The Chloroplasts
Tom Barkham, Jennifer Lindley, Laura Neuder, and Julie Nap
LBS 145
Several tests were used in order to differentiate between the various preparation methods of frozen, cooked, and canned beans with respect to carbohydrates, pigments, and the presence of enzymes. Raw green beans were used as a control in this analysis. We predict that canned green beans would have the greatest similarities to raw green beans in comparison to cooked and frozen. To test the presence of carbohydrates the Barfoed’s, Selivanoff’s, and Iodine tests were used. The Barfoed’s test exhibited a presence of monosaccharides in cooked green beans while frozen, canned and raw green beans showed a presence of polysaccharides. The Selivanoff’s test showed all beans reacting within one minute indicating a presence of ketoses. The Iodine test indicated the presence of starch in all beans. Pigment absorption was tested using paper chromatography and the absorption spectrum test. Our data from the paper chromatography was inconclusive. The absorption spectrum showed the greatest absorbance in descending order: frozen, cooked, canned, and raw. The presence of enzymes was tested using the presence of polyphenol oxidase (PPO), and the presence of heat and pH on enzyme activity diagnostic tests. PPO was found in frozen and canned green beans while raw and cooked did not exhibit the enzyme. There was no significant difference in the presence of PPO when heat was added. Frozen and raw green beans had the highest presence of PPO when the pH of the solution was altered. Therefore, the preparation of frozen green beans closely resembles the characteristics of raw.
There are various
ways to prepare green beans. In this laboratory investigation, we
decided to determine which preparation method (cooked, canned or
frozen) most closely resembles the characteristics of raw green
beans (control). Our initial hypothesis stated canned green beans
most closely reflected raw green beans. We preformed a series of
different tests to look for carbohydrate, enzyme and pigment
variations in the four samples of green beans. Our results did not
confirm our hypothesis, instead, we found that frozen prepared green
beans are closely comparable to raw.
In our first experiment, tests were completed in order to determine the
different types of carbohydrates. Plants synthesize starch and
then stockpile it in the form of glucose, which is a major
cellular fuel. Since starch is stored energy in the plant, all
preparation types of beans should contain starch. Previous
experiments have shown that that heating process undergone by prepared
vegetables depletes the vegetable of starch. Therefore, we predicted
that frozen, canned and cooked samples would all have starch
present, but the amount would be less compared to raw (Nyman,
Svanberg and Asp, 1994). Using Iodine staining, we tested for the
presence of starch. Each type of bean (raw, cooked, canned and
frozen) varied in the color change, but no assumptions as to the
amount of starch could be drawn from the variations in color. As
expected, due to the importance of starch to a plant; raw, cooked,
canned and frozen beans all contained starch.
The Selivanoff's test determined whether any carbohydrates present were
ketoses or aldoses. Ketoses are sugars, which contain a carbonyl
group within a carbon chain. An aldose is a sugar, which contains
a carbonyl group on the terminal end of the carbon chain. As
stated above, galactose is a common sugar found in green beans,
which consist of aldoses. When green beans are heated, galactose
is diminished into the solution that the green beans are cooked or
canned in. Therefore, we predicted that prepared green beans would
have more ketoses than raw green beans, which would consist mainly
of aldoses (Nyman, Svanberg and Asp, 1994). Within one minute,
each of the green bean solutions formed a red/brown precipitate,
indicating the presence of ketoses. Our results showed that raw
green beans do not have a differing dominant functional group
carbohydrate (aldose) than the prepared methods of green beans..
Instead frozen, cooked, canned and raw all contained ketoses. This
does not mean that aldoses are nonexistent in all green beans.
Instead, the concentration of ketoses is higher than the
concentration of aldoses.
The Barfoed's test distinguished the presence of monosaccharides
(single sugars) versus polysaccharides (many sugars) within the
green bean samples. Once again, we predicted that the raw beans
would contain monosaccharides as oppose to polysaccharides as with
the processed beans because cooking and canning processes deplete
the beans of carbohydrates. "During wet heat treatment, as in
blanching, boiling and canning of vegetables and fruits, there is a
considerable loss of low molecular weight carbohydrates (i.e.
mono- and disaccharides)" (Effects of Food Processing on Dietary
Carbohydrates: Carbohydrates in Human Nutrition , 1997). The
observation of a precipitate among the green bean solutions
indicated that each sample contained monosaccharides sugars. The
results did not confirm our prediction that fresh beans do contain
monosaccharides whereas the other types did not contain them.
Instead, our results show that raw, cooked, canned and frozen
green beans all contain monosaccharides. Again, this does not mean
that polysaccharides are not present within the sample, instead,
their concentrations are lower than monosaccharides.
Our second experiment involved pigments and absorption rates within the
chloroplast. The chloroplast is the component of a plant cell,
which helps to produce chemical energy in the process of
photosynthesis. The pigments that are released from the
chloroplast include carotene, xanthophyll, chlorophyll a and
chlorophyll b. Using chromatography, the samples of green beans
were tested to see how much of each pigment was present. Our group
predicted that canned and cooked green beans would have smaller Rf
values for chlorophylls than raw because chlorophylls are depleted
when heat is added. Caretenoids would remain the same in
comparison to raw green beans (de la Cruz-Garcia, Gonzalez-Castro,
Oruna-Concha, Lopez-Hernandez, Simal-Lozano and Simal-Gandara, 1999).
In contrast, frozen green beans should have smaller Rf values than
raw green beans for chlorophylls and caretenoids because a
decrease in temperature depletes the presence of pigments
(Monreal, de Ancos, Cano, 1999). In this experiment, none of the
pigments were observed using paper chromatography. This is because
the samples of green beans are not soluble in the solutions that were
provided in the laboratory. This does not mean that carotene,
xanthophyll, chlorophyll a and chlorophyll b are not present in
the bean. Instead, the methods provided in the laboratory were not
capable of expressing these pigments. Therefore, our results are
inconclusive.
The absorption spectrum analysis test was performed as another way to
look at the pigments in each type of green bean. A solution was
prepared using each sample of green bean and placed in a
spectrometer in which the absorbance was determined. We predicted
that cooked and canned would have the closest absorption rate to
that of raw green beans. This is because caretenoids stay intact
when the green beans are heated during preparation. Therefore, the
spectrum would resemble the caretenoids still present within the bean.
Chlorophyll on the other hand is depleted when green beans are
heated and therefore would not be representitive in the spectrum
(de la Cruz-Garcia, Gonzalez-Castro, Oruna-Concha,
Lopez-Hernandez, Simal-Lozano, and Simal-Gandara, 1999). We also
predicted that frozen green beans would have a spectrum quite different
than raw. This is because the freezing process depletes the green bean
of both chlorophyll and caretenoids (Monreal, de Ancos, Cano,
1999). After experimentation, we actually found frozen green beans
to have the highest absorption rate followed by cooked, canned and
raw. This could be because of the chlorophyll degradation of
pheophytin is favored in acidic conditions. Frozen green beans were
found to be the most acidic in the enzyme portion of this lab,
therefore confirming these results (Cruz-Garcia, Gonzalez-Castro,
Oruna-Concha, Lopez-Hernandez, Simal-Lozano, Simal-Gandara, 1999).
The freezing process actually increases the absorption of pigments
within green beans because the absorption spectrum of frozen
samples are significantly higher than the absorption spectrum of
raw green beans. Considering that at 400nm, both had relatively the
same absorption, frozen surpassed raw green beans at 430nm.
Therefore, the acidic conditions as well as the process that the
frozen green beans are prepared allow for a greater absorption of
pigments.
For the third experiment, the activity of the enzyme polyphenoloxidase
(PPO) was determined using the effects of heat and pH. First, we
determined if PPO was present in green beans. We could not find
any previous documentation to whether PPO is present in green
beans. Instead we hypothesized that it did contain the enzyme
because often green beans will discolor after harvesting. We found
that frozen and canned green beans had PPO because there was a slight
color change when 0.1% catechol was added. Next, we tested the effect
of heat on enzyme activity. Again the enzyme of study was PPO. We
predicted that cooked and canned green beans would have the
highest enzyme activity because heating increases the catalyzation
of enzymes (Kaack, 1995). Instead, we found that only cooked green
beans had a change in the catalyzation due to heat. The color
change was very slight indicating that a slight change in enzyme
catalyzation was undergone. When testing for the effects of pH, we
predicted that canned green beans would have the greatest presence
of enzymes. This is because we assumed that canned green beans are
in acidic solutions, as well as since they are heated during
preparation. Enzyme activity increases if an acidic pH is at
higher temperatures. On the other hand, a basic pH will decrease
the presence of enzymes when the green beans are heated, therefore
implying that cooked and canned green beans will have
significantly less presence (Delincee and Radola, 1974). Our
results contradicted our predictions, instead frozen green beans
have the highest presence of enzymes due to the effect of pH. When
we applied litmus paper to the green beans during the presence of PPO
tests, we noticed that frozen green beans had the most acidic pH.
Therefore, this information would correspond with the assumption
that frozen green beans have the highest enzyme presence due to pH.
Overall, due to carbohydrates, photosynthetic potential and enzyme
activity, frozen green beans most closely resemble raw green
beans. The carbohydrate diagnostic tests and the pigment
identification tests were inconclusive in this analysis. The
absorption spectrum of pigments as well as the effects of heat and
pH on enzyme activity lead us to conclude that the preparation of
frozen green beans closely resembles raw. This does not mean that
canned and cooked green beans are not comparable to raw green
beans. Although the conclusion was drawn that frozen beans are
most like the control, there was not an abundance of evidence
pointing in the direction that preparation highly affects green
beans content. Instead, frozen green beans are most comparable to
raw in general with the experiments done on sugars, photosynthesis and
enzymes. Testing different aspects of green beans, such as vitamins or
any other type of nutritional content may show another type of
processing most effective at preserving the values of a raw green
bean. Also, the fact that we used the same samples of green beans
each week, while refrigerating the samples when not in use, was
done to retain consistency. Forms of contamination as well as
spoiling could have altered our results. The time in which the
green beans were removed from their preparation method and were ground
down to a liquid like form could have altered the content of the
bean. Although the brand of beans used and the methods to obtain
analyzed materials was only one way to gather results, it was the
only way in which we were able to use within the laboratory.
Therefore, in this analysis of green beans, we conclude that the
preparation of frozen green beans closely resembles raw green
beans. This conclusion was made due to the data that we collected
while analyzing carbohydrate, photosynthetic and enzyme content.
We do not condone the consumption of cooked and canned green
beans. They also offer their consumer excellent tasted and nutrients.
But ultimately because of this analysis, we propose that frozen green
beans are the best substitute for raw.
Table 1. Carbohydrate Test Results. Each of the following tests have a specific characteristic that help to determine which carbohydrate is exhibited in cooked, raw canned and frozen samples. The Barfoed’s test distinguishes between monosaccharides (reduced red cupric ions) and polysaccharides (blue). Selivanoff’s test distinguishes between aldoses and ketoses (ketoses will react red within one minute while aldoses will react after several). The Iodine test identifies starches; it will turn a substance a blue-black color when starches are present. (three test tubes refers to each of three trials done)
|
Selivanoff’s |
Barfoed’s |
Iodine |
Cooked 25% |
3 test tubes reacted within 1 minute, with
a red/brown precipitate. |
3 test tubes reacted which showed a slight
red precipitate. |
3 test tubes resulted in a light gray color. |
50% |
3 test tubes reacted within 1 minute, with
a red/brown precipitate. |
3 test tubes reacted which showed a slight
red precipitate. |
3 test tubes resulted in a light gray color. |
Raw 25% |
3 test tubes reacted within 1 minute, with
a red/orange precipitate. |
3 test tubes reacted staying a blue color
with a very small red precipitate. |
3 test tubes resulted in an orange color. |
50% |
3 test tubes reacted within 1 minute, with
a red/orange precipitate. |
3 test tubes reacted which showed red
precipitate. |
3 test tubes resulted in a light gray color. |
Frozen 25% |
3 test tubes reacted within 1 minute, with
a dark red/brown precipitate. |
3 test tubes reacted which showed a red
precipitate. |
3 test tubes resulted in a dark purple color. |
50% |
3 test tubes reacted within 1 minute, with
a dark red/brown precipitate. |
3 test tubes reacted which showed a red
precipitate. |
3 test tubes resulted in a dark purple color. |
Canned 25% |
3 test tubes reacted within 1 minute, with
a light red/brown precipitate. |
3 test tubes reacted which showed no change
staying a blue color. |
3 test tubes resulted in a darker purple
color. |
50% |
3 test tubes reacted within 1 minute, with
a light red/brown precipitate. |
3 test tubes reacted which showed no change
staying a blue color. |
3 test tubes resulted in a darker purple
color. |
Control (+/-) |
(Deionized-Water) showed no change. |
(Sucrose) reacted to show no color change
(blue) |
(Starch) resulted in a dark purple color |