
➡️樣品中如有抗性澱粉(RS2和RS4)、低聚果糖Fructo-oligosaccharides (FOS)，難消化性麥芽糊精Resistant maltodextrin(RMD)可能會影響此套組之實驗結果
➡️低分子量水溶性膳食纖維(LMWSDF)需透過高效液相層析(HPLC)分析後，方可定量
➡️高分子量膳食纖維(HMWDF)=非水溶性膳食纖維(IDF) + 高分子量水溶性膳食纖維(SDFP)
➡️樣品中的總膳食纖維(Total dietary fiber ,簡稱TDF)=高分子量膳食纖維(HMWDF)+低分子量水溶性膳食纖維(LMWSDF)
AOAC Method 2009.01➡️此方法可分別檢測➡️高分子量膳食纖維(HMWDF)、低分子量水溶性膳食纖維(SDFS)
AOAC Method 2011.25➡️此方法可分別檢測➡️高分子量水溶性膳食纖維(SDFP)、低分子量水溶性膳食纖維(SDFS)、非水溶性膳食纖維(IDF)
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樣品之酵素分解步驟 (ENZYME DIGESTION OF SAMPLES)
(AOAC Methods 2009.01 and 2011.25)
Blanks
With each assay, run two blanks along with samples to measure any contribution from reagents to residue.
Samples
(a) Weigh duplicate.— 1.000 ± 0.005 g samples accurately into 250 mL Fisherbrand® soda glass, wide mouth bottles [B(b)].
(b) Addition of Enzymes.— Wet the sample with 1.0 mL of ethanol and add 40 mL of pancreatic α-amylase/AMG mixture [C(e)] to each bottle. Cap the bottles. Transfer the bottles to a Grant OLS 200 shaking incubation bath (or similar) [B(g)] and secure the bottles in place with the springs in the shaker frame. Alternatively, use a 2mag Mixdrive 15® submersible magnetic stirrer [B(g)] with 7 x 30 mm stirrer bars (Figures 3 and 4, page 18).
(c) Incubation with pancreatic αα-amylase/AMG.— Incubate the reaction solutions at 37°C and 150 rpm in orbital motion in a shaking water bath [B(g)]; or at 170 rpm on a 2mag Mixdrive 15® submersible magnetic stirrer (to ensure complete suspension) for exactly 16 h (e.g. 5.00 pm to 9.00 am).
(d) Adjustment of pH to approx. 8.2 (pH 7.9-8.4), Inactivation of αα-amylase and AMG.— After 16 h, remove all sample bottles from the shaking water bath and immediately add 3.0 mL of 0.75 M Tris buffer solution [C(i)] to terminate the reaction. (At the same time, if only one shaker bath is available, increase the temperature of the shaking incubation bath to 60°C in readiness for the protease incubation step). Slightly loosen the caps of the sample bottles and immediately place the bottles in a water bath (non-shaking) at 95-100°C, and incubate for 20 min with occasional shaking (by hand). Using a thermometer, ensure that the final temperature of the bottle contents is > 90°C (checking of just one bottle is adequate).
(e) Cool.— Remove all sample bottles from the hot water bath (use appropriate gloves) and cool to approx. 60°C.
(f) Protease treatment.— Add 0.1 mL of protease solution [C(f)] with a positive displacement dispenser (solution is viscous). Incubate at 60°C for 30 min.
(g) pH adjustment.— Add 4.0 mL of 2 M acetic acid [C(j)] to each bottle and mix. This gives a final pH of approx. 4.3.
(h) Internal standard.— Add 1.0 mL of D-sorbitol internal standard solution (100 mg/mL) [C(o)] to each bottle and mix well.
(i) Proceed to step [G(a)] for determination of HMWDF/ SDFS (AOAC Method 2009.01) or to step [H(a)] for determination of IDF/SDFP/SDFS (AOAC Method 2011.25).
- DETERMINATION OF HMWDF (IDF plus SDFP)
(AOAC Method 2009.01)
(a) Precipitation SDFP.— Pre-heat the sample to 60°C and add 192 mL (measured at room temperature) of 95% (v/v) EtOH (or IMS) pre-heated to 60°C. Mix thoroughly and allow the precipitate to form at room temperature for 60 min.
(b) Filtration setup.— Tare crucible containing Celite® {from [B(c)], page 6} to the nearest 0.1 mg. Wet and redistribute the bed of Celite® in the crucible, using 15 mL of 78% (v/v) EtOH or IMS from wash bottle. Apply suction to crucible to draw Celite® onto fritted glass as an even mat (see Figure 11, page 22).
(c) Filtration.— Using vacuum, filter precipitated enzyme digest [G(a)] through crucible. Using a wash bottle with 78% (v/v) EtOH (or IMS) [C(b)], quantitatively transfer all remaining particles to crucible. Retain filtrate and washings and proceed to step [I(a)] on page 15 for determination of SDFS.
(d) Wash.— Using a vacuum, wash residue sequentially with two 15 mL portions of the following: 78% (v/v) EtOH (or IMS), 95% (v/v) EtOH (or IMS) and acetone.
(e) Dry crucibles containing residue overnight in 105°C oven. If a forced air oven is used, loosely cover the crucibles with aluminium foil to prevent loss of dried sample.
(f) Cool crucible in desiccator for approx. 1 h. Weigh crucible containing dietary fiber residue and Celite® to nearest 0.1 mg. To obtain residue mass, subtract tare weight, i.e. weight of dried crucible and Celite®.
(g) Protein and ash determination.— The residue from one crucible is analysed for protein and the second residue of the duplicate is analysed for ash. Perform protein analysis on residue using Kjeldahl or combustion methods (Caution should be exercised when using a combustion analyser for protein in the residue. Celite® volatilised from the sample can clog the transfer lines of the unit). Use 6.25 factor for all cases to calculate mg of protein. For ash analysis, incinerate the second residue for 5 h at 525°C. Cool in desiccator and weigh to nearest 0.1 mg. Subtract crucible and Celite® weight to determine ash.
(h) Determination of HMWDF.— Subtract ash and protein from average residue weight and proceed to step [ J ] for calculation of HMWDF.
- DETERMINATION OF IDF and SDFP SEPARATELY
(AOAC Method 2011.25)
IDF
(a) Filtration setup.— Tare crucible containing Celite® {from [B(c)], page 6} to nearest 0.1 mg. Wet and redistribute the bed of Celite® in the crucible, using 15 mL of 78% (v/v) EtOH (or IMS) [C(b)] from wash bottle. Apply suction to crucible to draw Celite® onto the fritted glass as an even mat (see Figure 11, page 22).
(b) Filtration.— Using vacuum, filter the enzyme digest from step [F(h)] through the crucible. Using a wash bottle with 60°C deionised water, rinse the incubation bottle with a minimum volume of water (approx. 10 mL) and use a rubber policeman (spatula) to dislodge all particles from the walls of the container. Transfer this suspension to the crucible. Wash the bottle with a further 10 mL of water at 60°C and again transfer to the crucible. Collect the combined filtrate and washings and adjust the volume to 70 mL and retain this for determination of SDFP [H(f)] and SDFS [I(a)].
(c) Wash.— Using a vacuum, wash the residue successively with two 15 mL portions of the following: 78% (v/v) EtOH (or IMS), 95% (v/v) EtOH (or IMS) and Acetone. Discard the washings.
(d) Dry crucibles containing residue overnight in 105°C oven.
(e) Cool crucibles in desiccators for approx. 1 h. Weigh crucible containing IDF residue and Celite® to nearest 0.1 mg. To obtain residue mass, subtract tare weight, i.e. weight of dried crucible and Celite®. Calculate IDF; step [ J ], as shown on page 17.
SDFP
(f) Precipitation of SDFP.— Pre-heat the filtrate of each sample (approx. 70 mL) to 60°C and add 280 mL (measured at room temperature) of 95% (v/v) EtOH (or IMS) [C(a)] pre-heated to 60°C and mix thoroughly. Allow the precipitate to form at room temperature for 60 min.
(g) Recovery of SDFP and SDFS.— Proceed according to step [G(b)] to [G(h)] on page 13.
(h) For determination of SDFS.— Proceed according to step [I(a)] to [I(f)] on pages 15-16.
- DETERMINATION OF SDFS
Note 1: Since the development and publication of the INTDF method, an improved procedure for measurement of TDF has been developed, namely, the Rapid Integrated TDF method (AOAC Method 2017.16; Megazyme cat no. K-RINTDF). We strongly recommend the use of this method to simplify analyses and obtain more physiologically relevant analyses.
Note 2: Proper deionisation is an essential part of obtaining quality chromatographic data on SDFS.— To obtain familiarity regarding the appearance of salt peaks in the SDFS chromatograms, dissolve 10 mg of sodium chloride in 9 mL of deionised water and add 1 mL of 100 mg/mL LC internal standard [C(o)] and proceed to step [I(c)] at “Transfer the solution to a 10 mL disposable……”. To ensure the resins being used are of adequate deionising capacity, dissolve 10 mg of sodium chloride in 1 mL of deionised water. Add 1 mL of 100 mg/mL LC internal standard [C(o)], and proceed to step [I(b)] at “Transfer 2 mL of this solution to the top of.....”. The LC chromatogram of this solution should show no peaks in the time range corresponding to carbohydrates of DP3 or greater.
(a) Filtrate recovery and concentration.— {Set aside the filtrate from one of the sample duplicates [G(c)] to use in case of spills or if duplicate SDFS data is desired}. Transfer one half of filtrate [G(c)] of the other sample duplicate to a 500 mL evaporator flask and evaporate to dryness under vacuum at 60°C.
(b) Deionisation of sample.— Add 5 mL of deionised water to the evaporator flask and swirl the flask for approx. 2 min to dissolve the sample. Transfer the solution to a sealable polypropylene 20 mL container. Transfer 2 mL of this solution to the top of the Bio-Rad disposable column [B(c)] containing 4 g each of freshly prepared and thoroughly mixed, Amberlite FPA 53 (OH-) [C(s)] and Ambersep 200 (H+) [C(s)] (see Figure 7, page 22). Elute the column at a rate of 1.0 mL/min into a 100 mL Duran® bottle. When the sample has entered the resin, add 2 mL of distilled water to the resin and allow this to percolate in. Then add approx. 20 mL of deionised water to the top of the column and continue to elute at a rate of 1.0 mL/min. Transfer the eluate to a 250 mL round bottom rotary evaporator flask and evaporate to dryness under vacuum at 60°C. Add 2 mL of deionised water to the flask and redissolve the sugars by swirling the flask for approx. 2 min. Using a Pasteur pipette, transfer the solution to a polypropylene storage container.
(c) Preparation of samples for LC analyses.— Transfer the solution to a 10 mL disposable syringe [B(bb)] and filter through a 0.45 µm filter [B(y)]. Use a 100 µL LC glass syringe [B(cc)] to fill the 50 µL injection loop on the LC [B(t)]. Perform this analysis in duplicate. Column: Waters Sugar-Pak® (6.5 x 300 mm). Solvent: distilled water containing Na2Ca-EDTA (50 mg/L). Flow rate: 0.5 mL/min. Temperature: 90°C.
(d) Determine the response factor for D-glucose.— Since D-glucose provides an LC refractive index (RI) response equivalent to the response factor for the non-digestible oligosaccharides that make up SDFS the LC is calibrated using D-glucose, and the response factor is used for determining the mass of SDFS. Use a 100 µL LC syringe to fill a 50 µL injection loop for each standard D-sorbitol/D-glucose solution. Inject in triplicate. Obtain the values for the peak areas of D-glucose and internal standard from the 3 chromatograms. The reciprocal of the slope obtained by comparing the ratio of peak area of D-glucose/peak area of D-sorbitol internal standard (y-axis) to the ratio of the mass of D-glucose/mass of D-sorbitol (x-axis) is the “response factor”.
Determine the average response factor (typically 0.97 for D-sorbitol). Response factor (Rf) = (PA-IS) / (PA-Glu) x (Wt-Glu / Wt-IS)
where:
PA-IS = peak area internal standard (D-sorbitol).
PA-Glu = peak area D-glucose.
Wt-Glu = mass of D-glucose in standard.
Wt-IS = mass of D-sorbitol in standard.
(e) Calibrate the area of chromatogram to be measured for LMWSDF.— Use a 100 µL LC syringe [B(cc)], to fill the 50 µL injection loop with retention time standard [C(p)]. Inject in duplicate. Determine demarcation point between DP 2 and DP 3 oligosaccharides (disaccharides maltose versus higher oligosaccharides) (see Figure 10, page 22).
(f) Determine peak area of SDFS (PA-SDFS) and internal standard (PA-IS) in chromatograms of sample extracts.— Inject sample extracts [I(c)] on LC. Record area of all peaks of DP greater than the DP2/DP3 demarcation point as PA-SDFS. Record the peak area of internal standard as PA-IS.
NOTE: An in-line desalting procedure has been published.17 This procedure is more convenient, but the disposable desalting cartridges are costly and relatively few samples can be desalted with one cartridge.
商品特色
商品規格
- 商品規格(100個檢測反應)
- 100個檢測反應套組(100 determinations assay kit)
Bottle 1:
Concentrated pancreatic α-amylase (E-PANAA); 4 g, 75,000 Ceralpha Units/g.
Stable for > 5 years when stored dry below -10°C.
Bottle 2:
Amyloglucosidase (E-AMGDF) (20 mL, 3,300 Units/mL).
Stable for > 3 years at 4°C.
Bottle 3:
Purified protease (E-BSPRT) (10 mL, 350 tyrosine units/mL).
Stable for > 3 years below -10°C.
Bottle 4:
LC Retention Time Standard [maltodextrins plus maltose (4:1 ratio), approx. 5 g].
Stable for > 3 years; store sealed at room temperature.
Bottle 5:
D-Sorbitol (approx. 12 g, dry).
Stable for > 3 years; store sealed at room temperature.
