- The Rapid Total Starch (RTS) Method. Determination of starch in cereal and food products not containing resistant starch (Recommended Procedure; all incubations at pH 5.0).
1. Mill cereal, plant or food product to pass a 0.5 mm screen.
2. Accurately weigh ~ 100 mg of test sample, in duplicate (one as a sample blank) into Corning culture tubes (16 x 120 mm) [C(q)]. Record the exact weight. Tap the tube so that sample drops to the bottom of the tube.
3. To both tubes add 10 mL of buffer [B(a)] [Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM)] using a bottle top dispenser [C(m)] to increase throughput. Stir the tubes vigorously on a vortex mixer for 5 sec.
4. To one of the tubes (sample tube) add 0.1 mL of bottle 1 (thermostable α-amylase) [A(1)] using a HandyStep® dispenser [C(l)] with 5 mL tip. To the second tube (sample blank) add 0.1 mL of buffer [B(a)] [Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM)].
5. Vortex the tubes for 3 sec, cap the tubes loosely and immediately transfer them to a boiling water bath and start the timer. After approx. 2 min, tighten the caps and mix the tube contents vigorously on a vortex mixer. After a further 5 and 10 min, vortex the tube contents again for 5 sec and return the tubes to the boiling water bath. After 15 min (from addition of α-amylase), remove tubes from the boiling water bath and mix the contents vigorously for 5 sec on a vortex mixer. Place the tubes in a water bath at 50°C and allow them to equilibrate to temperature over 5 min.
6. To one of the tubes (the sample tube), add 0.1 mL of bottle 2 (Amyloglucosidase) [A(2)] using a HandyStep® dispenser with 5 mL tip and vortex for 3 sec. To the second tube (the sample blank) add 0.1 mL of buffer [B(a)] [Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM)]. Incubate the tubes at 50°C for 30 min with no further mixing.
7. Remove the tubes from the water bath and allow them to cool to room temperature for 10 min. Invert the tubes a few times to ensure condensed water on the inside of the lid is mixed with liquid in the tube.
8. Transfer 2.0 mL of each solution (sample and sample blank) to microfuge tubes [C(o)] and centrifuge the tubes at 13,000 rpm for 5 min (Retain the remaining 8.2 mL of incubation solution and refer to the NOTE below). Using a Gilson Pipetman dispenser, accurately transfer a 1.0 mL aliquot of the supernatants to 12 x 120 mm tubes containing 4 mL of buffer [B(a)] [Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM)] and mix the contents.
9. Accurately transfer duplicate 0.1 mL aliquots of each sample to the bottoms of 16 x 120 mm glass test tubes. Also transfer a single 0.1 mL aliquot of sample blanks to a 16 x 120 mm glass test tube.
10. Add 3.0 mL of GOPOD reagent and incubate the solutions at 50°C for 20 min and measure absorbance against the reagent blank at 510 nm. Concurrently incubate: Glucose controls: 0.1 mL of bottle 5 (glucose standard solution, 1.0 mg/mL) plus 3.0 mL of GOPOD reagent, in quadruplicate. Reagent Blank: 0.1 mL of buffer [B(a)] [Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM)] with 3.0 mL of GOPOD reagent in duplicate.
11. Calculate starch content (see Section F, page 14).
NOTE: For this extraction protocol, the final Extract Volume (EV) = 10.2
NOTE 1: If the GOPOD absorbance values for samples are less than 0.100, analyse the centrifuged sample solution (step 8) without further dilution. If absorbance values are greater than 1.0, then add 1.0 mL of the centrifuged sample solution to 10.0 mL of buffer [B(a)] [Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM)], mix well and remove 0.1 mL aliquots for analysis using GOPOD reagent. For samples containing less than 1% (w/w) starch content, increase sample size to 500 mg and analyse 0.1 mL of undiluted sample solution. Dilution (D) = 1, 5 or 11
NOTE 2: When fibrous samples such as grasses and silage are being analysed, grind the sample using a Nutri Bullet PRO 900 blender (See K-TSTA video). Grind for approx. 60 sec (until the sample is homogeneous).
- Determination of total starch content of samples containing resistant starch (RTS-NaOH Procedure - Recommended).
1. Accurately weigh 100 mg of test sample, in duplicate, into Corning culture tubes (16 x 120 mm) [C(q)]. Record the exact weight. Tap the tubes so that sample falls to the bottom of the tubes.
2. Add 0.2 mL of 80% v/v aqueous ethanol and stir the tubes on a vortex mixer to completely wet and disperse the sample (this step is very important in aiding the complete dissolution of samples with a high starch content).
3. Add 2 mL of cold 1.7 M sodium hydroxide solution [B(d)] using a HandyStep® dispenser with 25 mL tip and stir the tubes contents on a vortex mixer for 15 sec. Place the tubes in a rack in an ice-water bath over a magnetic stirrer and stir for 15 min (Figure 2, page 17). During this time, intermittently stir the contents of the tube vigorously on a vortex mixer 2-3 times. Ensure that there are no lumps in the sample slurry.
4. Add 8 mL of buffer [B(c)] [Sodium acetate buffer (600 mM, pH 3.8) plus calcium chloride (5 mM)] using a bottle top dispenser [C(m)] to increase throughput. Stir the tubes on a vortex mixer. Ensure that that the pH is ~ 5.0.
5. Immediately add 0.1 mL of bottle 1 (thermostable α-amylase) [A(1)] using a HandyStep® dispenser with a 5 mL tip to one of the tubes (sample tube). Then add 0.1 mL of bottle 2 (Amyloglucosidase) [A(2)] using a Handy Step® dispenser with a 5 mL tip to the same tube. To the second tube (sample blank), add 0.2 mL of buffer [B(a)] [Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM)]. Cap both tubes and vortex the contents for 3 sec.
6. Incubate the tubes at 50°C for 30 min.
7. Remove the tubes from the water bath and allow them to cool to room temperature. Invert the tubes a few times to ensure condensed water on the inside of the lid is mixed with liquid in the tube.
8. Proceed to step 8 of method (a) on page 8.
NOTE: For this extraction protocol, the final extraction and dilution values are:
Extract volume (EV) = 10.4
Dilution (D) = 1, 5 or 11
- AOAC Official Method 996.11- Determination of starch in cereal and food products not containing resistant starch, D-glucose and/or maltodextrins.
1. Mill cereal, plant or food product to pass a 0.5 mm screen.
2. Add milled sample (~ 100 mg; weighed accurately) to a Corning culture tubes (16 x 120 mm) [C(q)]. Tap the tube to ensure that all of the sample drops to the bottom of the tube.
3. Add 0.2 mL of aqueous ethanol (80% v/v) [B(f)] to wet the sample and aid dispersion. Stir the tube on a vortex mixer.
4. Immediately add 3 mL of diluted thermostable α-amylase [A(1) Note] (see page 5 for preparation details). Incubate the tube in a boiling water bath for 6 min, vortexing vigorously after 2, 4 and 6 min (to ensure complete homogeneity).
5. Place the tube in a bath at 50°C; add 4 mL of buffer [B(b)] [Sodium acetate buffer (200 mM, pH 4.5) plus calcium chloride (5 mM)] followed by 0.1 mL of bottle 2 (Amyloglucosidase) [A(2)]. Stir the tube on a vortex mixer and incubate at 50°C for 30 min.
6. Transfer the entire contents of the tube to a 100 mL volumetric flask (with funnel to assist). Use a wash bottle to rinse the tube contents thoroughly. Adjust to volume with buffer [B(b)] [Sodium acetate buffer (200 mM, pH 4.5) plus calcium chloride (5 mM)] and mix the contents thoroughly.
7. Transfer 2.0 mL of each solution to a microfuge tube [C(o)] and centrifuge the tube at 13,000 rpm for 5 min.
8. Accurately transfer duplicate 0.1 mL aliquots of each sample to the bottoms of 16 x 120 mm glass test tubes.
9. Add 3.0 mL of GOPOD reagent and incubate the solutions at 50°C for 20 min and measure absorbance against the reagent blank at 510 nm. 10. Calculate starch content (see Section F, page 14).
NOTE: For this extraction protocol, the final extraction and dilution values are:
Extract volume (EV) = 100 (see calculations on page 14)
Dilution (D) = 1
- Determination of total starch content of samples containing resistant starch but no D-glucose and/or maltodextrins (DMSO Format - AOAC Official Method 996.11).
1. Mill cereal, plant or food product to pass a 0.5 mm screen.
2. Add milled sample (~ 100 mg, weighed accurately) to a Corning culture tubes (16 x 120 mm) [C(q)].
3. Wet with 0.2 mL of aqueous ethanol (80% v/v) [B(f)] to aid dispersion and stir the tube on a vortex mixer.
4. Immediately add 2 mL of dimethyl sulphoxide (DMSO) and stir the tube on a vortex mixer. Place the tube in a vigorously boiling water bath and remove after 5 min.
5. Add 3 mL of diluted thermostable α-amylase [A(1) Note] to each tube immediately as it is being removed from the boiling water bath and mix the contents vigorously for 20 sec on a vortex mixer. Incubate the tube in a boiling water bath for 6 min, vortexing vigorously after 2, 4 and 6 min (to ensure complete homogeneity).
6. Proceed from step 5 of method (c) on page 11.
NOTE: For this extraction protocol, the final extraction and dilution values are:
Extract volume (EV) = 100;
Dilution (D) = 1
- Suggested procedure for the determination of starch in samples which contain D-glucose and/or maltodextrins- removal of D-glucose and maltodextrins with alcohol washing.
[Note: solubility of maltodextrins in aqueous ethanol is critically dependent on the DP range of the maltodextrins, the final ethanol concentration and the temperature of the solution].
1. Mill cereal, plant or food product to pass a 0.5 mm screen.
2. Add milled sample (~ 100 mg, weighed accurately) to a glass centrifuge tube (16 x 100 mm; 14 mL capacity) [C(p)].
3. Add 5.0 mL of aqueous ethanol (80% v/v) [B(f)] and incubate the tube at 8085°C for 5 min. Mix the contents on a vortex stirrer and add another 5 mL of 80% v/v aqueous ethanol.
4. Centrifuge the tube for 10 min at 3,250 rcf (~ 4,000 rpm) on a bench centrifuge [C(b)]. Discard the supernatant.
5. Resuspend the pellet in 5 mL of 80% v/v aqueous ethanol [B(f)] and stir on a vortex mixer. Add another 5 mL of 80% v/v aqueous ethanol and mix by inversion. Centrifuge as in step 4 and carefully pour off the supernatant.
6. Proceed to step 4 of method (c) on page 11. Extract volume (EV) = 100; Dilution (D) = 1
Alternatively: If the sample contains resistant starch, proceed to step 4 of method (d) on page 11.
Extract volume (EV) = 100; Dilution (D) = 1
- Determination of starch in samples in which the starch is present in a soluble or suspended form
1. Mix the sample contents thoroughly by stirring on a vortex mixer or by inversion. If necessary, heat or homogenise the sample to obtain a homogeneous suspension.
2. Immediately transfer 5 mL aliquots in duplicate (sample and sample blank) into two Corning culture tubes (16 x 120 mm) [C(q)] using a positive displacement dispenser. To each tube add 5 mL of buffer [B(b)] [sodium acetate buffer (200 mM, pH 4.5) plus calcium chloride (5 mM)] using a bottle top dispenser [C(m)]. Stir the tubes vigorously on a vortex mixer for 5 sec.
3. Proceed according to method (a) from step 4, page 8 for sample and sample blank.
4. Calculate starch content (see section F, page 15 for liquid samples).
Diluted Sample Volume (DSV) = 10.2 mL
Dilution (D) 1, 5 or 11; Sample Volume (SV) = 5 mL
- Determination of the starch content of samples containing resistant starch in a suspended form.
1. Mix the sample contents thoroughly by stirring on a vortex mixer or by inversion. If necessary, heat or homogenise the sample to obtain a homogeneous suspension.
2. Immediately transfer 2 mL aliquots in duplicate into two Corning culture tubes (16 x 120 mm) [C(q)] using a positive displacement dispenser. Add 2 mL of cold 1.7 M sodium hydroxide solution [B(d)] using a HandyStep® dispenser with 25 mL tip and stir the tube contents on a vortex mixer for 15 sec. Place the tube in a rack in an ice-water bath over a magnetic stirrer and stir for 15 min (Figure 2, page 17). During this time, also intermittently stir the contents of the tube vigorously on a vortex mixer 2-3 times. Ensure that there are no lumps in the sample slurry.
3. Proceed according to method (b) from step 4, page 10 for sample and sample blank.
Diluted Sample Volume (DSV) = 12.2 mL
Dilution (D) x 1, 5 or 11; Sample Volume (SV) = 2 mL
- Determination of enzyme resistant starch.
Resistant starch is accurately measured using the Resistant Starch assay kit (K-RSTAR) or the Rapid Resistant Starch assay kit (K-RAPRS) supplied by Megazyme. Results obtained using either K-RSTAR or K-RAPRS closely simulate those obtained under in vivo conditions.14 The Resistant Starch assay procedure (K-RSTAR) has been successfully subjected to interlaboratory evaluation (37 labs, 16 samples) to become AOAC Official Method 2002.0215 and AACC Method 32-40.01.
商品特色
ANALYSIS OF SAMPLES - EXAMPLE METHODS:
商品規格
- 商品規格(100個檢測反應)
Bottle 1:
Thermostable α-amylase
(10 mL, 3,000 U/mL on Ceralpha reagent* at pH 6.5 and 40°C or 2,500 U/mL on Ceralpha reagent* at pH 5.0 and 40°C).
Store at 4°C or alternatively below -10°C. See individual label for expiry date.
Bottle 2:
Amyloglucosidase
(10 mL, 3,300 U/mL on soluble starch or 200 U/mL on p-nitrophenyl β-maltoside*) at pH 4.5 and 40°C.
Store at 4°C or alternatively below -10°C. See individual label for expiry date. *
Full assay procedure is available at “www.megazyme.com”.
Bottle 3:
GOPOD Reagent Buffer.
Buffer (50 mL, pH 7.4), p-hydroxybenzoic acid and sodium azide (0.09% w/v).
Store at 4°C. See individual label for expiry date.
Bottle 4:
GOPOD Reagent Enzymes.
Glucose oxidase plus peroxidase and 4-aminoantipyrine. Freeze-dried powder.
Store at or below -10°C. See individual label for expiry date.
Bottle 5:
D-Glucose standard solution (5 mL, 1.0 mg/mL) in 0.2% (w/v) benzoic acid.
Store sealed at room temperature. See individual label for expiry date.
Bottle 6:
Standardised regular maize starch control.
Starch content shown on vial label.
Store sealed at room temperature. See individual label for expiry date.
