Carbohydrate, Bacteria, and Various Other Tests Reveal Only Minute Differences In Organic and Conventionally Grown Fruits
By:
The Luckie Charms
Kelly Eggan
Kayla Raak
Jeremy Speight
Sarah Warner
LBS 145 Cell and Molecular Biology
Sunday 6:00-9:00pm
Dr. Douglas Luckie
March 4th, 2005
Abstract:
Revised for Final Draft by Jeremy Speight
Our group conducted experiments in order to determine a selection of the differences between organically and conventionally grown fruits. We tested for differences in; types of carbohydrates, concentration of protein, photosynthetic pigments, amount of vitamin C, and the amount of bacteria. From each group, we tested oranges, grapefruits, lemons, and limes. We used Benedict’s test and found that all of the juices contained free aldehydes or ketones. We then performed Barfoed’s test and found that they all contained monosaccharides. We used Selivanoff’s test and found that organic fruits contain monosaccharide ketoses, while conventionally grown fruits contain disaccharide ketoses. Using Bial’s we found that there was no difference in ring structures between the two growth types. We then conducted the Iodine test and found that no starch was present in either growth type. Our group then performed a Bradford Assay on the fruits and found no discernable difference in protein concentrations between the two groups. We also tested for a difference in the photosynthetic pigments using paper chromatography and we found no substantial differences in the types of pigments present. We then performed a vitamin C test and found that neither growth type contained more vitamin C than the other growth type. Using our results from the aforementioned tests, we found there to be no substantial differences between organic and conventionally grown fruit.
Figure 3: This is a picture of our results from the Selivanoff’s test. The solutions are in this order read from left to right: Organic lime, organic orange, organic lemon, organic grapefruit, xylose (positive control), water (negative control), conventional lime, conventional orange, conventional lemon, conventional grapefruit. The results of this test are determined by the time in which they change from a clear color to a dark red color, as seen in the picture above. The organic fruits changed around 50 seconds with a red color change. This helped to show the presence of ketoses. Whereas the conventional ones changed around 1minute and 45 seconds or didn’t have a color change at all. This showed that the solution contained aldoses.
Discussion:
Revised for Final Draft by Kelly Eggan
By comparing prices of organically grown fruits at Foods for Living ( 2655 E. Grand River Ave. in East Lansing Michigan) and conventionally grown fruits from Meijer (2055 W Grand River Ave in Okemos Michigan) we realized that organic fruits cost, on average, $0.50 more than conventional fruits. We decided to perform various experiments on organically and conventionally grown citric fruits to see if there was a significantly large difference between the two production types. After the experiments were complete we could either suggest that one could save his or her money by buying conventional fruits or we could suggest that one should eat healthier by purchasing organic fruits.
We studied the properties of four citric fruits: grapefruits, lemons, limes and oranges, to see whether or not there were differences in carbohydrate and protein composition, pigments, and the amounts of vitamin C and bacteria contained in each production type. To decipher if organic fruits are different from conventionally grown fruits, we performed numerous experiments on each fruit, from each production type. These experiments included a Benedict, Selivanoff’s, Barfoed’s, Bial’s, Iodine, Bradford, paper chromatography, vitamin C, and bacteria test. The Benedict, Selivanoff’s, Barfoed’s, Iodine, and Bial’s experiments were performed to test for the presence of different carbohydrates. The Bradford assay determined protein concentration. We performed a paper chromatography experiment to see if there was a difference in pigments between the production types. Although most of the photosynthetic processes are carried out in leaves, we thought some of the photosynthesis would be carried out on the fruit itself. We used paper chromatography to determine a difference in the type of pigments present in the chloroplast, such as carotene, xanthophyll, chlorophyll a and chlorophyll b. We ran vitamin C and bacteria tests because we thought they would be particularly interesting. We believed the peels of organically grown fruits would contain a lesser coating of chemicals and will therefore absorb more sunlight, carbon dioxide and nutrients needed for growth. Also the soil for organic fruits will have fewer chemicals, and will provide a better breeding ground for the fruits. Because of these assumptions we hypothesized that the photosynthesis process would be greater in organic fruits compared to fruits grown from a conventional method. We therefore predicted that organic fruits would have different types of carbohydrates, more protein, photosynthetic pigments, and more vitamin C than conventional fruits. Because pesticides were not used on organic fruits, we believed that the amount of bacteria would be higher on organic fruit than on conventionally produced fruit.
The first experiment we ran on the fruit was called the Benedict’s test. This test is used to determine whether or not the carbohydrates present in the fruit contain free aldehyde and ketone groups. In the reaction, free aldehydes and ketones are present if copper is reduced and a red precipitate is formed. (Krha et al, 2004) We predicted that the precipitate would form in every fruit, both organically and conventionally grown, but the amount of precipitate would vary. We believe that organically grown fruits would produce more precipitate; meaning organic fruits would contain more free aldehyde or ketone groups than conventional fruits. The positive control for this experiment was xylose. Xylose produced a very dark red precipitate. This indicated many free ketone or aldehyde groups. The negative control, distilled water, produced no color change, meaning no free aldehyde or ketone groups are present in distilled water. The organic oranges and organic grapefruits formed a dark red precipitate, which denotes positive results for free aldehyde and ketone groups. The conventional oranges and grapefruits formed rusty orange/red precipitate which suggested that there was a lower concentration of free aldehyde and ketone groups present in conventional oranges and grapefruits than in organic fruits. The lemons, both conventionally and organically grown, showed no changes in color; therefore our lemons seemed to have no or very little amounts of free aldehyde or ketone groups. When Benedict’s reagent was added to both conventional and organic limes we noticed a very slight rust color. This led us to believe that limes contain a very small amount of free aldehyde or ketone groups. After the entire experiment our group concluded that due to the darker red color, organic oranges and grapefruits seem to have a higher amount of free aldehyde and ketone groups than conventional oranges and grapefruits. This supported our hypothesis stated earlier that organic fruits contain higher amounts of free aldehyde and ketone groups. Since both lemons and limes of organically and conventionally grown fruits showed such little changes in colors, our hypothesis could neither support nor refute the higher percentages of free aldehyde or ketone groups in these fruits. We are led to believe that lemons and limes contain little or no free aldehyde or ketone groups.
Barfoed’s test is used to distinguish between mono-, di-, and polysaccharides using lower pH conditions and shorter incubation time. Monosaccharides are the only saccharides that can react fast enough to reduce copper ions. (Krha et. al, 2005) If a monosaccharide is present, a rusty or brownish colored precipitate will form whereas di- and polysaccharides will either produce a slight color change or no color change at all. (Krha et. al, 2005) Before performing this experiment our group had tasted both organic and conventional grapefruits, lemons, limes, and oranges. Organic fruits tended to taste sweeter than conventional fruits. Glucose and fructose, both monosaccharides, were common sugars known to taste sweet. We hypothesized that the organic fruits would contain more monosaccharides than the conventional fruit. The positive control for this experiment was xylose. Xylose produced a very dark rust colored precipitate. The negative control, distilled water produced no color change. Distilled water does not contain monosaccharides. We also noticed that organic oranges and grapefruits showed dark rust colored precipitate. Conventional oranges and grapefruits produced a rust color slightly lighter than the color produced by the organic oranges and grapefruits. We believe this indicates different monosaccharides in conventional oranges and grapefruit compared to organic oranges and grapefruits. The lemons and limes of both conventional and organic types showed no color change. We believe this indicates that lemons and limes do not contain monosaccharides, or possibly too little an amount of monosaccharides to indicate a color change. It makes sense that lemons and limes lack monosaccharides because both lemons and limes taste bitter rather than sweet.
Another experiment we preformed on the fruit was the Selivanoff’s test. This test is helpful in differentiating ketoses and aldoses. A red color will appear if ketoses or aldoses are detected. Ketoses react more readily than aldoses. (Krha et al, 2005) To distinguish if ketones or aldoses are presents, we timed how long it took for the solution to change to a red color. If a red color appears within one minute, ketoses are present. If a red color appears after a few minutes, aldoses are present. We hypothesized that there was to be a difference between the conventional and organic fruits. We believed that either organic fruits contained ketoses and conventional fruits contained aldoses, or vice versa. Xylose, our positive control produced a very dark red precipitate within 30 seconds. This indicated the presence of ketones. The negative control, distilled water produced no color change, even after 10 minutes. This means distilled water does not contain either aldehydes or ketoses. The organic oranges turned red after about 50 seconds. This indicated that oranges contained ketoses. The organic lemons turned red after about one minute and fifteen seconds. Although this was barely over one minute we figured this meant the lemons contain aldoses, because ketoses indicate a red color almost immediately, usually less than one minute. The organic limes turned red after about one minute and 20 seconds. This timing was much like the lemons. The limes also contain ketoses. The organic grapefruit juices turned red, at about one minute. Organic grapefruits contain ketoses as well. The conventionally grown oranges turned red around one minute. This means that conventional oranges must contain ketoses. We found that conventional lemons took around one minute and 40 seconds to turn red. This is a longer time frame that organic lemons, but it still indicates aldoses. The same applied for conventional limes. It took each lime around one minute and 45 seconds. The conventional limes, as well as organic limes, both indicate aldoses. The conventional grapefruits turned a red color after one minute. This indicates that conventional grapefruits contain ketoses. To sum this up both conventional and organic oranges and grapefruits contain ketoses while both organic and conventional lemons and limes contain aldoses. From the information we have collected there is no difference in the types of sugars; ketoses or aldoses; among the different production types. Our hypothesis was wrong.
Another carbohydrate experiment we preformed was the Bial’s test. This test helped to show the presence of either a five or six carbon ring structure. (Pentose furanose or hexose furanose, respectively). Pentose furanose rings react with Bial’s solution to produce a green color. Hexose furanoses react with Bial’s solution to produce an olive brown color. (Krha et al, 2005) We predicted that the different fruits would contain different types of rings. Either organic fruits would contain pentose furanoses and conventional fruits would contain hexose furanoses or vice versa. Our positive control was sucrose. Sucrose produced a very dark olive precipitate indicating that it contained hexose furanose rings. The negative control, distilled water produced no color change, indicating that distilled water has neither hexose- nor pentose- furanoses. After testing oranges and grapefruits, both organic and conventional, we noticed that an olive color appeared. This means that both conventional and organic oranges and grapefruits contain hexose furanose rings. The lemons and limes of both organic and conventional types turned a light green color denoting pentose furanoses. With these results it is suggested that there is no difference in the types of carbon rings in organic and conventional fruits. Our hypothesis was wrong.
One important test used to observe the presence of starch was the Iodine test. A blackish-blue color change indicates a presence of starch. (Krha et al, 2004) Our group hypothesized that both the organic and conventional fruits would show a bluish color change but organic fruit would present a darker blue, almost black color, indicating more starch. Our positive control was potato extract. Once Iodine was added to the potato mixture, it immediately produced a black precipitate, indicating a large amount of starch. The negative control, distilled water produced no color change, indicating that distilled water does not contain starch. Each fruit was tested and each fruit turned a yellow color, meaning starch is most likely not present in any of the fruits. Our hypothesis was wrong.
We used the Bradford assay to test for the amount of protein found in each fruit. Our group believed that organic fruits would contain more protein because the chemicals used during conventional growth may decrease the protein content. For this test we took the absorbance of each fruit juice. Our positive control was Bradford assay solution. The absorbency at 595 nanometers was 2.065. This indicated a very high concentration of protein. The negative control, distilled water gave an absorbency of 0.0 nanometers. This means that distilled water does not contain protein. The organic orange showed an absorbance reading of 0.008 nanometers (nm). Organic grapefruit had an absorbency reading of .01nm. The organic lemon had a slightly higher absorbency than the grapefruit with a reading of 0.03nm. The organic lime had the highest organic absorbency with an absorbance reading of 0.06nm. All of the absorbency readings for organic fruits were very low. Since all of the absorbencies were well below 1.0nm we assume that the organic fruits tested had a minuscule amount of protein. One nm usually indicates a substantial amount of protein. Conventional fruits showed an even lower absorbency than organic fruits. The absorbencies of the conventional fruits were negative. The conventional orange and lemon had the lowest absorbencies with readings of -0.06nm each. The conventional grapefruit absorbency read -0.05nm and the conventional lime had an absorbency of -0.02nm. The standard curve we created using Bradford reagent was much too large to compare the absorbency of the fruits. This indicated that the amount of protein in each fruit was too small to properly calculate. Organic fruits gave a slightly higher absorbency then did conventional fruits, therefore organic fruits could contain more protein, but since a proper standard curve could not be calculated, we cannot determine if that statement is entirely true. Both conventional and organic fruits contained very small traces of protein. There was a very small difference between conventional and organic fruits, therefore our hypothesis was wrong.
The next topic that our group experimented on was the pigments present on the peels of each fruit. We hypothesized that there will be a difference in pigments located in the peels. Because organic fruits do not contain a coating of chemicals, unlike conventional fruits, organic fruits may have different pigments. We tested for differences in pigments with the use of the fruit peels, turned into a solution. We used paper chromatography because our fruits are not green. Paper chromatography is the best pigment test to use with colors other than green. Our positive control was spinach leaves. Four distinct colors were shown on the filter paper containing the spinach leaf juice: orange yellow, pale yellow, blue-green, and pale green. These colors represent a presence of carotene, xanthophyll, chlorophyll a and chlorophyll b respectively. (Krha et al, 2004) The Rf values were calculated for the positive control. They were as follows: 115/172, 18/23, 37/76, and 71/76 for carotene, xanthophyll, chlorophyll a and chlorophyll b respectively. The negative control was distilled water. No color change appeared on the filter paper, indicating that photosynthesis does not occur in distilled water. The paper chromatography did not work for any of our fruits. No color appeared on the filter paper after running the experiment. Testing for pigments is best when using green leaves. Since our fruits lacked these leaves, we had to use peels, which did not give accurate results. We can neither support nor refute our hypothesis that organic fruits have greater pigments than conventional fruits.
Vitamin C was also an important test in our research. After tasting organic and conventional grapefruits, lemons, limes and oranges, we noticed a sweeter taste in the organic fruits. We hypothesized that the sweeter taste may indicate a more sugars and therefore a higher level of vitamin C. A pink color change of fruit solution brought on by the addition of indophenol indicated the presence of vitamin C contained in each fruit. (Alpert, unknown) The positive control for vitamin C was a 1000mg vitamin C tab, distributed by the company “Origin.” Only 4 drops of indophenol produced a red color on the tab. The negative control was distilled water. Indophenol did not change the color of distilled water; meaning distilled water does not contain vitamin C. The organic oranges needed, on average, about 10 drops of indophenol to change to a pink color. The organic grapefruits averaged 25 drops of indophenol to produce a pink hue. Organic lemons needed an average of 13 drops of indophenol and organic limes needed 29 drops of indophenol. Based on these results, oranges have the most vitamin C followed by lemons, grapefruits, and limes. Conventional oranges needed an average of 12 drops of indophenol to produce a pink hue. Conventional grapefruits needed about19 drops of indophenol for a color change. The conventional lemons needed an average of 14 drops of indophenol and the conventional limes needed 31 drops of indophenol to produce a color change. The order for highest vitamin C content in conventional fruits follows the same order of vitamin C contained in organic fruits: Oranges had the most vitamin C followed by lemons, grapefruits, and limes. There was only a small difference in the amount of indophenol used among the different fruit types. Conventional oranges, lemons, and limes needed more droplets of indophenol than organic oranges, lemons, and limes, but conventional grapefruit needed less droplets of indophenol than organic grapefruits. Based on this data we can reject our hypothesis. Organic fruits and conventional fruits probably contain equal amounts of vitamin C.
The last test conducted was a bacterium test. Before the experiment our group hypothesized that organically grown fruit would contain more bacteria than conventionally grown fruits. We came to this prediction because we know that organically grown fruits are grown without the use of any kind of chemicals, including beneficial chemicals such as antibiotics and pesticides. (Healy, 2004) Our positive control was E. coli bacteria. E. coli produced 200 colonies of bacteria. Our negative control was distilled water, which, surprisingly enough, produced one very small colony of bacteria! This means that either distilled water does contain some bacteria, or we may have accidentally exposed the plate to another source of bacteria. When testing the fruit we wiped the bacteria using two different methods. In the first method we wiped the fruit three times in the exact same spot. The second method we wiped the entire top half of the fruit peel with the swab. The organic lemon was the first fruit we tested. It produced one colony of bacteria using method one. Using method two the organic lemon produced four colonies of bacteria. Next we tested the organic lime. Using method one, 14 colonies of bacteria were produced, and using method two, only one colony of bacteria was produced. After testing organic oranges, we noticed that the first method did not produce any bacteria. Method two only produced one colony. The last organic fruit we tested was an organic grapefruit. Using method one 57 colonies of bacteria were produced. Using method two 53 colonies were produced! Next we tested conventional fruits. The conventional lemon produced 41 colonies of bacteria on method one. Method two of the conventional lemon produced only three colonies. The conventional lemon produced much more bacteria than the organic lemon in method one, but one colony less in method two. Next we analyzed the results of the conventional lime. The conventional lime produced 27 colonies in method one and 23 colonies in method two. The conventional lime contained much more bacteria than the organic lime. When we tested the conventional orange, no bacteria were produced in either method. This was very similar to the organic orange. The organic orange contained only one more colony of bacteria than the organic orange. The conventional grapefruit did not produce any bacteria also. This made us very curious, because we know bacteria is just about everywhere. How could no bacteria show up on conventional oranges and conventional grapefruits? Are group hypothesized that we may have done a number of things wrong during this experiment which will be discussed in the next paragraph. In some respects conventional fruits did contain more bacteria than organic fruits such as in limes. At the same time, some conventional fruits contained fewer bacteria colonies than organic fruits such as the grapefruit. The amount of bacteria in each sample also varied from each method. This was true among the lemons. Organic lemons showed more bacteria than conventional lemons, in method two, but in method one conventional lemons showed more bacteria than organic lemons. From this data we can reject our initial hypothesis that organic fruits would contain more bacteria than conventional fruits.
Every experiment contains a certain amount of error. In our experiments there is the possibility of much error. For example, we bought the fruits on the same days, in the same stores, in the same bins, but one store may have had their fruit on the shelves for a longer period of time. Or the fruit in each bin may have varying ages. The ages of the fruits were not exactly the same, and that could cause much error because an older fruit may lose nutrient content faster than a fresher fruit. There could have also been differences in the way the fruit was handled causing there to be more bacteria on one type of fruit or other such related differences. There is also possible error in the each experiment we run. For example the absorbance machine might not have calibrated the same each time it was ran. Also there is also the possibility of human error in every experiment. The same person did not conduct every test. There could have been slight differences in the way they were run, such as different area of the fruit being swabbed for bacteria, which could alter the outcome. There is also a lot of possible error with the use of pipettes. If the pipettes were off by even one decimal the correct amount of solution would not be used in the experiment. Bubbles form easily in the pipettes. This could cause a lesser amount of solution to be drawn up into the pipette. The Iodine tests indicated no starch in each fruit. Each fruit may have contained starch, but it was too small to show up in an iodine test. If we had known of a different method, we may have noticed a change in starch. Running a more specific paper chromatography, Bradford assay, and iodine tests, without errors, could have proven or disproved our hypothesis. The bacteria test had a lot of potential for possible error, such as open plates being exposure to air, or other surroundings, that carry bacteria. The swabs we used may not have been completely sterile. Or the interval of time used to allow bacteria to grow in the Petri dishes may not have been adequate. As always, time is a constraint. We only ran each test three times. The greater amount of tests completed gives a smaller percent error. There are always fluke incidents in which a test tube wasn’t cleaned properly or an insufficient amount of solution was made. These instances can produce values far different from the average values. With time to run only each test three times may mean a much greater percent error than an experiment run more than three times.
From the observations that our group made, it is easy to say that based on carbohydrate, paper chromatography, vitamin C and bacteria tests, organic fruits and conventionally grown fruits have many more similarities than differences. Organic fruits may vary from conventional fruits because they seem to contain different monosaccharides and ketoses than conventional fruits, because of the darker color produced with organic fruit solutions, but the shades were very similar and there were not enough trials run to get an accurate portrayal. When comparing equal fruit types, for example conventional grapefruits to organic grapefruits, conventional and organic fruits showed different results based only on the types of fruit, indicating no difference based on production type. This was true for the Benedict’s, Selivanoff’s and Bial’s tests. In the Benedict’s test both organic and conventional oranges and grapefruits indicated free aldehydes and ketones. Limes of both production types indicated different free aldehydes and ketones, and lemons of each production type seemed to show no presence of free aldehydes or ketones. Selivanoff’s test showed that oranges and grapefruits, both organically and conventionally grown, contain ketoses and lemons and limes of each production type contain aldoses. The Bial’s test was very similar in that oranges and grapefruits of each production type contained hexose furanoses and lemons and limes of each production type contained pentose furanoses. The iodine test failed for every fruit so there is no difference in the amount of starch based on production type. The Bradford Assay for protein resulted in such little protein in each fruit that no accurate comparison between conventional and organic fruits could be made. Our paper chromatography experiment indicated that organic and conventional fruits might have similar pigments. The vitamin C test also indicated a similarity. When comparing equal fruit types, for example conventional grapefruits to organic grapefruits, conventional and organic fruits seemed to need about the same amount of indophenol to indicate vitamin c content. Lastly, the bacteria test did not indicate if organic fruits have more bacteria than conventional fruits or vice versa. We believe that more trials may have indicated a difference.
Based on all of the results from our experiments, we recommend saving money and purchasing conventional fruits, at least until further research is completed, for the differences between the conventionally and organically grown fruits are minuscule.