FOOD3002 Practical: Food Polysaccharides

Polysaccharides are macro-constituents of many foods, where they make major contributions
to water-holding and texture properties. They also play important roles in digestibility and
nutritional value. This practical will examine some of the analytical methods used for plant-
derived food polysaccharides.

Please ensure you have read the Risk Assessment and Safety Data Sheets for this class,
which are available on Canvas.

Learning outcomes
At the end of this practical you should be able to describe:
• How to determine the amylose content of starch
• The pasting and viscosity properties of starch
• How the structure of β-glucan differs from other glucose polymers found in foods
• The role of lichenase and β-glucosidase in determining β-glucan content
• The importance of using standard methods in determining the quantity of individual
food constituents

Exercise 1: Determination of amylose in potato starch

Amylose content of starches
Amylose content determines the suitability of starch for numerous end-uses. Low-amylose
starches are best suited when high-solubility, soft gels and thickeners are required, whereas
high-amylose starches are associated with higher levels of resistant starch and slower
digestibility. The low solubility of amylose, and variability of amylose and amylopectin
structures, present difficulties for the accurate determination of amylose content.

One of the most common methods used to measure amylose content is based on the
differences in absorption maxima of iodine complexes formed by amylose and amylopectin.
After defatting, solubilised starch is incubated with a solution of I2 in KI. The amylose complex
with iodine absorbs light maximally at 620 nm, compared to about 570 nm for the amylopectin-
iodine complex. In this experiment, the protocol of Chrastil (1987) is applied to potato starch,
which can be analysed without the defatting step required for samples with higher fat content.

1. Weigh 20 mg of a potato starch sample (with unknown amylose content) into a 10-mL
glass tube.

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 2. Weigh 20 mg of three calibration standards into separate 10-mL glass tubes:
a. 0% amylose/100% amylopectin
b. 25% amylose/75% amylopectin
c. 50% amylose/50% amylopectin.
3. To the sample and standards, add 2 mL of 1 M NaOH, cap the tubes and heat in a
95°C water bath with occasional shaking until the starch is completely dissolved (20-
30 min). Cool and add 4 mL of deionized water with vortex mixing.
4. Take 0.1 mL of each solution into separate (uncapped) glass test-tubes (from your
cupboard) and add 5 mL of 0.5% trichloroacetic acid (TCA) with mixing.
5. Add 0.05 mL of I2-KI solution (0.13% I2 and 0.3% KI).
6. Mix and incubate at room temperature (not in a water bath) for 30 min.
7. Read the absorbance of the sample and standards at 620 nm against a water blank.
8. Calculate the percentage amylose in your potato starch sample for Q1 in the Quiz (also
see video on the practical page).

Exercise 2: Determination of β-glucan in cereals

b-Glucan content of cereals
You will have the choice of several cereal products for this exercise. Please select one of these
products for the β-glucan determination. The class aim is to have a representative number of
results for each of the cereal products at the end of the practical, and you will need the results
from one of your peers for Q2 in the Quiz.

Oat and barley have relatively high levels of (1→3), (1→4)-β-glucan, commonly referred to as
β-glucan. From a nutritional point of view, β-glucan is seen as a favourable food constituent
as it has been associated with reducing cholesterol absorption; indeed, diets rich in β-glucan
have been recommended for people wishing to lower their cholesterol levels. Relatively high
levels of β-glucan are not looked upon favourably by the brewing industry, however, because
they are associated with chill haze (cloudy beer) and reduced production efficiency in beer
making. Diets of pigs, poultry and companion animals should contain only limited amounts of
β-glucan to avoid nutritional and environmental problems.

Due to its importance in food processing and nutrition panel labelling, there is a need for quick
and accurate methods to determine the β-glucan content of cereal grains and their processed
products. The method used in this practical is the accepted, or standard, method for β-glucan
determination (AACC-I Method 32-23, AOAC Method 995.16, EBC Methods 3.11.1, 4.16.1,
8.11.1 and ICC Standard Method 166). In this method, samples are hydrated in sodium

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phosphate buffer and the enzyme lichenase is added to hydrolyse the β-glucan into glucose
oligosaccharides. The oligosaccharides are then hydrolysed into glucose monomers using β-
glucosidase. The glucose monomers are quantified using a glucose oxidase/peroxidase
(GOPOD) reagent specific for D-glucose.

The principal steps in the method are shown below:

Lichenase hydrolyses these

bonds to produce gluco-
4
G-4G oligosaccharides

3
G-4G-4G G-G + G-G

3
G-4G G G

Gluco-oligosaccharides

β-glucosidase hydrolyses
the oligosaccharides into
free glucose

G G G G G G
1. Grind the cereal samples (each around 2.0 g) separately into flour using a mortar and
pestle.
2. Weigh 100 mg ± 1 mg of both flours into separate 15-mL centrifuge tubes.
3. Add 0.2 mL of 50% ethanol to the tubes and swirl to mix.
4. Add 4.0 mL sodium phosphate buffer (20 mM, pH 6.5), vortex gently and then place in
a boiling water bath for 60 s.
5. Vortex the tube gently and return to the boiling water bath for 2 min. Remove the tube
and vortex again.
6. Place the tube in a 50°C water bath for 5 min to equilibrate.
7. Add 0.4 mL of lichenase and swirl to mix the contents of the tube. Seal the tube with
Parafilm and incubate at 50°C for 30 min. Mix the contents of the tube 2–3 times during
the incubation.
8. Add 5.0 mL of sodium acetate buffer (200 mM, pH 4.0), vortex and incubate at room
temperature for 5 min.
9. Remove the Parafilm and centrifuge the tubes at 3,000 rpm for 10 min (ensure tubes
are ‘balanced’ when placed in the centrifuge). Keep the supernatant (sample).

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 10. Transfer two 0.1-mL aliquots of each sample to two clean test tubes (i.e., four tubes in
total). Add 0.1 mL of β-glucosidase to one of the tubes and 0.1 mL sodium acetate (50
mM, pH 4) to the remaining tube to serve as the control. Incubate the tubes at 50°C for
10 min.
11. Prepare a glucose standard curve by adding 0, 0.05, 0.1, 0.15 and 0.2 mL glucose (1
mg/mL) to five tubes. Make to volume (0.2 mL) with 50 mM sodium acetate (pH 4).
12. Add 3.0 mL of GOPOD reagent to all the tubes (standards, step 11, and samples, step
10) and incubate at 50°C for 20 min.
13. Read the absorbance of the samples and standards at 510 nm against a water blank.
14. For Q2 of the Quiz, using information you obtain from the scientific literature, compare
the value you determined for the β-glucan content of your product to the β-glucan
content obtained by another student for a different cereal product. Are these values
within the expected ranges for these two products, respectively.

Exercise 3: RVA analysis of various starches

Pasting properties of starch
The Rapid Visco Analyser (RVA) is an instrument used in many research and food company
laboratories around the world to determine the viscoelastic properties of starch pastes and
gels and to predict the processing and cooking behaviour of foods and food ingredients.

A demonstration of an RVA analysis of starch will be performed during the class. For Q3 of the
Quiz, clearly label the following five properties on the viscosity trace that will be made available
on Canvas: peak, breakdown, setback and final viscosities, and the pasting temperature. For
Quiz Q4, briefly discuss the practical information for food manufacturers that may be obtained
from the determination of each of these RVA properties.

Assessment (6%)
Assessment for Food Polysaccharides consists of the following:
• Answers to the Quiz written individually (12 Questions). Do the Quiz on Canvas
(6%). Some of the questions are on the practical class. Other questions are on
polysaccharides in foods more generally and include some calculation questions.

Deadline for submission of the Food Polysaccharides Quiz:

Odd Group: 16 March 2023
Even Group: 23 March 2023

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