Lab 03: Determination of the content of reducing sugar using Benedicts test

2007 Al-Bio

Lab 3: What has happened to the banana?

Problem:
Why yellow bananas are sweeter than green bananas?
Hypothesis:
Yellow bananas are sweeter than green bananas because some starch is converted into reducing sugar during
ripening.
Aim:
To prove the hypothesis by comparing the amount of reducing sugar and starch in bananas of different degree of
ripeness.
Principle:
Extraction of chemicals from bananas
To grind up the tissue, so as to destroy the cell membrane and release chemicals inside for quantitative
analyses of reducing sugar with Benedicts solution and starch with iodine solution.
Iodine test
Since a blue-black colouration is formed when adding iodine solution into starch, the intensity of blue-black
colour can be used to determine the quantity of starch in the sample bananas. The higher the colour intensity
indicates a higher concentration of starch in the sample.
Benedicts test
Boiling the mixture of reducing sugars and Benedicts solution gives brick red precipitate. In quantitative test of
reducing sugar, if Benedicts solution is in excess, the amount of ppt. formed is proportional to the amount of
reducing sugar in the sample.
Pre-experimental trials will be carried out to determine the volume ratio of banana extracts to Benedicts
solutions. A greenish or blue colour of the resultant solution after heat will be an acceptable ratio for the
experiment. This ensure complete reaction of reducing sugar with excess Benedicts solution.
Variables:
The independent variable is the the degree of ripeness of bananas ( i.e. colour of bananas)
In Benedicts test, the dependent variable is the amount of reducing sugar in bananas (according to the hypothesis,
the amount of reducing sugar depends on the degree of ripeness of bananas). This can be measured as the amount of brickred precipitate formed in the test.
In iodine test, the dependent variable is the amount of starch in bananas. This can be measured as the
intensity of blue-black colour resulted in the test.
Also there are some control variables should be kept as constant throughout the experiment. They are the mass
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Lab 03: Determination of the content of reducing sugar using Benedicts test

2007 Al-Bio

of food samples, volume of water for extraction, the volume ratio of extracts to Benedicts solution,
temperature of water bath, heating duration, the volume ratio of extracts to iodine solution.
Expected result
A trend of larger amount of brick-red precipitate and lower intensity of blue-black colour is found in banana
with a higher degree of ripeness. (i.e. an ascending amount of reducing sugar and a descending amount of starch
will be found from green banana to greenish yellow banana and than yellow banana.)
Assumptions:
1.
the only reducing agent for precipitate formation in the experiment is reducing sugar in the solutions
2. all sweet taste of the yellow banana comes from reducing sugar (e.g. not from sucrose)
3. No genetic variation among the banana samples will cause different in the testing biochemical change.
4. all chemical changes stopped during extraction and the whole experiment
5. the reaction of reducing sugar and starch are completed during tests.
List of apparatus ---Procedures
1. 25g of each food samples were weighted with electronic balance.
2.

3.

samples were minced with knife and then be ground for 5min. with 25 cm3 of 60oC water in mortar
separately.
(to prevent contamination, all apparatus were cleaned thoroughly with water before dealing with different
food samples. Hot water was used to dissolove the starch for easy extraction)
Boiling tubes were labeled as follow:
Boliling tube label

Content

filtered green
filtered greenish yellow
raw banana extract half-ripe banana extract

filtered yellow
ripe banana extract

4.

extracts were filtered through 2 layers of muslin cloth into the above three labeled boiling tubes 1, 2 and 3
respectively

5.

After pre-experimental trials, the ratio of banana extracts to Benedicts solutions was decided. By use of
measuring cylinder and pipettes, the filtered extracts, water, Benedicts solution and iodine solution were
transferred into the following labeled test tubes respectively. (Pipette should be erect and be read at the
same level as eyes to prevent reading error)
Test tubes for Benedicts test and controls of Benedicts test
Test tube label

B1

B2

sample

green raw
banana

greenish yellow yellow ripe Nil

half-ripe banana banana
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B3

B4

Lab 03: Determination of the content of reducing sugar using Benedicts test

2007 Al-Bio

volume of Benedicts 10
solution (cm3)

10

10

10

volume of sample
extract (cm3)

volume of water
(cm3)

Total volume of
content (cm3)

15

15

15

15

* Test tube B4 was the control to show no contamination in water and reagent and the
formation of precipitate is due to the reaction between chemicals in extracts and the
Benedicts solution.
Test tubes for Iodine test and controls of Iodine test
Test tube label

I1

I2

sample

green raw
banana

greenish yellow yellow ripe Nil

half-ripe banana banana

volume of Iodine
solution (cm3)

volume of sample
extract (cm3)

volume of water
(cm3)

Total volume of
content (cm3)

I3

I4

Test tubes I4 was the control to show no contamination in water and iodine
solution and the colour change of iodine solution is due to the presence of starch
in extracts.

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Lab 03: Determination of the content of reducing sugar using Benedicts test

2007 Al-Bio

6.

Record the initial observations of Benedicts test and the colour changes of iodine test.

7.

the test tubes for Benedicts test (B1-B3) and control of Benedicts test (B4) were boiled in water bath for
5 mins. (water bath for even heating; 5mins. was required as sufficient time for completely reaction of
reducing sugar or Benedicts solution; safety glass was used to protect the eyes form Bunsens flame and
the spitting out of mixture from test tube during heating )

8.

the test tubes were removed from water bath and were allowed to stand for 20 mins in test tube rack. (to
allow sedimentation of all precipitates)

9.

The amount of brick-red precipitate was measured as the height of the precipitate deposited at the
bottom of the test tubes with a ruler.

Results:
Table 1: Observations and amount of precipitate of sample extracts before and
after heating in Benedicts test
Test tube label

B1

B2

B3

B4

content

green raw
banana
and
Benedicts
solution

greenish
yellow halfripe banana
and
Benedicts
solution

yellow
ripe
banana
and
Benedicts
solution

water and
Benedicts
solution

Initial
observation
(before heating)

blue
blue solution
solution
with no red
with no
precipitate
red
precipitate

blue
solution
with no
red
precipitate

blue
solution
with no
red
precipitate

Observation
after heating

greenish
greenish
solution
solution with
with red
red precipitate
precipitate

greenish
solution
with red
precipitate

blue
solution
with no
red
precipitate

0.2

1.0

0.0

Height of
precipitate at
bottom of test
tube after 20
mins. (cm)

0.4

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Lab 03: Determination of the content of reducing sugar using Benedicts test

2007 Al-Bio

Table 2: The Final observation of sample extracts in Iodine test

Test tube label

I1

sample

Intensity of dark
blue color

I2

I3

I4

green raw greenish

banana
yellow halfripe banana

yellow
ripe
banana

Nil

high

low

medium

* Iodine solution is originally brown in colour.

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no dark
blue
colour
obtained

Lab 03: Determination of the content of reducing sugar using Benedicts test

2007 Al-Bio

Discussion
Result interpretation
From result table 1, the greenish colour of the mixtures in tubes B1, B2 and B3 shows that Benedicts solution
was in excess and complete reaction of reducing sugar in these tubes occurred.
According to the principle, the amount of reducing sugar proportional to the height of precipitate formed. When
comparing the height of precipitate in tubes B1, B2 and B3, it is found that yellow ripe banana (B3) contains the
greatest amount of reducing sugar (Height of precipitate = 1.0 cm), greenish yellow half-ripe banana (B2)
contains lesser amount of reducing sugar (Height of precipitate = 0.4 cm) and the green raw banana contains the
least amount of reducing sugar (Height of precipitate = 0.2 cm).
That means the amount of reducing sugar increase with the degree of ripeness of banana.
From result table 2, the intensity of dark blue colour decreases from tube I1 (high intensity) to tube I2 (medium
intensity) and then tube I3 (low intensity). As the intensity increases with the amount of starch in samples, thus
the amount of starch is highest in green raw banana (I1), lesser in greenish yellow half-ripe banana (I2) and the
least in yellow ripe banana
That means the amount of starch decrease with the increase in the degree of ripeness of banana.
The decrease in starch content and increase of reducing sugar as banana get ripening support the hypothesis
of the experiment. Yellow bananas are sweeter than green bananas because some starch is converted into
reducing sugar during ripening
Others
- other significant discussion (e.g. abnormalities, limitation, sources of error, wrong assumption, any new assumption, improvement
suggestion, suggestion of following experiment for further investigationetc.)

Conclusion
The hypothesis is supported by the experiment. The conversion of starch into reducing sugar during ripening
cause a sweeter taste in yellow banana than green banana.

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