Carbohydrate Tests on Apple Juices vs. Apples (Malus sylvestris)
Indicate Equal Nutritional Importance
By: Farhan Bhatti, Andy Hanson, Jana McNulty, Dan Nelson
ABSTRACT:
As an extension to a plethora of studies on apples (Malus sylvestris),
pressed red delicious apples were tested against Meijer apple juice, Robinette's
cider, and Odwalla organic juice for comparison of: nutritional content in
the form of carbohydrates, photosynthetic properties, and enzyme activity.
Similar results were expected for each liquid. Carbohydrate structures were
investigated with the use of three tests: Benedict's, Barfoed's, and Selivanoff's.
Both Benedict's and Barfoed's tests determine reducing sugar presence and
Selivanoff's test determines the presence of an aldose or ketose. Photosynthetic
properties of apple leaves were tested by using paper chromatography to search
for pigments such as chlorophyll, which play a large role in photosynthesis.
When experimenting with enzymes, a touch of juice was added to catechol, a
substrate that changes color when oxidized by the enzyme PPO, and data checking
for a change in absorbency over time was recorded. Enzyme activity was also
tested by measuring the effect varying pH values have on absorbency.
Regarding the presence of carbohydrates, it was shown that the
pressed apples and apple liquids contained reducing sugars and had either
disaccharides or polysaccharides. Aldose sugars were present in all groups.
The presence for PPO also came up positive in all groups. The effect of pH
on absorbencies followed a direct relationship for each trial of apple skins
and leaves. From our analysis of nutritional content, convincing data is present
to support our hypothesis that red delicious apples have equal nutritional
value as Meijer apple juice, Robinette's cider, and Odwalla organic apple
juice.
Figure:
Figure 3. Results From Selivanoff's Test. This test was performed
on the pressed apple juice, the Meijer apple juice, the apple cider, and the
Odwalla apple juice. Both trials are pictured together. In order from left
to right, the tubes pictured are the second trial of the Meijer juice, the
first and second trials of the pressed juice, the first and second trials
of the cider, the positive control (galactose), the second and first trials
of the Odwalla juice, and the first trial of the Meijer juice. All liquids
changed color in less than one minute, which indicates the sugar is a monosaccharide
ketose.
DISCUSSION:
So what do the above results actually mean? Is there a significant
difference between juice from a pressed apple and apple juice purchased from
a store? To start, we predicted there would not be a difference, and our data
indeed helped support our claim. Juice companies cannot claim to be "100%
pure" or "All-Natural" unless they are confident their product,
after undergoing rigorous testing, will appear to be what they claim it to
be. And what they claim it to be is a pure substitute of eating apples.
We expected to find no difference in components of the juice,
and the first experiment we performed to test our claim was Benedict's test.
Benedict's test determines whether or not there is a free aldehyde or ketone
group in the carbohydrate (Luckie, et.al, 2003). A red precipitate formed
during all trials for the juice from the red-delicious apple and the store-purchased
liquids. This indicates they all have free aldehyde or ketone groups, and
thus, they are reducing sugars. We saw a color change when performing Barfoed's
test on the pressed apple, a test which indicated if the apple contained either
disaccharides or polysaccharides. The color changed because only monosaccharides
can react quickly enough to reduce copper ions (Luckie, et.al, 2003). When
this experiment was repeated on the other apple liquids, the same results
were obtained. This led us to believe the sugars are monosaccharides, which
coincides with our results from Selivanoff's test. We cannot conclude from
this test that the sugars in both are the same, but only that the sugars in
both are monosaccharides. Selivanoff's test, the test to distinguish between
a ketose and aldose, showed very similar results for all liquids. Resorcinol
in concentrated HCl, when heated with a sugar, produced furfural or hydorxymethylfurfural,
which further reacted in a boiling water bath to yield a RED color. If this
happens in less than one minute, one can conclude it is a ketose. Anything
over one minute is an aldose. We used one positive control - galactose. We
tested the four apple liquids twice, and each trial yielded the same result
- a uniform color change within thirty seconds of heating. Hence, our results
lead us to believe we were dealing with monosaccharide ketoses (Table 3).
The purpose of organic juice is not to deliver a product with
different nutritional content, but rather, to deliver a product fertilized
with organic matter instead of manufactured chemicals (Sullivan, 2001).The
Odwalla juice company even places a "shelf life" on its products
to ensure purity and cleanliness. The "expiration date" of our Odwalla
apple juice was the day after we performed the original carbohydrate experiments,
January 22nd. This juice was refrigerated for four weeks after its expiration
date and allowed to ferment. At this point, we repeated the carbohydrate tests
and found no change in the sugar composition of the juice. The only noticeable
difference: the smell.
We thought there would be identical pigments found in the juices
as well. The paper chromatography for the apple leaves yielded orange-yellow
and blue-green pigments. Using a chromatogram strip, and by placing drops
of our apple juice onto a strip, the pigments could be identified. The individual
pigments in the leaves were Carotene, Xanthophyll, Chlorophyll a, and Chlorophyll
b. The individual pigments in the peel were Carotene and Xanthophyll (Table
5).
The absorption spectrum test was executed to investigate which
wavelengths of light the apple leaves and the apple peel absorb and reflect.
For the leaves, during both trials, we observed that they absorbed light in
the blue and red regions of the UV Light Spectrum, but reflected green light.
The pigment chlorophyll absorbed the most light energy in the blue spectrum.
For the apple peel, during both trials, we observed that the peel absorbed
light in the blue and green regions of the UV Spectrum, but reflected red
light. We tested the apple peel to see if it had any effect on the photosynthetic
process. Since the peel reflected red light, however, we were not able to
establish a relationship between apple peels and photosynthesis based on our
data.
Two experiments were performed to test the enzymes. The first
was the PPO test. PPO is the enzyme that ultimately causes apples to turn
brown (Billaud et. al, 2002). Because we were dealing with liquids, we faced
a minor challenge. It would have been very difficult to observe any changes
had we added catechol to the different liquids, as the procedure in the lab
manual suggests. The color of freshly squeezed juice from the apple is much
lighter than the brown apple juice sold at stores, and since catechol turns
substances with PPO brown, we would have seen no noticeable difference. Thus,
instead of adding a bit of catechol to the various liquids, we added a bit
of the various liquids (0.5ml) to 2.5ml of catechol and observed any change
in absorbency over time. If the solution did change absorbency, then we could
conclude PPO was active. Out of the four liquids, the only one which did not
have a constant absorbency was the pressed apple juice. Fresh apples contain
active PPO (Billaud et. al, 2002), however, the enzyme was not active inside
the three other apple liquids for a few reasons. First, the PPO in the three
purchased apple liquids had already oxidized the pulp that was used to make
the three juices. Also, the optimal temperature for enzyme treatment in apples
is 30°C (NCBE Journal, 2000). Thus, although an additional reaction of
the enzyme was originally expected, an additional reaction did not occur when
the apple liquids were added to the catechol.
We found the pH was 4 for the pressed apples and the cider,
and 5 for the Odwalla and Meijer juice, hence, the former two liquids were
slightly more acidic than the latter two liquids. Next, we tested the effect
of pH. Thirty-six test tubes were prepared for this experiment, with varying
pH buffers in the test tubes. We observed as the pH buffer grew in size, for
the pressed apple juice only, the general trend was an increase in absorbency.
This is because the enzyme PPO was still active in this juice (NCBE Journal,
2000). The absorbency of the cider, Odwalla juice, and Meijer juice varied,
but only slightly and not by a substantial amount. This data supports our
reasoning that PPO is present, but not active in the three purchased liquids.
Some potential weaknesses or problems we encountered were we
did not record the temperature the apples were pressed at, and did not know
what other additives were placed in the apple juice. We tested the apples
when they were freshly squeezed, however, and thus, the juice was still clear.
We didn't use fresh apples; instead we used the ones we purchased at the beginning
of the experiment, so the freshness of the apples was in jeopardy. Even after
purchasing the apples from a grocery store, we had no way of telling how old
the apples were that we had purchased. Another error we encountered came during
our test for the effect of pH. The spectrophotometer displayed negative absorbency
values for some of the liquids. Catechol and the juice was used as a blank,
and the reading was taken at 480 nm. In hindsight, we should have used a more
concentrated solution to ensure our values were not hovering around 0 absorbency.
Further trials should have been performed to check this, however, due to time
constraints, we were only able to perform one trial of this experiment.
Further research on this topic could be performed. Suggested
research includes not only testing freshly pressed apples vs. apple juice,
but different brands of juices. Information about any preservatives added
to the juice would need to be researched. One could also test other apple
products, such as apple sauce, to see if these products uphold the same nutritional
value as ripe apples.
As is the case in many scientific experiments, we performed our experiment
with a healthy amount of background information, and we obtained results that
seem to be in accordance with what we expected. The Food Pyramid does not
distinguish between fruit and its juice counterpart, and we can assume that
if the true intentions of the Food Pyramid are to make us healthier, then
most if not all of the benefits one receives from eating fruit are indeed
preserved when the fruit becomes fruit juice. Important similarities were
shown at the molecular level between juice from a pressed apple and various
apple drinks. With improvements in technique and additional knowledge on the
topic, we believe the same results can be obtained and be portrayed more convincingly
to the general public.
REFERENCES:
Billaud, Brun-Mérimee, Maraschin,, Nicolas, Roux. 2002.
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solutions and Maillard reaction product model systems on polyphenoloxidase
from apple. II. Kinetic study andmechanism of inhibition. Food Chemistry.
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"Enzymes in fruit juice production." In a Jam and
out of Juice.
http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/INAJAM/PDF/JAM01.pdf
NCBE Journal. (December 2000).
Luckie, Maleszewski, Sayed, Wilterding. LBS-145 Cell and Molecular
Biology: Lecture/Lab, Spring 2003, Course Packet. pp. 63-66. MSU Printing
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Luckie, Maleszewski, Sayed, Wilterding. LBS-145 Cell and Molecular
Biology: Lecture/Lab, Spring 2003, Course Packet. pp.72-73. MSU Printing Services.
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Sullivan, Dan. 2001 Organic Apple Outshine Conventional Ones.
Organic Gardening. Vol. 48 p. 16