Sugar presence and higher protein levels found by carbohydrate and protein tests cause human preference of sweet corn

By: The Kelvinators

Kristen Keck

Polina Frolov

Chuck Stahlmann

Blake Rice

Abstract

We posed the question of why humans traditionally prefer sweet corn to dent corn. We predicted different carbohydrate compositions, photosynthetic properties, and variations in protein content, although we were not sure which breed of corn would be higher and lower.

Carbohydrate tests including Benedict’s, Barfoed’s, Selivanoff’s, Bial’s, and Iodine were conducted. Benedict’s test revealed more reducing sugars in sweet corn, based on the amount of precipitate the test yielded, and Barfoed’s test indicated monosaccharide reducing sugars in sweet corn. Selvinaoff’s test indicated that sweet and dent corn contain ketoses in the form of monosaccharides. Bial’s test revealed a pentose furanose ring in sweet and a furanose configuration in dent corn. The iodine test suggested a starch content in both types of corn.

The photosynthesis tests used were paper chromatography and absorbance spectrum. The chromatography test determined pigments present in both husks and kernels. We predicted a lower pigment concentration in dent corn, supported in both the husk and kernel chromatography tests, and absorption spectrum test.

To test enzyme reactions we used the Bradford Assay and salivary amylase. The Bradford indicated a higher protein concentration in the sweet corn. The salivary amylase test combined with the Iodine test suggested higher starch content in dent corn.

These experiments supported our predictions of different sugar contents and protein concentrations in sweet and dent corn resulting in its more appealing taste to humans, although we were unable to determine which performed more photosynthesis because of the timing of the growing season.

Discussion

Traditionally, humans eat sweet corn, and give dent corn to livestock. While the exact etiology of this practice is unknown, it is clear that dent corn has a much harder and thicker endosperm than sweet corn, (Peet, 2001) which may be the reason why humans do not consume dent corn. However, we believe that the human preference of sweet corn over dent corn is for a deeper reason than the toughness of the dent corn kernel. Sweet corn is a mutant type of corn that differs from field or dent corn by a mutation at the sugary (su ) locus. The sweet corn (su) mutation causes the storage area of the seed to accumulate about two times more sugar than field corn (Schultheis, 1998). The demand on energy produced from photosynthesis increases with an increase in sugar production. We predict then, that the lower monosaccharide content, and thus less photosynthetic pigments, with a larger amount of protein in dent corn compared to sweet corn causes dent corn to have a less appealing taste.

To test our hypothesis, we ran several carbohydrate tests beginning with Benedict’s. Benedict’s test reveals the presence of reducing sugars, which contain free aldehyde or ketone groups, by the formation of a precipitate and a color change to red (Krha, 2003). Our results showed that reducing sugars are present in both sweet and dent corn. However, a larger amount of precipitate in the sweet corn solution suggests that more reducing sugars are present. This may mean that there are more complex carbohydrates, such as starch, in the dent corn because of the fewer free aldehyde or ketone groups. If this is true, it would support our hypothesis that less of the sugar present in the dent corn exists as monosaccharides. Or, the fewer reducing sugars found in the dent corn may be attributed to its lower overall sugar content. While it is evident that reducing sugars are found in both types of corn, only speculation can be made about the significance of the amount of precipitate.

To further evaluate the presence of reducing sugars that are monosaccharides in the two types of corn, Barfoed’s test was conducted. A red precipitate is a positive result for the presence of reducing sugars that are monosaccharides, and no color change represents di- or polysaccharides (Krha, 2003). Positive results for the sweet corn were obtained, further supporting our hypothesis. No color change was observed for the dent corn, although a white precipitate was formed. While the cause of the white precipitate is unknown, it is apparent from our results that reducing sugars that are monosaccharides could not be detected in the dent corn. As fructose, a monosaccharide, is the sweetest sugar (Bruice, 2001), an increased presence of fructose in sweet corn could account for a sweeter, more appealing taste, than dent corn which is more likely to contain di- or polysaccharides.

(2A) (2B)

Figure 2 Barfoed’s Test: the purpose of the Barfoed’s test is to distinguish what types of sacchrides the two types of corn contained. This test is very similar to the Benedict’s test. The solution that was initially put into the test tubes before heating was blue. Results were determined by color change. (A) Figure 2A displays our test results (n=3) for dent corn. No color change was observed for the dent corn, although a white precipitate was formed. While the cause of the white precipitate is unknown, it is apparent from our results that monosaccharides could not be detected in the dent corn. (B) Figure 2B displays our test results (n=3) for sweet corn. As you can see, the sweet corn reacted to create a great deal of red precipitate. This indicates the presence of monosacchrides .

Selivanoff’s test can differentiate between sugars. Monosaccharide ketoses change color in under a minute, disaccharide ketoses change color in approximately one minute, and aldose sugars change color after one minute. For both the sweet and dent corn, monosaccharide ketoses were detected with color changes in 22 and 26 seconds. Although monosaccharide ketoses were found in both types of corn, the quantity was not able to be determined from this test. In addition, the test did not indicate what the monosaccharide ketose was, but fructose is one possibility. Fructose is the sweetest of all sugars (Bruice, 2001), and much of the larger amount of sugar found in sweet corn (Schultheis, 1998) may be fructose, accounting for a sweeter taste.

In Bial’s test, a greenish color appears if sugar is a furanose, green/olive if the sugar is a pentose-furanose, muddy brown if it is a hexose (or higher)- furanose, or no color change if it is a pyranose (Krha, 2003). For all trials of both sweet and dent corn, an olive green color was observed indicating pentose-furanose sugars. Pentose-furanoses are five-carbon five-membered ring sugars. Ribose, a component of DNA and RNA (Bruice, 2001), is such a sugar. The pentose-furanoses detected in both types of corn may be from the sugar-phosphate backbone of DNA or RNA. Alternatively, because sweet corn is a mutation of dent corn (Schultheis, 1998), it can be assumed that many of the same types of sugars will be found in both sweet and dent corn.

Starch is a major product of photosynthesis in most plants, and we therefore expected to find starch in both types of corn. To test for the presence of starch, an Iodine test was conducted. If starch is present, the solution should turn a blue-black color. A brown color is a negative result (Krha, 2003). The dent corn solution reacted with the iodine-potassium iodide to produce a bluish-black color, signifying the presence of starch. Sweet corn, however, turned a brownish-black color perhaps indicating a small starch presence. Dent corn is commonly used for industrial purposes, like making corn starch, while sweet corn is typically consumed directly (Schultheis, 1998). While we were unable to test for the total concentration of starch, the common use of dent corn to make cornstarch suggests that there is a higher concentration of starch in the kernels of dent corn than sweet corn. Starch has a bland taste, and could contribute to the preference of sweet corn over dent corn. The sweet corn, having twice as many sugars as dent corn (Schultheis, 1998), may then be composed mostly of sweet monosaccharide sugars, such as fructose.

Sugars are a product of photosynthesis, and with sweet corn having twice as many sugars as dent corn (Schultheis, 1998), we predicted sweet corn would have more photosynthetic pigments. A xanthophyll called zeaxanthin gives corn kernels their bright yellow color (Freeman, 2002). To see if xanthophyll could be detected, paper chromatography tests on the kernels of both types of corn were conducted. After two trials of a paper chromatography test using both sweet and dent corn kernels, however, we were unable to obtain any results. An absorption spectrum analysis of both corn types was also conducted, giving inconclusive results when compared to the absorption of xanthophyll . According to our results, the kernels absorb wavelengths of 400 nm best, while the peak absorbency for xanthophyll is around 500 nm (Freeman, 2002). Although xanthophyll is known to be present in corn, it may not be readily detectable from a paper chromatography test. Either the plant leaves, or the husks covering the kernels, then, would be the expected location of most photosynthetic reactions.

Because corn leaves were not obtainable, paper chromatography tests using a husk solution were conducted using husk from both types of corn. The dent corn did not give any results, and the sweet corn gave only a faint appearance of pigments. Spinach was used as the control for the paper chromatography experiments and would have been used to compare Rf values. Spinach is a good specimen to use as a control because of its easily identifiable Rf values for all four of the pigments. The pigments for the sweet corn did not separate, and instead, the dot of solution placed on the paper enlarged to a lighter green color. The pigments did not travel up the paper, thus presenting no Rf value. Because of the time of year, the corn may have lost much of its pigment. We were unable to use freshly picked corn, and instead had to freeze the corn for several weeks. In the time since the corn had been harvested, the chloroplast may have died, causing undetectable pigments.

An absorption spectrum was then used to determine the absorption of both of the husk solutions. Spinach was used as the control for this part of the experiment as well. As with the spinach absorption, a peak absorption was found at 670 nm as well as 400 nm for the sweet corn and corresponds to the chlorophyll a, chlorophyll b, and carotenoid pigments. The dent corn, however, had a peak only at 400 nm and instead declined in absorbance as the wavelength increased. The color of the dent corn husks were a good indication that we would probably not obtain any relevant results. Because dent corn is harvested later than the sweet corn, the husks of the dent corn had lost most of its green color. The time of year, then, had a major effect on our photosynthesis results. Further photosynthesis tests from husks harvested at the same time, when both sweet and dent corn are actively growing, are needed to come to any conclusions regarding the difference in photosynthetic pigment differences in sweet and dent corn. Age of the husks may have also played a role in the inconclusive nature of the paper chromatography experiments.

Differences in protein content of sweet and dent corn plays a significant part in both taste and texture of the corn. The Bradford Assay was performed to determine the total protein concentration in both sweet and dent corn kernels. Concentrations of 10, 30, and 50 microliters were compared to the BSA standard curve. Negative concentrations were observed in both the sweet and dent corn. However, the sweet corn did have higher protein concentrations than dent corn at each of the corn solution concentrations. A significant systematic error may have occurred to account for the negative concentrations. Although, comparisons can still be made between the relative concentrations of sweet and dent corn because the error equally affected both measures. This error is due to proteins that were discovered in the cuvettes used to determine the absorbance standard curve and thus, the protein concentration. Because the BSA sat in the cuvettes for approximately 15 minutes longer than the corn solutions did, the BSA had time to bind to the protein in the cuvette, skewing the results. The same blank was used for both the BSA curve and the corn solutions, and also sat for the extra 15 minutes, accounting for the negative concentrations. Still, sweet corn has higher protein concentrations compared to dent corn. We had predicted that dent corn would have a larger concentration of protein based on the thickness of the outer coating. It may be that the thick coating is not made up of proteins, and is instead some other tough substance. The higher concentration of proteins in sweet corn, though, provide amino acids which meet the basic needs of practically all healthy people (Freeman, 2002) and may contribute to the tradition of eating sweet corn instead of dent corn.

PPO is an enzyme found in many plants that accounts for the browning color of damaged fruits and vegetables. A polyphenyloxidase (PPO) experiment was not performed because the group “Grilled Chesse Sandwiches” in the Spring of 2003 had already performed PPO on corn and did not obtain any results. (Yancy, 2003). Further, we did not expect to find PPO in the corn based on our observations of damaged portions of corn that did not turn brown.

Salivary amylase is an enzyme found in saliva and begins the digestion of carbohydrates (Freeman, 2002). Knowing that dent corn has a much harder kernel than sweet corn, we tested the effect our own saliva containing salivary amylase would have on the corn kernels. The saliva did not seem, at first, to have a large effect on either type of corn. To examine the breakdown of the carbohydrates in the corn kernels, both solutions were once again tested for starch using the Iodine test. The dent corn, originally detected as containing starch, gave a negative result for the iodine test after the addition of the saliva. This is most likely because the starch had been broken down into its component sugars. The sweet corn, however, gave a positive result. The reasons for this are unclear, however, the amount of saliva used for each of the types of corn was not measured. Because of this, it is likely that not enough salivary amylase was used to break down the starch. The lack of starch before the presence of salivary amylase and the presence of starch after the addition of saliva may be attributed to the dilutions of the solutions. A much higher concentration of corn was used in the original Iodine test than in the post-saliva Iodine test. Having less corn in solution could have allowed for easier access of IKI to the starch present in the sweet corn and may indicate a low concentration of starch in sweet corn, requiring dilution for detection.

Throughout the experiments, human error is likely to have occurred. Improperly washed lab materials, mismeasured solutions, and inadequate variance of trial concentrations are all possibilities. Further tests could be conducted, such as quantitative sugar tests, more accurate pigment identification tests, and the Bradford Assay with non-protein containing cuvettes.

Sweet corn was identified as having a different composition than dent corn. Sweet corn contained more reducing sugars, monosaccharides, and less starch than the dent corn contributing to the sweeter taste. In terms of photosynthesis, pigments were identifiable for the sweet corn husks and no results could be obtained for the dent corn. Barring error due to age or otherwise, this supports our hypothesis that the presence of more sugars in sweet corn is a result of more photosynthetic pigments. The protein concentration was higher in the sweet corn than in the dent corn, adding to the nutritional value. In addition, the energy for protein synthesis ultimately comes from photosynthesis, further supporting the theory that sweet corn contains more photosynthetic pigments. The salivary amylase test reinforced the higher starch concentration in dent corn because of its evidence of the breakdown of starch. In conclusion, the presence of reducing sugars, monosaccharides, and less starch, and higher protein content, and subsequent sweeter taste and higher nutritional value of sweet corn may account for its human preference over dent corn.