Photosynthetic and Sugar Analysis of Green Beans (Phaseolus vulgaris) Confirm EDTA as Best Preservative

Vortex Genies

Danielle DeLand, Jessica Grover, Shannon Slavsky, Kari Storm

Abstract

Preservatives are natural or chemical components used to maintain shelf life of foods.Ê An interest was raised as to how different preservatives affect the breakdown of green beans.Ê During this investigation, the Iodine test, Selivanoffâs test, and Barfoedâs test were applied to four treatments of green beans.Ê Treatments included green beans in each of the following solutions: citric acid, sodium chloride, and EDTA.Ê The Iodine test confirmed starch was not present in all treatments.Ê Selivanoffâs test verified green beans with and without preservatives contained monosaccharide ketoses.Ê Barfoedâs test found green beans containing no preservatives had the greatest amount of monosaccharides present.Ê Citric acid and EDTA solutions produced the second and third largest amounts of monosaccharides.Ê Sodium chloride formed a precipitate that was inconclusive for monosaccharides.Ê Next, four treatments were placed into a spectrophotometer and an absorption spectrum was plotted.Ê All treatments had similar absorbencies that were high in the violet region then decreased across the spectrum.Ê Green beans without preservatives, however, presented a peak in the red region. ÊLastly, salivary amylase was applied to four treatments, and Selivanoffâs and Barfoedâs tests were reused.Ê Results were expected to be similar to those of the original carbohydrate tests.Ê Selivanoffâs test found results matched those of the initial test.Ê Barfoedâs test found citric acid and sodium chloride produced a greater amount of precipitate after the addition of salivary amylase.Ê Green beans treated with EDTA and no preservatives formed fewer monsaccharides than their initial tests. ÊThese investigations confirmed EDTA was the best preservative tested.

Discussion

The purpose of the investigation was to determine which preservative best prevented decomposition in green beans.Ê The preservatives tested were citric acid, sodium chloride, and EDTA.Ê It was hypothesized that EDTA would be the best overall preservative.Ê This was predicted because EDTA is a chelating agent that attaches to trace elements of metals such as copper, iron, and cobalt and causes these metals to become chemically inactive (Benarde, 1971).Ê In addition, previous studies have shown that chelating agents, such as EDTA, are effective in preventing bacteria and yeast from decomposing food (Brul and Coote, 1999).Ê A series of tests, including sugar analysis and absorption spectrum tests, were performed on four treatments with three replications of each to confirm this hypothesis.Ê These treatments included green beans containing no preservatives and green beans preserved with citric acid, sodium chloride, and EDTA.

ÊÊÊÊÊÊÊÊÊÊÊ In the analysis of sugars present in green beans, the Iodine test, Selivanoffâs test, and Barfoedâs test were carried out on each of the four treatments.Ê The Iodine test was expected to confirm that all treatments and their replications contained starch based on the idea that plants manufacture and store starch for cellular fuel (Margareta et al., 1994).Ê In the course of experimentation, all samples of green beans were concluded to have a negative result for starch.Ê Although starch is created and stored in the plantâs tissue, the starch content is absent in the green bean, which is the seed of the plant.Ê Green bean plants are known chemically to contain starch because they are sugar producing plants.Ê This amount of starch may have been negligible in the green bean treatments because the preservatives used were not efficient in stopping the decomposition of the long starch chains.Ê Starch, the positive control, reacted with I₂KI to produce a bluish black color indicating the presence of starch.Ê Distilled water, the negative control, did not result in this color.Ê These controls indicated the I₂KI reagent was functioning properly.Ê

ÊÊÊÊÊÊÊÊÊÊÊ To differentiate between ketoses and aldoses, Selivanoffâs test was used.Ê Galactose, a sugar found in green beans, is composed of aldoses.Ê When green beans undergo reactions, such as decomposition, galactose becomes diminished (Margareta et al., 1994).Ê It was predicted that green beans containing preservatives would test positive for aldoses, while green beans containing no preservatives would be comprised of ketoses.Ê This prediction was not validated because all samples tested presented a reddish color change within 30 seconds indicating the presence of monosaccharide ketoses.Ê The results showed the majority of the concentration to be ketoses.Ê This, however, did not suggest that aldoses were not present in the green bean solutions.Ê It did imply that the concentration of ketoses was larger than that of aldoses.Ê Fructose was used as the positive control and displayed a reddish color within 30 seconds indicating it was a monosaccharide ketose.Ê The negative control, distilled water, resulted in no color change within 2 minutes.Ê This information verified that Selivanoffâs reagent was working correctly.

Barfoedâs test was used to distinguish monosaccharides from di- and polysaccharides.Ê The assumption was that with added preservatives, the green beans would be slowed or stopped in the decomposing stage, keeping the specimen comprised of di- and polysaccharides versus monosaccharide sugars.Ê It was anticipated that green beans in EDTA solution would contain the fewest monosaccharides.Ê EDTA is a chelating agent and binds with elements, such as copper, thus rendering them inactive and unable to react with monosaccharides (Benarde, 1971).Ê Barfoedâs reagent contains copper ions that EDTA would adhere to and not allow to react with monosaccharides. ÊHowever, through results of the experiment it was concluded that green beans preserved with EDTA produced the second lowest amount of monosaccharide precipitate with 0.007 grams.Ê The concentration of EDTA in the solution used was 13.4%, while the favored concentration to use was 26.9%.Ê Overall, the solution of EDTA was not as concentrated as preferred for best use as a preservative.

The green beans preserved with sodium chloride were anticipated to contain the second smallest amount of monosaccharide precipitate.Ê This was expected because sodium chloride depletes moisture from food, thus reducing bacteriaâs ability to attack due to high moisture content (Benarde, 1971).Ê In the findings of the experiment, it was shown that sodium chloride actually contained the least average amount of precipitate with 0.0037 grams.Ê This precipitate, however, was a bluish green color and not the reddish color that indicates monosaccharides.Ê An additional side reaction between negligible amounts of sodium chloride and Barfoedâs reagent may have produced this precipitate.Ê This data was unable to be compared with the results from the different treatments because of the discoloration of the precipitate.Ê

Citric acid was hypothesized to contain the third largest average amount of monosaccharide precipitate.ÊÊ The reasoning behind this assumption was that when citric acid is used alone it has little effect as a preservative.Ê The products will brown after one day (Pintauro, 1974).Ê This hypothesis was confirmed because the citric acid precipitate weighed 0.009 grams, making it the third largest average amount of monosaccharides present.Ê

Green beans containing no preservatives were expected to have the greatest average amount of monosaccharide precipitate because there is no natural or chemical preservative to protect it from spoiling.Ê The average amount of monosaccharide precipitate found for this treatment was 0.0127 grams.Ê This data supported our hypothesis of green beans without preservatives would produce the largest average amount of monosaccharides.Ê Glucose, the positive control, formed a red precipitate verifying it was a monosaccharide.Ê Distilled water, the negative control, did not form this precipitate and confirmed that Barfoedâs reagent was functioning properly

The next assay performed involved placing four treatments and their replications into a spectrophotometer and an absorption spectrum plotted.Ê Rotted green beans without preservatives were expected to be a brown color because of the destruction of chlorophyll resulting from chemical changes (Pintauro, 1974).Ê These green beans were hypothesized to have low absorptions in the red, yellow, orange, green, and blue regions resulting in the brown color.Ê The spectrum for green beans in citric acid was expected to show absorptions in the same regions as green beans without preservatives.Ê This is because citric acid alone is inefficient as a preservative and that products are expected to brown after one day (Pintauro, 1974).Ê The only exception was the absorption levels for the corresponding colors were not as low as the absorption levels for those same colors in green beans not containing preservatives.Ê Green beans in sodium chloride and EDTA solution were anticipated to have absorbencies in the red and blue regions, which resulted in the reflected color green to be seen.Ê This was predicted because sodium chloride and EDTA hinder the chemical changes that result in the destruction of chlorophyll (Pintauro, 1974).Ê Our research showed that green bean solutions treated with preservatives had high absorbencies in the violet and blue regions of the spectrum.Ê These absorbencies then steadily decreased through the green, yellow, and orange regions.Ê They continued to gradually decrease through the red region of the spectrum as well.Ê The treatments containing no preservatives also showed high absorbencies in the violet and blue regions of the spectrum.Ê Likewise, these absorbencies steadily decreased from the green to orange region.Ê However, in the red region, the absorbency slightly increased to form a peak and then steadily decreased.Ê This data suggested that preservatives affected absorbency in the red region of the spectrum.Ê Preservatives absorbed within the green beans may have contained chemicals that reflected light within this region.Ê Also, the spectrophotometers used may have resulted in inaccurate absorbencies.Ê These machines were not as reliable as preferred because they are of an older technology.Ê

ÊÊÊÊÊÊÊÊÊÊÊ Lastly, Selivanoffâs test and Barfoedâs test were applied to four treatments which contained the addition of salivary amylase.Ê The predicted results for the reapplication of Selivanoffâs test was expected to show similar results to those hypothesized for that original sugar analysis test (Margareta et al., 1994).Ê This was expected because salivary amylase is effective in breaking down macromolecules but not changing the structures (Kulp, 1975).Ê It was found that all treatments and their replications reacted to give a red color within 1 minute.Ê This indicated the presence of monosaccharide ketoses.Ê Even though these outcomes showed the concentrations to be mainly comprised of ketoses, this however did not suggest that aldoses were not present in the treatments.Ê The concentration of ketoses was shown to be greater than that of aldoses.Ê The positive control, fructose, presented a reddish color change within 30 seconds while the negative control, distilled water, did not indicate any color change within 2 minutes.Ê This confirmed that Selivanoffâs reagent was working adequately.

ÊÊÊÊÊÊÊÊÊÊÊ Barfoedâs test was anticipated to show the same results that were predicted for that original sugar analysis test.Ê However, a greater amount of monosaccharides were expected to be produced for each of the four treatments.Ê This was due to the catalytic effect that salivary amylase plays on the breakdown of green beans (Kulp, 1975).Ê The greater the presence of monosaccharide precipitate signifies the preservative to be less efficient at maintaining the di- and polysaccharide structures.Ê Our experimental data discovered that green beans preserved in citric acid produced the largest average amount of monosaccharide precipitate with 0.0127 grams.Ê Natural lemon juice contains trace amounts of sugar (Anonymous, 2004).Ê The lemon juice that was applied to the green beans may not have been completely removed before being tested, which may have added to the amount of precipitate formed.Ê When this data was compared to the results of the original Barfoedâs test, it was found that citric acid preserved green beans treated with salivary amylase produced greater amounts of monosaccharides.Ê This confirmed our hypothesis.

ÊÊÊÊÊÊÊÊÊÊÊ Green beans preserved with sodium chloride produced 0.010 grams, making it the second largest average amount of precipitate found.Ê However, the precipitate found was to be of a bluish green color and not the expected red color which indicates monosaccharides.Ê This precipitate could have resulted from a possible side reaction between traces of sodium chloride in the samples and Barfoedâs reagent.Ê When this amount of precipitate was compared to that of the original sugar analysis, it was found that this sample treated with salivary amylase contained larger amounts of precipitate, which was expected.Ê Overall, this data could not be compared with the results of different treatments because of the discoloration of the precipitate.Ê

ÊÊÊÊÊÊÊÊÊÊÊ The treatments containing no preservatives were discovered to contain the third largest average amount of monosaccharide precipitate with 0.009 grams.Ê This value was also less than the amount of monosaccharide precipitate produced in the original Barfoedâs test.Ê Of these results, neither was expected.Ê The freshness of the green beans tested was not as consistent as preferred.Ê The green beans used in the original sugar analysis test had a presence of brown spots on their surface.Ê This indicated they were further along in decomposition than those used in the independent study.Ê Due to this additional breakdown of the green beans used in the original Barfoedâs test, more monosaccharides may have been produced from that experiment.

ÊÊÊÊÊÊÊÊÊÊÊ EDTA preserved green beans contained the smallest average amount of precipitate with 0.006 grams, which was hypothesized.Ê However, when compared to the original Barfoedâs test, these samples treated with salivary amylase contained fewer monosaccharides.Ê The salivary amylase may have been greatly diluted by the EDTA solution making it less efficient in catalyzing the breakdown of the green beans.Ê This also could have occurred in the citric acid treatments but our data suggests this may have not happened.Ê The reaction of Barfoedâs reagent with the positive control, glucose, formed a red precipitate, confirming monosaccharides were present.Ê The negative control, distilled water, produced no red precipitate.Ê This validated that Barfoedâs reagent was functioning correctly.

Overall, the Barfoedâs tests, with and without salivary amylase, were most useful in concluding that EDTA was the best preservative studied.Ê For both of these experiments, EDTA preserved green beans produced the fewest amount of monosaccharides during the time frame allowed for decomposition.Ê Data from the Iodine test concluded that none of the green bean treatments tested positive for a presence of starch.Ê Selivanoffâs tests, with and without salivary amylase, determined that all samples of green beans were mainly comprised of monosaccharide ketoses.Ê The absorption spectrum showed that all green bean treatments had high absorbencies in the violet and blue regions which steadily decreased through the green, yellow, and orange regions.Ê In the red region of the spectrum, treatments containing no preservatives slightly increased to form a peak and then slightly decreased.Ê Treatments containing preservatives continued to gradually decrease in this region.Ê A possible factor that could have altered results was that the temperature the green beans were stored at may not have been constant. ÊAlso, measurement errors using pipettes and scales could have caused discrepancies in our data.Ê Extensions of this experiment would include changing different variables.Ê These include changes in preservatives used, the amount of preservatives, and different combinations of preservatives.Ê In addition, tests could be done on the nutritional content of green beans, such as vitamins and minerals.Ê Finally, another approach to extending the experiment would be to vary the temperature and time the green bean sample were left to decay.Ê

Fig. 8.Ê Comparison of monosaccharide precipitates with salivary amylase to those without salivary amylase.Ê The precipitates massed from the original Barfoedâs tests and the final Barfoedâs tests were compared to determine if the presence or absence of salivary amylase had an impact on the amount of precipitate created.Ê It was found that the citric acid solutions and sodium chloride solutions treated with salivary amylase formed a larger amount of monosaccharide precipitate compared to treatments in the original Barfoedâs test.Ê But sodium chloride cannot be compared with the other three treatments because its precipitate was bluish green and cannot be verified as a monosaccharide.Ê The green bean treatments with no preservatives and EDTA formed more monosaccharide precipitates than the ones treated with salivary amylase.Ê