As research continues to be performed to determine what
combination of vitamins, minerals, macromolecules, and other nutrients helps a
person live the healthiest life, many trends persist. A recent low-carbohydrate craze has focused
on a high intake of protein. Protein can be obtained from many foods, but the
culinary and botanical genre of the nut offers a high level of protein and
other beneficial nutrients. This study
examined peanuts and walnuts to find out if one offered higher nutritional
benefits than the other.
The
peanuts and walnuts were tested for sugars, amino acids, and proteins, but
before this could be done, they had to be dissolved into an aqueous
solution. Due to the fact that “like
dissolves like” and peanuts and walnuts contain a significant amount of
non-polar lipids, water was not a sufficient solvent. A mixture of non-polar or amphiphilic
methanol, acetonitrile, and Triton X-114 was used to aid the dissolution of all
molecules into solution (Delgado-Zamarreño et al., 2002). The tests performed after the completion of
the extraction were: Barfoed’s test, Selivanoff’s test, the iodine test for
coiled polysaccharides, the
Amino
acids are organic acids that can be bound together in seemingly infinite
configurations to form proteins, which are necessary for life. A large variety of amino acids is ideal
because all 20 are used to create proteins (Freeman, 2005). While the body can synthesize 12 amino acids,
the other 8 must be obtained from food (Freeman, 2005). A TLC test was performed on the samples to
determine which amino acids were present.
Since amino acids can combine in any order, proteins can have a large
disparity in mass, and a nut may contain many different proteins. HPLC was employed to determine the number of
different proteins present in the nuts, and the
The
carbohydrate compositions of peanuts and walnuts were also examined. Carbohydrates, commonly known as sugars, are
broken down to provide the body with ATP.
The three main groupings of carbohydrates are monosaccharides,
disaccharides, and polysaccharides (Krha, 2006). A combination of different types of sugars is
necessary for the body to have a constant supply of energy. The Barfoed’s test was used to verify the
presence of monosaccharides (Krha, 2006).
Monosaccharides can be classified as either aldoses or ketoses based on
the placement of the carbonyl group (Freeman, 2005). This distinction, identified using the
Selivanoff’s test, can help determine which monosaccharides are present (Krha,
2006). While the first two carbohydrate
tests identified monosaccharides or their distinguishing characteristics, the
last test, the iodine test for coiled polysaccharides, signaled the presence or
absence of starch in the nuts (Krha, 2006).
Starch is a coiled polymer of glucose and has similar benefits to fiber
(anonymous-11, 2006).
Although both
peanuts and walnuts can be part of a healthy diet, it was hypothesized that
peanuts offer more nutritional benefits than walnuts based on their higher
concentration and variety of proteins, and larger amino acid and carbohydrate
diversity. The results of the
experiments performed did not necessarily support this hypothesis. Overall, the carbohydrate portion of the
hypothesis, which stated that peanuts will have a larger variety of
carbohydrates than walnuts, was refuted because the results for all three of
the carbohydrate tests were the same for both samples. Peanuts and walnuts were both found to lack
monosaccharides but contain starch, and the Selivanoff’s test results were not
consistent with any of the controls, leaving the presence of aldoses or ketoses
in question. From the TLC test for amino
acids, walnuts were determined to contain cysteine, histidine, lysine,
arginine, and proline, while peanuts indicated the presence of serine,
methionine, valine, tyrosine, and phenylalanine. While the amino acids determined to be
present in both nuts were some of the amino acids expected to be found, many
suspected amino acids were missing, and the same amino acids were not present
in each sample. Walnuts were found to
have 10 different types of proteins and a protein concentration of 190.
mg/mL. Peanuts contained 4 different
proteins and had a protein concentration of 188 mg/mL. These protein concentrations determined from
the
Since
both peanuts and walnuts were found to contain starch, but no monosaccharides,
it was concluded that all of the sugars in the nuts have been bound together by
glycosidic linkages to form polysaccharides.
While each sugar that makes up a polysaccharide is still an aldose or a
ketose, the Selivanoff’s test failed to break apart the glycosidic linkages to
form monosaccharides, and therefore the results did not give conclusive data
(Krha, 2006). The findings of research
performed by Basha on the sugar composition of peanuts support the conclusion
that peanuts do not contain monosaccharides, as the simplest sugar identified
was sucrose, a disaccharide. Holding
true to the hypothesis that both nuts contain polysaccharides, the Iodine test
indicated the presence of starch, a polymer of glucose. Due to the fact that glucose is an aldose, if
the polysaccharides had broken down, both peanuts and walnuts should have been
found to contain aldoses (Krha, 2006).
The positive test for starch strengthens the argument that nuts are a
good part of a balanced diet because research performed by J.M. Goldring in
2004 finds starch to promote good health.
The carbohydrate analysis did not show any difference between peanuts
and walnuts, and therefore it was determined that both nuts provide an equal
amount of nutritional variety. Future
research should include quantitative tests to determine which nut offers more
energy.
The only
significant problem that was encountered during the carbohydrate tests occurred
with Selivanoff’s test. The color
changes observed for the peanut and walnut samples did not coincide with either
the positive or negative controls or the mock trial. This problem most likely occurred because one
of the chemicals in the extraction procedure reacted unfavorably with the
Selivanoff’s reagent.
The
amino acid TLC test proved to be more challenging than initially assumed. While none of the three trials performed gave
clear data, possible dots of amino acids were observed on the third attempt
under UV light. Based on the assumption
that these spots were truly amino acids, a larger variety of amino acids was
observed in the walnuts. Previous
research shows aspartic acid, threonine, serine, glutamic acid, proline,
glycine, alanine, valine, methionine, isoleucine, lecine, tyrosine,
phenylalanine, histidine, lysine, and arginine to be present in walnuts
(Savage, 2001). The only amino acids
suspected to be in walnuts from this study were cysteine, histidine, lysine,
arganine, and proline. Savage found all
of these amino acids except cysteine.
The amino acids: tryptophan, threonine, isoleucine, leucine, lysine,
methionine, cysteine, phenylalanine, tyrosine, valine, arginine, histidine,
alanine, aspartic acid, glutamic acid, glycine, proline, and serine are found
to be in peanuts (anonymous-12, 2005).
The results from the TLC showed that tryptophan, methionine,
phenylalanine, tyrosine, valine and serine were in the peanut sample. Therefore, although all-inclusive lists of
amino acids may not have been compiled from this study, the likelihood of the
determined amino acids being present is very high. The overall variety of amino acids cannot be
analyzed from these data collected with TLC, but they support parts of other
more thorough studies that uphold the hypothesis, as mentioned above.
Multiple errors
occurred throughout the TLC experiments.
The first trial was unsuccessful because the most updated procedure was
not performed. Both the amino acid standard
and a nut sample were placed on one TLC plate.
The sample was painted on, while dots of the standard were pipetted
on. This procedure resulted in plates
that showed very good standard marks (circles), but unobservable samples. Other methods did not produce standard plates
that were as easily observed, but this was contributed to the fact that the
sample mistakenly placed on the standard pate somewhat would have beneficially
diluted the standard. The second trial
of amino acid TLC failed because the incorrect mobile phase was added. Therefore, the plate was developed in such a
way that pigments, if present, would have appeared. The third trial was run correctly, but the
data were still unclear. Under UV light
some spots that appeared to be amino acids were observed, but there was no way
to know whether this assumption was correct.
A few of the solvent fronts appeared to approach the edge of the TLC
plates, and this could have been a problem caused by a lack of equipment and
the need to use very narrow plates.
Circles were drawn around the observed spots and the Rf values were
found. Some of them correlated with
known Rf values, but others did not (Krha, 2006). The known Rf values did not exceed 0.500, but
spots observed in trial three and on the standards of trial one had Rf values
greater than this number. These spots
were either false readings, or the samples traveled farther than expected. The TLC plates could have failed to develop
properly because one of the solvents used in the extraction procedure inhibited
the normal process. Triton X-114 is the
most likely candidate because the least information is understood about
it. Another possible problem was that the
lipids present in the nuts could have interfered with the TLC procedure. An alternate extraction procedure including
the solvent hexane was used to break apart the lipids, but the protocol
employing acetonitrile, methanol, and Triton X-114 had to be utilized to
dissolve the sample into solutions.
Therefore, if one of the original extraction chemicals was the problem,
this method would not solve the dilemma.
The
The
variety of proteins in walnuts revealed through HPLC was found to be larger
than that of peanuts. The data showed 10
peaks on the walnut chromatograms, while only 4 peaks appeared on the peanut
chromatograms. These peaks were assumed
to represent individual proteins in the samples. The chromatograms consist of absorbance
profiles at the wavelengths of 214 and 280 nm, at which peptide bonds (amide
bonds) and phenyl groups (six-carbon rings like those found in phenylalanine,
tyrosine, and tryptophan) absorb, respectively (Watson, 2006). Because the HPLC instrument was calibrated
with a ribonuclease (14kDa) and bovine serum albumin (BSA) standard (66kDa),
only the proteins with affinities within the range of these standard proteins
would appear on the chromatogram (Watson, 2006). The chromatogram at 214 nm was analyzed to
determine the number of proteins, and the chromatogram at 280 nm was used to
back up these data. Some of the peaks on
the 280 nm chromatogram may represent substances that contain a phenyl group
but are not proteins. Peptide bonds are
present in all proteins so the data from the 214 nm chromatogram is more
reliable. But there still could be
another substance in the samples that also has an amide bond that would give a
false peak. The health benefits of these
different types of protein cannot be determined because their concentrations
are unknown. It is only possible to
assume that in this case, the larger variety of proteins equates to a high
protein concentration because the
The HPLC test ran
smoothly, but an extraction procedure unlike than the one used for the other
experiments was necessary to prepare the samples in such a way that they would
not damage the machine. While this
alternate extraction method did not alter the amount of proteins obtained, if
further tests were run, the results would not be based on the constant
conditions throughout. Therefore, all of
the data would be hard to compare.
Another source of possible interest with the HPLC procedure was that the
peanuts and walnuts were not diluted to the same concentrations. Since only the presence of different proteins
was measured, and the original concentrations were unknown, this did not skew
the data.
Although
data were obtained from the
The
most difficult part of all of the research was finding and implementing an
extraction method. The general procedure
was obtained from Delgado- Zamarreño and colleagues, but it did not contain any
specific volume or ratio of chemicals to use.
Upon experimentation, 5 mL each of acetonitrile, methanol, and Triton
X-114 were found to be adequate, but a different ratio may have been more
appropriate. It may also have helped
some of the tests such as Selivanoff’s test and TLC to have given better
results.
The
analysis of the nutritional value of peanuts and walnuts could have been much
more beneficial with more time. If this
study were to continue into the future many new things would be attempted. First, new methods of extraction would be
applied to samples undergoing Selivanoff’s test to determine if an adverse
reaction with one of the extraction chemicals was the problem. Tests to quantify the amount of starch
contained in each nut would also be helpful in distinguishing between peanuts
and walnuts. Multiple new processes
would be undertaken to improve the results obtained from amino acid TLC; the
first procedure attempted would be hydrolysis.
Hydrolysis would allow the molecules in the nuts to react with water
over a long period of time in order to break them apart as thoroughly as
possible (Delgado- Zamarreño, 2001). If
hydrolysis does not work, a chemistry expert would need to be consulted to
determine if the acetonitrile, methanol, or Triton X-114 are capable of causing
all of these problems. As for the
protein portion of the analysis, the samples used in the