“Which has a higher sugar content when comparing bottled juices that claim ‘no sugar added’ to

freshly extracted fruit juice for orange, apple and grapefruit juice?”

Biology IA

Details
Candidate name: Aashna Kotwani
School: King George V School
Tutor Group: R03S
Research Question: “Which has a higher sugar content when comparing bottled juices that
claim ‘no sugar added’ to freshly extracted fruit juice for orange, apple and grapefruit juice?”

Introduction:

The comparison of sugar content in extracted fresh natural juice from a fresh fruit to
commercially bottled fruit juice with “no sugar added” label will be explored in depth with a
scientific link to the amount of reducing sugars present in each by using the benedicts test. The
purpose of this experiment is to test and determine whether the sugar content of bottled juice that
claim “100% fruit juice” with “no sugar added” is equivalent to fresh extracted juices from fresh
organic fruits. The fruit and fruit juices that were tested were orange, apple and lemon. Both the
bottled juice and fresh juices were tested by the benedicts reagent. and measured the absorbance
of the red precipitate solution using a colorimeter, and mass of precipitate after filtering the
mixture was measured using an electronic balance. I decided to experiment on reducing sugars as
most juices contain reducing sugars as the main component. I am personally interested in this
investigation because my family drinks juices on a daily basis, and my grandma has diabetes.
Therefore, in order to take my family’s precaution for the future and be sure to live a healthy life,
I would like to avoid foods with high amounts of sugar.

Research Question:

The research question “Which has a higher sugar content when comparing bottled juices that
claim ‘no sugar added’ to freshly extracted fruit juice for orange, apple and grapefruit juice?”
involves investigating the absorbance of remaining solution after obtaining precipitate mass
using a colorimeter to compare the sugar content of reducing sugars between freshly extracted
fruit juice and manufactured juice. The absorbance shows the amount of light that has been
absorbed by the filtered solution after the Benedict’s test with the juice. The darker the color, the
higher the absorbance, meaning the higher the amount of reducing sugars present, hence
containing high glucose. The mass of precipitate gives you the amount of sugar present, while
the absorbance supports the data by indicating the level of sugar present depicted by the intensity
of the color. The inspiration for this investigation comes from a daily habit in my family where
we drink fruit juices. Many people have fruit juices every day. They are healthy, however they
may have a high amounts of sugar. Although sugar in fruits such as fructose are natural, if
consumed in high quantities, it is not healthy. Too much sugar could have a negative effect on
health and lead to diabetes, obesity, heart disease etc. Companies have been advertising fruit
juices with “no sugar added” to raise concern of the recommended amounts of sugar that should
be consumed. I did this experiment because it is important to know whether the bottled juices
approximately contain the same amount of sugar content in freshly extracted juice, which would
help people to reconsider their choices on which commercially bottled juice they should
consume.

Background Information:

Orange belongs to the genus Citrus of species of Citrus x Sinensis in the family called Rutaceae.
Apple belongs to the genus Malus of species of Malus pumil in the family of Rosaceae.
Grapefruit belongs to the genus Citrus of species of Citrus x paradise in the family of Rutaceae.
The bottled juices used were the brand Florida’s Natural for apple, orange and grapefruit juice
that claimed to be 100% squeezed with ‘no sugar added’. These were ‘from concentrate’ juices
because if you are comparing it to freshly extracted juice, it is purely the juice, without diluting it
and adding water.

All monosaccharides and disaccharides, except sucrose, are reducing sugars. Reducing sugars are
substances that reduce copper sulfate, which produces a brick red precipitate of copper oxide
when heated in a hot water bath. The benedicts reagent is a blue colored aqueous solution of
copper sulfate, sodium carbonate and sodium citrate. Benedict’s solution is used to identify the
presence of reducing sugars. In alkaline solutions at high temperatures, reducing sugars reduce
blue copper ions to brick red copper oxide precipitate.

Variables

Table 1: Table of independent variables and dependent variables

Category Variable Method of measuring

Independent Flavor of Different flavor of fruit juices is used in two different forms in the
variable fruit juice, way it is stored – bottled juice and freshly extracted juice. The
and type of flavor of fruit juices used are orange juice, apple juice, and
juice. grapefruit juice. For each juice, the sugar content in bottled juice
and fresh extracted fruit juice will be determined, and to see
whether the amount of reducing sugars present in fresh extracted
fruit juice is the same as the amount in bottled fruit juice that
claimed “no sugar added”.
Dependent Absorbance The first dependent variable is the mass of the precipitate which
variable of the will be measured using the electronic balance after collecting the
remaining precipitate on the filter paper. The second dependent variable is
solution after the absorbance which will be measured using a colorimeter. This
collecting the serves as an indicator of the extent of sugar content in the fruit
precipitate, juices, as it shows the amount of light absorbed by the solution.
and the mass This data can then be deduced to compare results with bottled fruit
of precipitate. juices and freshly extracted fruit juice. The colorimeter will be set
to absorb the blue light using a filter of 635ppm, which I have
chosen because it is the wavelength for detecting red light, which
is the color closest to the wavelength of brown light. Before
putting the cuvettes in, the colorimeter is calibrated with distilled
water to get the accurate results as possible.
Table 2: Table of controlled and uncontrolled variables
Control variab le Possible impact
(1=lowest, 5=highest)
Volume of fruit 5: The volume of fruit juice may affect
juice (2cm3) the time taken and color for precipitate
to form when Benedict’s solution is
added. This impact on the absorbance
would allow anomalies to be
introduced, having a negative effect on
the experiment.
Time each test 4: If boiling tubes stayed in water for
tube stayed in different times, it may affect the color
water baths (five and amount of precipitate formed.
minutes)
Volume of 4: The volume of Benedict’s solution
Benedict solution added to the fruit juice may affect the
added amount of Copper Sulfate ions
reduced, leading to anomalies in the
result.
How it is controlled/minimized
For each test tube, 10cm3 of
distilled water is measured using
a measuring cylinder of ±
0.1cm3.
A clock or stopwatch is used to
measure the time each boiling
tube is in the water baths – should
be for five minutes – until
precipitate forms
The volume of Benedict’s
solution is limited to 4cm3,
measured with a 10cm3
measuring cylinder with an
uncertainty of ±0.10cm3.

Hypothesis
H0: The sugar content of bottled 100% fruit juice is equivalent to the sugar amount in freshly
extracted fresh juice. Therefore, implying there is no difference.
H1: The sugar content of bottled fruit juice that claim “no sugar added” is greater in amount that
freshly extracted fresh juice. The more reducing sugar there is present, the more precipitate will
be formed, and a darker color of the precipitate will be shown, concluding that it is less desirable
for consumption.

Method
Table 3: Table of Required Apparatus
Apparatus Units and uncertainty Required number

Boiling tube - 30
Boiling tube rack - 4
100 cm3 Beaker cm3 ± 12.50 1
Bunsen burner - 1
Heat proof mat - 1
Gauze - 1
Balance g ± 0.01 1
Pipette - 1
 Knife
Bottled orange juice (100% - 1 bottle
with “no sugar added”)
(Florida’s Natural)
Bottled apple juice (100% - 1 bottle
with “no sugar added”)
(Florida’s Natural)
Bottled juice (100% with “no - 1 bottle
sugar added”) (Florida’s
Natural)
3
10 cm measuring cylinder cm3 ± 0.1 1
Orange (Citrus × sinensis) - 1
Grapefruit (Citrus × paradise) - 1

Apple (Malus) - 1
Mortar and pestle - 1
Glass rod - 5
Colorimeter Arbitrary units 1
Cuvettes - 30
Filter funnel - 1
Filter paper - 30
Grater - 1

1) Obtain an orange. Cut its tips with a sharp knife, then cut lengthwise to split it in half.
2) Squeeze the orange into 100cm3 beaker, then extract 2cm3 into a boiling tube. (For apple,
using a mortar and pestle to crush more, then transfer 2cm3 into a boiling tube)
3) Using a measuring cylinder, measure out 2cm3 of bottled orange juice into a boiling tube.
4) Add 4cm3 of benedict’s reagent in each of the boiling tubes with the juices, and note
down the initial colour.
5) Place these two boiling tubes in a boiling tube rack.
6) Prepare a water bath by heating half a beaker of water on a Bunsen burner at low flame.
7) Make sure to label the boiling tubes.
8) Place both the boiling tubes in a beaker of water bath for five minutes (until the
precipitate turns red)
9) Carefully remove the tubes from the boiling water and place it in a rack.
10) Using a clean dry glass rod, stir the contents of each tube
11) Observe and note colour changes
12) If reducing sugars are present, the solution will turn green, yellow, orange or brick red
(precipitate) depending on sugar concentration. (refer to colour palette later)
13) Repeat steps 1-9 for apple juice and grapefruit juice.
 14) Filter the mixture using filter paper and filter funnel to obtain the precipitate. Weigh the
precipitate using the balance for each freshly extracted juice and bottled juice for each
flavour.
15) After filtering, use the remaining solution in the test tube to fill ¾ of the cuvettes.
16) Put water in the cuvette first in the colorimeter to calibrate it. Then put the cuvettes with
the solution in colorimeter and note down the absorbance value measured in arbitrary
units.
17) Repeat whole process five times.

Table for recording results:

Grapefruit juice
Trial Mass of precipitate (g) Absorbance
Bottled juice Fresh juice Bottled juice Fresh juice

Table 5: Table of safety, ethical and environmental considerations.

Safety/ethical/ Possible impact
environmental
considerations
Orange, Apple Apples, oranges and 2
and Grapefruit grapefruit is wasted as a
is used. possible food source.
A knife is used Sharp edge of knife could 5
for cutting. result in cuts to the skin and
fingers.
Glass ware Potential danger that glass 3
may shatter as there is a lot
of washing of the glass
equipment. It can cause cuts
in the skin with its sharp
broken edges.
Impact Method of managing/minimization
(1 =
lowest,
5 =
highest)
Compost the waste of the
remaining fruits. Or cut half of the
fruit and extract the juice from one
half, keep the other half for
yourself to eat later.
To avoid injuries, tightly hold the
knife with a firm grip and keep
your fingers away while cutting.
Goggles. Hold the glass ware
tightly and having a strong grip to
avoid dropping. This prevents any
damage to glassware leading in
injuries. In addition, new glassware
will be needed to replace the
broken ones.

Data Analysis

Calculating the mean: = The sum of the absorbance in arbitrary units, and the
Formula: mass of precipitate.
N = The number of repeats (observations) conducted for
each fruit juice
6
= The mean of the absorbance in arbitrary units and the
mass of precipitate measured in grams.
Example:
1.445 + 1.565 + 1.398 / 3 = 1.469
The mean is the arithmetic average of a group of data. In this experiment, it allows for valid
comparison of absorbance in arbitrary units and mass of precipitate for bottled juice and freshly
extracted juice. The mean helps increase the accuracy of the results. It also helps to identify
anomalies. However, when calculating the mean, anomalies are not included.

Calculating the sample standard deviation:

Formula:
s = sample standard deviation
= the mean
x = the individual absorbance of remaining solution after
precipitate is filtered.
n = The number of repeats (observations) conducted for a
time.
Worked example:

(6 - 7.000)2 + (7 – 7.000)2 + (8 – 7.000)2

=1
3-1
The standard deviation measures how distributed the values of the absorbance of remaining
solution are. The smaller the standard deviation, the more precise the measured data is, and the
more reliable the experimental method is. Standard deviation will be used as the source of error
bars on the graph.

Raw and processed data:

Table 6: Table of mass of precipitate formed and absorbance of each fruit flavor.
Orange juice Apple juice
Mass of Mass of
Trial precipitate (g) Absorbance Trial precipitate (g) Absorbance
Bottled Fresh Bottled Fresh Bottled Fresh Bottled Fresh
juice juice juice juice juice juice juice juice
1 2.26 2.63 0.239 1.046
1 1.84 2.54 0.272 0.077
2 2.58 2.33 0.211 0.768
3 2.55 2.22 0.546 0.14 2 1.98 2.41 0.138 0.07
4 3.25 2.31 0.517 0.151 3 1.88 2.35 0.223 0.145
5 2.91 2.31 1.114 0.117 4 2.09 2.56 0.201 0.129
5 2.23 2.27 0.189 0.057
 Grapefruit juice
Trial Mass of precipitate (g) Absorbance
Bottled juice Fresh juice Bottled juice Fresh juice
1 3.38 2.34 0.598 0.225
2 2.49 2.17 0.69 0.138
3 3.59 2.26 0.109 0.2
4 2.93 2.21 0.105 0.208
5 2.59 2.91 0.112 0.221

Juice Mean Mass Mean Standard dev of Standard error

in (g) absorbance absorbance (3 s.f.) (3 s.f.)
(Au)
Orange Bottled 2.71 0.565 0.103 0.0460
juice
Fresh 2.36 0.165 0.059 0.0263
juice
Apple Bottled 2.00 0.205 0.0489 0.0219
juice
Fresh 2.43 0.0956 0.0389 0.0174
juice
Grapefruit Bottled 3.00 0.323 0.295 0.132
juice
Fresh 2.38 0.198 0.0352 0.0157
juice

This procedure was repeated five times to ensure reliability. This set of experiment was then
repeated for the two other fruit juices. The results of this test revealed that all fruit juices contain
reducing sugars. The intensity of the color determined which one had most amount of reducing
sugar. The results showed that apple contained a larger amount of reducing sugars than
grapefruit juice and orange juice, as it formed a brick red precipitate, while orange and grapefruit
formed orange color precipitate. After collecting the precipitate, I used the remaining solution to
determine the absorbance using a colorimeter. The results revealed that the solution with the
darkest color, showed the highest absorbance value, concluding that that had the largest amount
of reducing sugar present.

Qualitative Data

Image 1: Color palette (shades of brown) for reference (“Template: Shades of Brown,” 2017)
 As the reaction progresses, the color of the mixture changes gradually from blue to green,
yellow, orange and then red. The color depends on the amount of precipitate formed and the
amount of reducing sugars present. A green solution formed depicts traces of reducing sugars are
present. A color palette is used to make qualitative results more clear. Image 2 shows the
precipitate formed for the benedicts test with freshly extracted orange juice, resulted in a
‘fulvous’ color. The test with bottled orange juice resulted in a ‘ochre’ color, suggesting bottled
orange juice has slightly higher amounts of reducing sugar as ochre is darker than fulvous.

Qualitative Data: Boiling tubes Cuvettes

Cuvette rack
3
250cm
beaker

Food waste - oranges

Gauze

Benedict’s
solution

Lighter Image 3: The cuvettes containing the

remaining solution after obtaining the
Tripod precipitate of orange juice.
Bunsen burner Heatproof mat

Image 2: The boiling tubes are put in a

hot water bath for red precipitate to
form.
Graph 1: Graph 2:
Graph showing the average mass of precipitate (g) Graph showing the average absorbance value
after benedict’s test for all measured in Arbitrary units (Abu) after benedict’s
juices: test for juices:
Conclusion and Evaluation

Trends and patterns:

From the graph above, it can be seen that for grapefruit and orange juice, the sugar amount in the
bottled juice is higher than freshly extracted juice. For apple juice, it can be seen that the sugar
amount in the freshly extracted juice is higher than the amount in bottled juice. This is because
apple naturally contains high amounts of sugar. However, we are unsure of what type of sugar is
in high amount ie: glucose, sucrose or fructose. Therefore, we can conclude that it may be
healthier to drink Orange and Grapefruit bottled juice, but there is no real significance in the
difference of sugar content in both as the results are similar. The alternative hypothesis should be
accurate, as there is no significant difference in the sugar content of bottled juice and freshly
extracted juice.

Addressing the hypothesis:

The graph of recorded data rejects the null hypothesis and supports the alternative hypothesis,
due to it indicating there is equal amounts of sugar in bottled juice and freshly extracted juice.
There is no significant difference between the two. The line of best fit conforms to the graph.
Although for Apple juice, there were high amounts of sugar in the freshly extracted juice.

Scientific Explanation:

Fructose is a sweet sugar and a main component of flower and nectar, it is the sugar found in
fruits. Fructose is a fruit sugar, and is a bonded to glucose to form the disaccharide sucrose. It is
the sweetest of all naturally occurring carbohydrates. It has the formula of C6H12O6. Glucose is
the main energy source for most living things. It is one of the carbohydrates produced in
photosynthesis and forms building blocks of many carbohydrates. It is a simple sugar with the
formula C6H12O6. It is made of six carbon atoms, twelve hydrogen atoms, and six oxygen
atoms. Sucrose is table sugar. It is a disaccharide made by two monosaccharides – glucose and
fructose. It is produced naturally in plants. It has the formula C12H22O11. Excessive amounts of
fructose consumption can lead to negative health effects such as obesity, diabetes and bowel
disturbances.

Literature review:

Graph 2: Graph showing a similar investigation of varying temperature on the enzyme activity.
The results of a similar investigation are shown
on Graph 2. The graph shows the sugar
concentration of different fruit juices in freshly
extracted natural and bottled by doing the Brix
test using a refractometer. Comparing Graph 1
and Graph 2, both graphs suggest that for
orange and grapefruit juice, the sugar contents
in bottled juices are higher than naturally
extracted juice, and that for apple juice the
sugar contents in naturally extracted juice are higher than bottled juice.
Evaluation of results:

In order to measure the mass, the precipitate is filtered out and is put on an electronic balance. In
addition, to make my data results more accurate, after filtering out the precipitate, the
concentration of the remaining solution can be measured using a colorimeter, which determines
its absorbance. This will tell me how much Benedict’s solution has been used up allowing me to
estimate the concentration of the reducing sugar in the original sample. A colorimeter can be
used to make more accurate measurements. A reading is taken of percentage light transmission
or absorbance. In my case, I measured the absorbance. The solution is in a colorimeter where the
absorbance is measured.

The more copper sulfate that has been used in the Benedict’s test, the less light will be blocked
out in the sample, and less absorbed. Therefore, the reading gives a measure of the amount
reducing sugar based on the Benedict’s reaction. To measure the concentration of reducing sugar
in the solutions, I used a colorimeter. This moves this practical from qualitative to quantitative.

However, multiple factors could’ve affected these results. They could have been affected by the
country of origin of the fruit, the surrounding climate, brand of fresh fruits, which could’ve lead
to different absorbance and precipitate values.

Conclusion:

We can see from the table that the amount of reducing sugar present in the bottled juice of
orange juice is 2.71g on average, and 2.36g of freshly extracted orange juice. The difference is
0.35g. Similarly, for apple juice, the difference between bottled and fresh juice is 0.43g. For
grapefruit juice, the difference between bottled and fresh juice is 1.38. We can conclude that
there is not a significant in the sugar contents of freshly extracted fruit juiced and 100% pure
bottled fruit juice in all three flavors. Therefore, it is most reasonable for a consumer to be better
off with respect to a balanced diet if she consumes fresh squeezed fruit juice.

Even though fructose is naturally found in fruit, it is not necessarily healthier than sucrose and
glucose. It can be equally harmful. Consuming large amounts of fructose can lead to negative
health effects. It is a primary cause of symptoms in certain patients with diabetes, obesity etc. It
is important to know whether the bottled juices claiming “no sugar added” truly represent the
sugar content of freshly extracted juice. If the sugar content in bottled fruit juice with the “no
additional sugar” label is higher than that of freshly-extracted fruit juice, people may want to
reconsider the amount of commercially-bottled fruit juice they consume.

Improvements:

Table 8: Table of weakness and possible improvements

Weakness Potential impact Improvement

The method of After each trial, the same water bath For each trial, refill the water
measuring the was used, this meant that the water bath with normal distilled
temperature of the would still have been warm, therefore water and then heat the water
water bath has a large when the boiling tube was kept in the bath.
uncertainty. water bath in for 5 minutes, the
temperature was not constant and
could have affected the results. And
since the water started to boil quickly,
the boiling tube was removed from
the water bath before 5 minutes. This
has an impact on control variables of
both the temperature and time.
When squeezing the It would have affected the amount of Use a cheesecloth to squeeze
fruit juice out of the sugar content in the fruit juice, the freshly extracted juice to
fruits, there could’ve resulting in inaccurate results of mass avoid any bits in it.
been bits present. of precipitate and absorbance value.

Further Investigation:

Nutrient content of fresh and manufactured fruit juices can also be compared, looking at amounts
of carbohydrates and electrolytes. Another study has done an investigation and results
demonstrated the differences in the nutrient content. After analyzing the results, it was found that
sucrose was more frequent in manufactured fruit juices. Fresh fruit juices had a more neutral pH
level than manufactured fruit juices, demonstrating a higher acidic. Manufactured juices derived
from apple, guava, orange, pear, and pineapple had a higher solute concentration than
corresponding fresh juices. All tested fresh juices demonstrated lower sodium levels than
commercial versions. A majority of the fresh fruit juices contained higher levels of nutrients such
as: potassium, phosphorus and magnesium than the commercial juices Therefore, when there is
choice available, it is reasonable to suggest that a consumer is likely better off with respect to a
balanced diet if they consume fresh squeezed fruit juice.

References:

Scientific Research Publishing, J. S. (2012, November 08). Comparison of Sugar Content in Bottled 100%
Fruit Juice versus Extracted Juice of Fresh Fruit. Retrieved from
https://www.scirp.org/journal/PaperInformation.aspx?PaperID=24482

K. (2012, November 19). Benedict's Test - Qualitative Test in Carbohydrates. Retrieved from
https://www.youtube.com/watch?v=TDFbtEwbmz0

Benedict's Test : Principle, Reagent Preparation, Procedure and Interpretation. (2017, July 21). Retrieved
from https://laboratoryinfo.com/benedicts-test-principle-reagent-preparation-procedure-interpretation/

(n.d.). Retrieved from https://www.quora.com/Why-did-the-orange-juice-turn-brick-red-after-the-

Benedict%E2%80%99s-test-for-reducing-sugars

Thelen, A. (n.d.). Retrieved from https://msu.edu/course/lb/145/luckie/inquiriesF2003/ABCD.html

Food Tests - Benedict's Test for Reducing Sugar. (n.d.). Retrieved from
http://brilliantbiologystudent.weebly.com/benedicts-test-for-reducing-sugars.html
Orange (fruit). (2018, April 09). Retrieved from https://en.wikipedia.org/wiki/Orange_(fruit)