PHAT FOUR - PINEAPPLE RESEARCH

The Molecular Differences found between two Ananus comosus
by Carbohydrate, Photosynthetic, and Enzyme Assays.

By: THE PHAT FOUR

Carrie Barton
Meggan Johnson
Benjamin Turnwald

Abstract
Is there a difference at the molecular level between the Smooth Cayenne and the Golden Supreme pineapple? Our hypothesis was that there would be a difference in the pineapples. The first tests we performed were sugar tests. Benedict's test served in identifying whether the pineapples were composed of free aldehyde or ketone groups. Both pineapples had aldehyde groups present. Barfoed's test was similar, it showed whether the two samples were made up of monosaccharides or polysaccharides, in our case we found monosaccharides. Selivanoff's test distinguished whether the sugars present were ketose's or aldose's. Our data indicated that both pineapples contained sugars that were ketoses. Bial's Test assayed for a furanose ring, which was present in both. We also did a taste test. For all of the sugar tests, the two pineapples presented similar data but with minor alterations. The second group of tests showed the absorption spectrum of the chloroplasts found in the leaves of the two samples and the specific chloroplasts present. The Golden Supreme had a higher absorption rate than the Smooth Cayenne. Both pineapples had carotene, xanthophylls, chlorophyll a, and chlorophyll b in different concentrations. We also tested pH, which resulted slightly differently. We tested for the presence of polyphenoloxidase (PPO) that came out negative for both. Since our enzyme test was negative, we did a backup PPO inhibitor test that was positive for both pineapples. The results of these experiments support a noticeable difference, on a molecular level, between both varieties of pineapples.

Discussion
Is there a difference at the molecular level between the Smooth Cayenne and Golden Supreme pineapple? We predicted that there would be differences. If two different varieties of pineapple are tested for molecular differences then significant differences will be found. This research did support our hypothesis. Although for some of the tests the results were similar, for many the two varieties had obvious differences.
Let us first discuss the carbohydrate tests. Benedict's test assayed solutions for the presence of reducing sugars. Benedict's test expressed results that were similar. Both varieties and all concentrations yielded a copper precipitate indicating the presence of reducing sugars. However, the Golden Supreme solutions turned a greenish blue color while the Smooth Cayenne solutions stayed a blue color. This can be simply explained. Pineapple is a sweet fruit and it contains sugar. The green/blue color differences can be explained by the difference in pH between the two varieties. The more acidic Golden Supreme could have caused the solution to become greener at some point during the reactions. The second test, Barfoed's Test, tested for reducing sugars that were monosaccharides. The solutions of Smooth Cayenne expressed a slight precipitate along with the control group. The Golden Supreme solutions expressed slightly more precipitate. The Golden Supreme could have produced more precipitate because monosaccharides are more concentrated in that variety. This makes sense because the Golden Supreme is sweeter than the Smooth Cayenne (Dole Food Company, 2002). Selivanoff's test was performed next to differentiate between ketose groups and aldose groups. All of the concentrations of both varieties reacted under a minute, indicating the presence of a ketose group. The 15% concentration of the Golden Supreme reacted under a minute, but turned a lighter red. Only after sitting for a few minutes did it turn a dark red. The lighter red color could be possible because the Golden Supreme contains more aldose groups than Smooth Cayenne. There could have been plenty of ketose groups in the 25% dilution for the full red color to appear at around 30 seconds. That would also explain why the solutions turned dark red after a few minutes. The next carbohydrate test that was performed was Bial's test, which tests for the presence of a furanose ring. All of the concentrations of the two varieties and the control turned dark olive brown. That indicated the presence of a furanose ring. Since furanose is a sugar, this is not surprising. The last sugar test that we performed was a "taste test". The results were pretty clear that the Golden Supreme is considered "sweeter". This is because the Golden Supreme has a lower acid content (Dole Food Company, 2002). The carbohydrate tests all indicated the same conclusions and only varied slightly in color, if at all. The results of these tests, therefore, do not support our hypothesis.
The photosynthesis tests were performed next. The absorption spectrum tests yielded some slight differences. The two trials of each variety were unsurprisingly identical. However, the chloroplasts in the Golden Supreme appeared to absorb more light then the Smooth Cayenne. As stated before Golden Supreme is a sweeter fruit. As the plant absorbs more light, more sugar will be produced (Campbell and Reece, 2002). Therefore, these results make perfect sense. The paper chromatography tests yielded pretty consistent results. The Rf values were all about the same except for one Smooth Cayenne strip that expressed higher results. All strips expressed 4 color bands, which indicated the presence of carotene, xanthophyll, chlorophyll a and chlorophyll b (Maleszewski et al, 2002). The higher bands on the one Smooth Cayenne strip may possibly be an error. Ideally, we would have liked to do this test again to have more trials to compare, but since it is lengthy, time constraints did not allow it. The results from the photosynthesis tests were only slightly different. We found that Golden Supreme leaves appear to absorb more light because of the pigments present, but we cannot come to a conclusion of the comparison of Rf values due to inconsistency. The evidence that one leaf absorbs more light than another supports our hypothesis.
The enzyme tests were the last tests to be performed. These tests provided little results due to complications that will be discussed later. However, in a comparative pH test, the Golden Supreme tested more acidic. We expected Golden Supreme to be more acidic (Dole Food Company, 2002). This supported our hypothesis. The next test to be performed was for the presence of PPO. PPO was not found to be present. We expected PPO to be present in pineapples. We found research that claims that PPO is responsible for "blackheart injury", which is discoloration of the pulp days after harvest (Lubulwa et al, 1995). We also found research that suggests that pineapple contains a "nonvolatile organic acid" that inhibits the enzyme PPO (Wen and Wrolstad, 1999). The results from the next test support the inhibition hypothesis. We tested to see if PPO was inhibited by putting pineapple stock solution on potatoes (a PPO containing organism). The results were clear. The control group turned dark brown and the two groups with pineapple stock solutions on them did not experience any change. Therefore, our results support that pineapple flesh contains PPO inhibitors.
After completing this research, we realized that we had problems and many weaknesses in our experimental design. The sugar tests gave us clear answers for questions like, "Does this fruit contain ketose or aldose?" however; all of the results were different in a slight way. For example, in Barfoed's test, both varieties produced precipitate, indicating the presence of monosaccharides. The Golden Supreme varieties, however, produced more precipitate. What does that mean for sure? As stated before, we think that it means that more monosaccharides are present. How we could find this out for sure is by running more detailed carbohydrate tests. One problem that we had in performing the carbohydrate tests was the dilutions. We had originally planned on testing dilutions of 25%, 50%, and 75%, but the 50% and 75% dilutions contained too much pulp to measure accurate amounts with the pipettes. So, the dilutions that we ended up testing were 15% and 25%. What we could have done was filter the 50% and 75% dilutions through cheesecloth to filter out the pulp. The photosynthesis results, particularly the absorption spectrum test, provided some good contrasting results. If we could do the photosynthesis tests again we would add some more tests regarding light absorption to try and find more supporting evidence of differences between the two varieties. We obviously had problems with the enzyme tests since our pineapples did not test positive for PPO. We had planned on the testing the effects of heat and pH on the pineapple flesh. So, if we could do those experiments again, we would come up with better-planned PPO inhibition tests.
Many errors could have been made throughout the research as well. Since the research was done over seven weeks, the pineapples could have been acquired at different times of the growing season, which may alter the fruit. Also, we did not know when the pineapples had been harvested each time we purchased them. The Golden Supreme could have been harvested for 10 days at the time of the tests and the Smooth Cayenne, 5. That difference in harvest date could have affected the results of the tests. Another error that could have been made was with the test tubes. We at the beginning of the experiment did not wash the test tubes used. It was assumed that the previous group using them had properly washed them. If some tubes were not properly washed the material left in the tubes could have affected our results. With any of the tests, human error could have been possible and samples been mixed up. Although unlikely, it is possible.
In conclusion, our results support our hypothesis that if two different varieties of pineapple are tested for molecular differences then significant differences will be found. The sugar tests all showed that both varieties contained the same types of sugars. However, the appearances of the two varieties after undergoing the tests were clearly different. The absorption spectrum test also showed clear differences in light absorption between the two varieties. The pH values were different for the two varieties. Although the pigment test and the enzyme tests were similar, we feel like our results express enough evidence to support our hypothesis.

FIGURE 1: The absorption spectrum of chloroplasts

This figure shows the difference in absorption of chloroplasts between the leaves of the Smooth Cayenne and the Golden Supreme pineapples. The top black and pink lines indicate the absorbance of the Golden Supreme through two different trials. The yellow and green lines represent the Smooth Cayenne. The Golden Supreme had a higher absorbency rate than that of the Smooth Cayenne.

References

Dole Food Company, 2002. Pineapples. Available: http://www.dole5aday.com/FoodService/pdfs/Factsheet_Pineapples.pdf. Accessed: Oct 6, 2002.

Lubulwa, G., Underhill, S. and Davis, J. 1995. Pineapple Quality Improvement. Available: www.aciar.gov.au/downloads/publications/Workingpapers/wp20/wp20.doc. Accessed: Oct 6, 2002.

Wen, L. and Wrolstad, R.E. 1999. Characterization of enzymatic browning inhibitors in pineapple juice. Available: http://www.confex.com/ift/99annual/abstracts/3903.htm. Accessed: Oct 6, 2002.