Standard Operating Procedure
Total starch extraction procedure
Department: Agronomy
Created by: Gerald Sebuwufu
Laboratory: Crop Production & Physiology
Supervisor: Dr. Mark Westgate
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview: Lipid extraction of neutral lipids carried out using hexane.
Equipment:
1.
2.
3.
Glass test tubes (round bottomed; 16 x 120 mm or 18 x 150 mm).
Micro-pipettors, 100 μL (e.g. Gilson Pipetman® or Rainin EDP-2® motorized dispenser).
Positive displacement pipettor e.g. Eppendorf Multipette® - with 50 mL Combitip® (to dispense 3 mL aliquots
of bacterial α-amylase solution). - with 5.0 mL Combitip® (to dispense 0.1 mL aliquots of amyloglucosidase
solution).
4. Bench centrifuge (required speed 3,000 rpm; approx. 1,800 g).
5. Analytical balance.
6. Spectrophotometer set at 510 nm.
7. Vortex mixer (e.g. IKA® Yellowline Test Tube Shaker TTS2).
8. Thermostated water bath set at 50°C.
9. Boiling water bath with tube rack.
10. Stop clock
11. -20C freezer
Reagents
Total Starch Kit – http://secure.megazyme.com/Total_Starch_AA_AMG
Contains:
1. Bottle 1: Thermostable α-amylase (10 mL, 3,000 U/mL on Ceralpha reagent* at pH 6.5 and 40°C or 1600 U/mL
on Ceralpha reagent at pH 5.0 and 40°C). Stable for > 4 years at 4°C.
2. Bottle 2: Amyloglucosidase (10 mL, 3300 U/mL on soluble starch (or 200 U/mL on p-nitrophenyl β-maltoside*) at
pH 4.5 and 40°C. Stable for > 4 years at 4°C.
3. Bottle 3: GOPOD Reagent Buffer. Buffer (48 mL, pH 7.4), p-hydroxybenzoic acid and sodium azide (0.4 % w/v).
Stable for > 4 years at 4°C.
4. Bottle 4: GOPOD Reagent Enzymes. Glucose oxidase plus peroxidase and 4-aminoantipyrine.Freeze-dried
powder. Stable for > 5 years at -20°C.
5. Bottle 5: D-Glucose standard solution (5 mL, 1.0 mg/mL) in 0.2 % (w/v) benzoic acid. Stable for > 4 years at room
temperature.
6. Bottle 6: Standardized regular maize starch control. Starch content shown on vial label. Stable for > 4 years at
room temperature.
Other reagents needed
i. Sodium acetate buffer (100 mM, pH 5.0) plus calcium chloride (5 mM).
ii. Sodium acetate buffer (1.2 M, pH 3.8).
iii. Potassium hydroxide solution (2 M).
iv. Ethanol (80% v/v)
March/25/20131
Protocol:
Determination of total starch content of samples containing resistant starch, but no D-glucose and/or
maltodextrins (KOH Format - Recommended).
1. Mill bean seed (dried at 60oF for 48hrs) to pass a 0.5 mm screen.
2. Add milled sample (~100 mg, weighed accurately) to a glass tube (16 x 120 mm).
3. Wet with 0.2 mL of aqueous ethanol (80 % v/v) to aid dispersion, and stir the tube on a vortex mixer.
4. Add a magnetic stirrer bar (5 x 15 mm) and 2 mL of 2 M KOH to each tube and re-suspend the pellets (and
dissolve the RS) by stirring for approx. 20 min in an ice/water bath over a magnetic stirrer (Figure 1).
i.
ii.
iii.
NOTE:
Do not mix on a vortex mixer as this may cause the starch to emulsify.
Ensure that the tube contents are vigorously stirring as the KOH solution is added. This will avoid the
formation of a lump of starch material that will then be difficult to dissolve.
5.
Add 8 mL of 1.2 M sodium acetate buffer (pH 3.8) to each tube with stirring on the magnetic stirrer. Immediately
add 0.1 mL of thermo stable α-amylase (bottle 1) and 0.1 mL of AMG (bottle 2), mix well and place the tubes in a
water bath at 50°C.
6. Incubate the tubes for 30 min with intermittent mixing on a vortex mixer.
7. Quantitatively transfer the contents of the tube to a 100 mL volumetric flask (using a water wash bottle). Use an
external magnet to retain the stirrer bar in the tube while washing the solution from the tube with a water wash
bottle. Adjust to 100 mL with distilled water and mix well. Centrifuge an aliquot of the solution at 1,800 g for 10
min.
8. Transfer duplicate aliquots (0.1 mL) of the diluted solution to the bottom of glass test tubes (16 x 100 mm).
9. Add 3.0 mL of GOPOD Reagent to each tube (including the D-glucose controls and reagent blanks), and incubate
the tubes at 50°C for 20 min.
10. D-Glucose controls consist of 0.1 mL of D-glucose standard solution (1 mg/mL) and 3.0 mL of GOPOD Reagent.
Reagent Blank solutions consist of 0.1 mL of water and 3.0 mL of GOPOD Reagent.
11. Read the absorbance for each sample, and the D-glucose control at 510 nm against the reagent blank.
12. CALCULATIONS (Solid samples):
13. Starch, % = ΔA x F x FV/0.1 x 1/1000 x 100/W x 162/180
= ΔA x F/W x FV x 0.9
where:
ΔA = Absorbance (reaction) read against the reagent blank.
F = 100 (μg of D-glucose) (conversion from absorbance for 100 μg of glucose absorbance to µg)
FV = Final volume (i.e. equals 100 mL or 10 mL)
0.1 = volume of sample analyzed.
1/1000= Conversion from μg to mg.
100/W= Factor to express “starch” as a percentage of flour weight.
W = The weight in milligrams (“as is” basis) of the flour analyzed.
162/180= Adjustment from free D-glucose to anhydro D-glucose (as occurs in starch).
14. Starch % w/w (dry wt. basis):
= Starch % w/w (as is) x 100/100-moisture content (%w/w)
Personal Protective Equipment / Engineering Controls:
Eye protection (goggles & shield)
Skin protection (proper shoes, nitrile gloves, lab coat, etc.)
Ventilation system
Safety shower
Eye wash station
Hazard Controls & Storage Precautions:
1. α-amylase
Precautions for safe handling
Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
Provide appropriate exhaust ventilation at places where dust is formed.
Conditions for safe storage
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Keep container tightly closed in a dry and well-ventilated place.
2. Amyloglucosidase- from Aspergillus niger
Precautions for safe handling
User Exposure: Avoid contact with eyes, skin, and clothing.
Avoid inhalation. Avoid prolonged or repeated exposure.
Conditions for safe storage
Suitable: Keep tightly closed. Store away from heat.
Store at 2-8°C
3. p-hydroxybenzoic
Precautions for safe handling
Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
Provide appropriate exhaust ventilation at places where dust is formed. Normal measures for preventive fire
protection.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place.
4. Glucose oxidase/peroxidase reagent
Precautions for safe handling
Provide appropriate exhaust ventilation at places where dust is formed. Normal measures for preventive fire
protection.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place.
Recommended storage temperature: 2 - 8 °C
5. 4-aminoantipyrine
Precautions for safe handling
Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
Provide appropriate exhaust ventilation at places where dust is formed. Normal measures for preventive fire
protection.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place. hygroscopic Light sensitive.
6. D-Glucose standard solution
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place.
Recommended storage temperature: 2 - 8 °C
7. Ethanol
Precautions for safe handling
Avoid contact with skin and eyes. Avoid inhalation of vapour or mist.
Use explosion-proof equipment. Keep away from sources of ignition - No smoking. Take measures to prevent the
buildup of electrostatic charge.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place. Containers which are opened must be carefully
resealed and kept upright to prevent leakage. Hygroscopic.
8. Sodium acetate- Keep container tightly closed in a dry and well-ventilated place.
9. Potassium hydroxidePrecautions for safe handling
Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
Provide appropriate exhaust ventilation at places where dust is formed.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place.
Absorbs carbon dioxide (CO2) from air.
Air sensitive. Strongly hygroscopic
10. Sodium Azide
Precautions for safe handling
Avoid contact with skin and eyes. Avoid formation of dust and aerosols.
Provide appropriate exhaust ventilation at places where dust is formed.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place.
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Never allow product to get in contact with water during storage. Do not store near acids.
11.
Waste Disposal Procedures & Decontamination:
All excess solutions will be put in a safe bottle for disposal by EH&S. Discharge into the environment must be avoided.
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Health & Safety Info for Required Reagents:
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Chemical name
Sodium azide
Sodium acetate
Potassium hydroxide
3
Kit reagents
α-Amylase
Amyloglucosidase
p-hydroxybenzoic
Glucose
oxidase/peroxidase
reagent
4-aminoantipyrine
3
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3
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Target Organ
Heart, Central
nervous system,
Brain.
Skin, eyes
Tissue of the mucous
Membranes and
upper respiratory
tract.
Skin, Eyes, digestive
tract
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Incompatibilities
Nitro compounds, Organic
materials, Magnesium, Copper,
Water, reacts violently with:,
Metals, Light metals, Contact
with aluminum, tin and zinc
liberates hydrogen gas. Contact
with nitromethane and other
similar nitro compounds causes
formation of shock-sensitive
salts., vigorous reaction with:,
Alkali metals, Halogens,
Azides, Anhydrides
Skin, eyes
Oxidizing agents
3
Skin, eye, respiratory
Skin, eye, respiratory
Strong oxidizing agents
4
Skin, eye, respiratory
Strong oxidizing agents, Strong
acids, Acid chlorides, Acid
anhydrides
Strong bases, Strong oxidizing
agents, Strong reducing agents
2
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Alkali metals, Ammonia,
Oxidizing agents, Peroxides
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D-Glucose standard
solution
Ethanol
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Skin, eye, respiratory
 The above summary consists of guidelines for proper handling & disposal of chemicals used in this
procedure. You must read attached MSDSs for more specific information before using the procedure.
References
Megazyme International Ireland. 2011. Total starch assay procedure (Amyloglucosidase/ α- Amylase method)
http://secure.megazyme.com/files/BOOKLET/K-TSTA_1107_DATA.pdf
Sigma-Aldrich. 2012. Material Safety Data Sheets- http://www.sigmaaldrich.com
March/25/20135