Loss Of Photosynthetic Pigments, Vitamin
C, and Sugars in Brussel Sprouts Cooked at Various
Times
Team
Fusariums
By: Nathan Anthony Althaver, Nidhi Manu Atri,
Mallory Christine Nagarah, and, Gerald Henry Schuster
III
LBS 145 ö Cell and Molecular
Biology Spring 2005
Abstract
ÊThe main focus of our experiment was to
determine if brussel sprouts lost their nutritional
components after cooking. In completing this experiment, we sought to determine
when in the cooking process ascorbic acid, sugars, and proteins denature.Ê We also looked at the ability of the Brussels
sprouts to photosynthesize and the pigment color changes after being cooked.
Using brussel sprouts as our vegetable model, we
conducted our research by boiling the brussel sprouts
for 5, 10, 15 and 20 minutes and then analyzing the tissue.Ê Various tests were done in order to determine
the presence and types of sugars. We ran Benedicts test and it showed that for
every cooking time it produced a red precipitate showing reducing sugars.Ê Barfoedâs test
showed that the raw, 5, 10, and 15 minute solutions monosaccharides
and the 20 minute as a disaccharide. Selivanoffâs
test showed the same results for raw, 5, 10, and 15 minute solutions, which was
a ketose.Ê The
20 minute solution was shown to be an aldose.Ê The Iodine test showed as you cooked more,
the starch concentration decreased.Ê
Protein concentration determined in the Bradford Assay increased as
cooking took place. The paper chromatography test showed the pigment grows
fainter as the cooking took place.Ê The
ability of the brussel sprouts to photosynthesize
(Action Spectrum) and the amount of ascorbic acid present (Ascorbic Acid Assay)
decreased as the cooking time increased.Ê
Further research using the Hill test showed that the brussel
sprouts ability to undergo photosynthesis was reduced when cooked because the
absorbance values decreased over time. Finally, for the Vitamin C test,
ascorbic acid concentrations were reduced the more the brussel
sprouts were cooked.
Discussion
ÊÊÊÊÊÊÊÊÊ The subject of our
study was brassica oleracea L.
var. gemmifera
or more commonly known as brussel sprouts. We
examined the effects of cooking the brussel sprouts
for different amounts of time. We particularly looked at sugars, protein concentration,
photosynthetic ability, potassium, and ascorbic acid concentrations. The goal
of the study was to determine if cooking brussel
sprouts for a long amount of time would affect these important components that
would enhance oneâs diet. We hypothesized that the longer brussel
sprouts were cooked, the more components they would lose. We also sought out to
answer the question at what point in the cooking process are components lost in
the greatest amount.
We began our research by
performing the following carbohydrate tests: Benedictâs, Barfoedâs,
Selivanoffâs, and the Iodine test. Benedictâs test
tests for free aldehyde or ketone
groups. Barfoedâs test was done in order to
distinguish monosaccharides from di-
and polysaccharides. Selivanoffâs test differentiates
between ketoses and aldoses. The Iodine test
distinguishes polysaccharides from monosaccharides.
ÊFor Benedictâs test both the raw brussel sprouts and the brussel
sprouts that had been cooked for 5, 10, 15, and 20 minutes all produced a
precipitate that was varying shades of rusty orange. The rust-orange
precipitate indicated that reducing sugars were indeed present. The two
positive controls, fructose and galactose, both
yielded a red precipitate and the negative control of water produced no
precipitate at all meaning that the reagent worked correctly. The conclusion
that was yielded from the results found was that the cooking time has no effect
on the presence of reducing sugars.Ê
Based on a study conducted by Edward Baker and Peter Halloway,
it was known that brussel sprouts should come out to
be positive for free aldehyde and ketone
groups and this was supported by our results as well (Baker and Halloway, 1975).
To test for the presence of monosaccharides Barfoedâs test
was used. If monosaccharides are present a red
precipitate should form. The raw brussel sprout
solution yielded a yellowish-green precipitate while the five minute solution
had a precipitate that was slightly redder in color. The ten and fifteen minute
solutions both had the yellowish-green precipitate that was also seen in the
raw solution. Finally, for the twenty minute solution there was a red-green
precipitate. The positive control xylose has the red
precipitate indicating that it is a monosaccharide and the negative control of
water produced no precipitate as expected.Ê
All the brussel sprout treatments, including
the raw, formed a precipitate indicating the presence of monosaccharides.Ê The monosaccharide present in brussel sprouts is glucose (Heaney and Fenwick, 1980). So,
we were expected that positive results for monosaccharides
with Barfoedâs test would be produced and that is
what we found to be true. However, the color of the precipitate and the amount
varied among the brussel sprouts that were cooked for
different amounts of time.Ê This is suspected
to be attributed to the fact that the brussel spout
solution was green to begin with. Also since this test is a qualitative test,
we cannot be sure of the amounts of monosaccharides
that are present. Further research can be done to quantify this test, such as
weighing the amounts of precipitate formed. If the test was quantified, we
could have seen how much precipitate formed and would have been able to tell at
what time (5, 10, 15, 20 minutes) in the cooking process the most monosaccharides were present.
For Selivanoffâs
test, we recorded several results. The initial hypothesis was supported by the
results obtained; as the brussel sprouts are cooked
for longer, sugars are lost. This test was used to distinguish between ketoses
and aldoses. Both the raw brussel
sprouts and the brussel sprouts that were cooked for
5 minutes turned the solution red in less then a minute (45 and 50 seconds
respectively), indicating the presence of monosaccharide ketoses.Ê The brussel sprouts
that were boiled for 10 and 15 minutes turned the solution red in approximately
one minute, which indicates the presence of disaccharide ketoses.Ê Finally the brussel
sprouts that were boiled for 20 minutes turned the solution red in more then a
minute (75 seconds) which indicates the presence of aldoses.
The controls worked out as expected: fructose turned the solution red in less
than one minute, galactose took greater than a
minute, and water did not result in a change. From these results we can see
that as the brussel sprouts are cooked longer, they
lose their ketoses. It is possible that the ketoses are boiled off, indicating
that there is a breakdown as the cooking process is elongated. We know that brussel sprouts contain both aldoses
and ketoses when they are raw (Baker and Holloway, 1975). From this data it can
be concluded that after fifteen minutes of cooking the spouts they contain only
aldoses.
The results of the Iodine test
were uniform over the raw and five different cooking times.Ê It had been hypothesized that the starch
concentration would decrease because prior studies had indicated that enzymes
are broken up when heated. Despite being raw or boiled for 5, 10, 15 and 20
minutes, the color of the solution remained a yellowish brown color indicating
a negative test for starch.Ê We expected
these results because in the study preformed on broccoli, a close relative to brussel sprouts also had a negative result after performing
the starch test on their cooked and raw broccoli (Bale et al., 2002).Ê The positive control turned the solution a
bluish-black color and the negative control of water produced to color change.
When these results were yielded it was unexpected because this test showed that
there is in fact no starch present in brussel
sprouts.
A test for pigment
identification was also conducted using paper chromatography. The raw solution
for chlorophyll b had an average Rf value of 0.488, chlorophyll a was 0.703,
xanthophylls was 0.839 and carotene had an average value of 1.000. The five
minute solution had an average Rf value of
0.457 for chlorophyll b, an average of 0.547 for chlorophyll a, an average
value of 0.860 for xanthophyll, and carotene yielded
an average value of 1.000. The solution for the ten minute brussel
sprouts had a very faint chlorophyll b separation so it was too faint to
determine actual numbers. Chlorophyll a had an average of 0.585, xanthophyll had an average Rf
value of 0.835, and carotene had an average value of 1.000.Ê The fifteen minute solution separation was
very faint to determine pigment movement. However, one strip produced some
separation. On this strip chlorophyll b had a value of 0.488, chlorophyll a had
very faint separation, xanthophyll had a value of
0.832, and carotene had a value of 1.000. The twenty minute solution had such a
faint separation that it was too faint to determine pigment movement. The
control separation which was spinach yielded an average Rf
value of 0.548 for chlorophyll b, an average value of 0.754 for chlorophyll a,
an average value of 0.832 for xanthophyll, and
finally an average value of 1.000 for carotene. The Rf Êvalue indicates the rate of flow and it
was seen that there was indeed the presence of pigments in these brussel sprout solutions. It was seen that the pigments did
however fade as the cooking time increased. This is what was predicted based
upon the color of the solutions; the brightest colors were seen in the raw and
the lightest was the twenty minute solution.
The Hill Reaction measures the
ability of photosynthesis to occur. In this study it is measuring the ability
of brussel sprouts, specifically, to photosynthesize
when cooked for different amounts of time. The chloroplasts are only being
exposed to white light exposing the chloroplasts to white light because we know
that it contains both red and blue light. It was predicted that the brussel sprouts that were cooked the longest (20 minutes)
will have the highest absorbency when measured in the spectrometer and that the
rest of the groups would decrease as they were cooked for less and less time.
This means the solution will remain a darker color indicating that the cooked brussel sprouts are poor photosynthesizers
and possibly that photosynthesis did not take place at all.Ê The average absorption for the raw solution
was 2.655 for the one exposed to light and 2.610 for the one that was kept in
the dark. In the five minute solution the average absorbance yielded for the
light exposed solution was 2.870 and 2.700 for the one kept in the dark. The
ten minute solution had an average absorbance of 2.450 for the light solution
and 2.365 for the solution kept away from all light sources. For the brussel sprouts cooked for twenty minutes, the average
absorbance was 2.360 for the one kept in the light and 2.390 for that kept in
the dark. These results indicted that there may either been little
photosynthesis occurring or none at all. This went against the predicted
results that the ability of the brussel sprouts to
photosynthesize would go down as they were cooked for longer. However, after
research was conducted it was determine that this is because the photosynthesis
of brussel sprouts occurs in the leaf of the plant,
not in the actual sprout that we eat (Gunatillake,
2003).
Later in the study a test for
ascorbic acid was conducted. Ascorbic acid is associated with chloroplasts and
is present in green leafy vegetables (Reiss 1993). This means that there may be
a correlation between photosynthetic ability and ascorbic acid content of
cooked versus raw vegetables. There could be a connection indicating that as
Vitamin C content increases so does the photosynthetic ability and vice versa.
In the independent portion of
this experiment, a quantitative test for ascorbic acid was done to see if
amounts of ascorbic acid (vitamin C) are lost in the cooking process. It is
known that brussel sprouts contain ascorbic acid from
a list that contains all the phytochemicals present
in brussel sprouts (anonoymous-2, unknown). With this
information research was conducted in order to find an assay to be utilized in
order to further this study. One study showed a significant loss of potassium
in spinach due to cooking for a long period of time (anonymous-5 2002). Due to
this, it was suspected that ascorbic acid can be lost during the cooking
process as well. It was predicted that a drop in the ascorbic acid
concentration of the brussel sprouts would be soon as
they were cooked for longer times (Smith 2005).Ê
Our results illustrated that this was indeed the case. It was found
through experimentation that an average of 18 drops of raw solution was
required to be added to the corn starch solution until there was a color
change. The average amount of drops required for five minutes was 24, 26.67 for
the ten minute solution, 42 for the twenty minute solution, and 43.67 drops for
the twenty minute solution. As cooking time increased, the amount of drops
necessary to obtain the color change also increased. This indicated that as
time went on, less ascorbic acid was present, going in accordance with our
predicted results (anonymous-4, unknown).
Errors in our results can be
attributed to human error in performing the experiments. There can be errors in
measurements, mixing, documentation of results, and more.Ê This could be improved upon by emphasis on
sterile lab techniques and double checking each one of our steps in our
experiments. Also there can also be instrument error especially concerning the
tests that utilized the spectrometer. If it by some chance was not zeroed
correctly then all of the data would be thrown off. There is also the threat of
contamination. There are a number of things that may have been contaminated
from dirty beakers to contaminations of stock solutions of the different
reagents used in all of the tests. This is why it is essential to do as many
repetitions as possible and to document all data as accurately as possible.
ÊÊÊÊÊÊÊÊÊ In this study
several experiments were conducted in order to measure the presence of
concentration of several components in brussel
sprouts. Some resulted in data that supported the initial hypotheses while
others completely refuted it.Ê Benedictâs
test illustrated that there was a rise in reducing sugars before they fell. It
had been predicted that they would fall continuously as time went on and this
was clearly negated by the results yielded. The results for Barfoedâs
test went along with the predicted results that there were indeed monosaccharides present. The resulting data from Selivanoffâs test also supported the predicted hypothesis
that components are lost because the ketoses are lost as cooking time
increases. The Iodine completely contradicted the initial hypothesis that starch
content would decrease because it was discovered that there is n starch present
in brussel sprouts. The Bradford Assay was
inconclusive and no correlation was seen between protein concentration and
cooking time. It had been predicted that concentration would decrease but this
was not shown by the data. The paper chromatography test showed that brussel sprouts did indeed contain pigments and the results
supported the initial hypothesis that pigments are lost in the cooking
process.Ê The Hill Reaction was also a
surprise because through it was learned that brussel
sprouts do not photosynthesize themselves, it is the leaves of the plant that
do. As far as the independent experiment, the initial hypothesis was supported.
It was shown that as brussel sprouts are cooked for
longer periods of time, the amount of ascorbic acid decreases. The question as
to when the significant portion of components are lost could not be properly
assessed because more quantitative tests are necessary to determine this. A
good way to further this study would be to quantify all tests by possibly
weighing all yielding precipitates going further than that.Ê
Table 3:
ÊPaper
chromatography trial values and their averages.Ê When compared to the control, the average Rf values decrease compared
to their control value as the brussel sprouts are
cooked for longer amounts of time indicating a loss of chlorophyll as the time
increases.
|
Raw |
chlorophyll b
(Rf) |
chlorophyll a
(Rf) |
Xanthophylls
(Rf) |
Carotene (Rf) |
|
Trial 1 |
0.549 |
0.754 |
0.832 |
1.000 |
|
Trial 2 |
0.484 |
0.711 |
0.831 |
1.000 |
|
Trial 3 |
0.432 |
0.643 |
0.854 |
1.000 |
|
Average Rf |
0.488 |
0.703 |
0.839 |
1.000 |
|
|
|
|
|
|
|
5 minute |
|
|
|
|
|
Trial 1 |
0.432 |
0.556 |
0.897 |
1.000 |
|
Trial 2 |
0.469 |
0.586 |
0.813 |
1.000 |
|
Trial 3 |
0.471 |
0.581 |
0.869 |
1.000 |
|
Average Rf |
0.457 |
0.574 |
0.860 |
1.000 |
|
|
|
|
|
|
|
10 minute |
|
|
|
|
|
Trial 1 |
Too faint |
0.609 |
0.869 |
1.000 |
|
Trial 2 |
Too faint |
0.560 |
0.800 |
1.000 |
|
Trial 3 |
Too faint to
determine pigment movement |
|
||
|
Average Rf |
|
0.585 |
0.835 |
1.000 |
|
|
|
|
|
|
|
15 minute |
|
|
|
|
|
Trial 1 |
0.488 |
Too faint |
0.832 |
1.000 |
|
Trial 2 |
Too faint to
determine pigment movement |
|
||
|
Trial 3 |
Too faint to
determine pigment movement |
|
||
|
Average Rf |
0.488 |
|
0.832 |
1.000 |
|
|
|
|
|
|
|
20 minute |
|
|
|
|
|
Trial 1 |
Too faint to
determine pigment movement |
|
||
|
Trial 2 |
Too faint to
determine pigment movement |
|
||
|
Trial 3 |
Too faint to
determine pigment movement |
|
||
|
Average Rf |
|
|
|
|
|
|
|
|
|
|
|
Control
(Spinach) |
0.548 |
0.754 |
0.832 |
1.000 |